Methods and compositions for spinal fusion

Abstract

Provided herein are methods of using isolated vertebral skeletal stem cells (vSSCs) to promote osteogenesis. The compositions and methods disclosed herein can be used to improve outcomes of orthopedic procedures, e.g., during spine fusion procedures to maximize the fusion mass and thereby prevent pseudarthrosis.

Description

[0001] METHODS AND COMPOSITIONS FOR SPINAL FUSION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 624,680, filed January 24, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] TECHNICAL FIELD

[0005] Provided herein are methods of using isolated vertebral skeletal stem cells (vSSCs) to promote osteogenesis. The compositions and methods disclosed herein can be used to improve outcomes of orthopedic procedures, e.g., during spine fusion procedures to maximize the fusion mass and thereby prevent pseudarthrosis.

[0006] BACKGROUND

[0007] Vertebral bone is subject to a distinct set of disease processes from those of long bones, notably including a much higher rate of solid tumor metastases that cannot be explained by passive blood flow distribution alone. The basis for this distinct biology of vertebral bone has remained elusive.

[0008] Spine fusion is a common orthopedic procedure, and multiple cellular adjuncts have been studied for their ability to improve fusion outcomes with limited efficacy. However, the specific stem cell mediating spine fusion has thus far remained unknown. Therefore, there exists a need for cell-based compositions and methods for promoting osteogenesis and generating the osteoblasts mediating spine fusion, thereby improving the outcomes of spinal fusion procedures.

[0009] SUMMARY

[0010] Disclosed herein are compositions and methods for promoting osteogenesis and generating the osteoblasts mediating spine fusion. Here we identify a vertebral skeletal stem cell (vSSC), co-expressing the transcription factors ZIC1 and PAX1 together with additional cell surface markers, whose expression profile and function are markedly distinct from those of long bone skeletal stem cells (IbSSCs). vSSCs display formal evidence of sternness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. Lineage tracing of vSSCs confirms that they make a persistent contribution to multiple mature cell lineages in the native vertebrae. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and sternness. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed clinically in breast cancer. Specifically, when an organoid system is used to place both vSSCs and IbSSCs in an identical anatomic context, vSSC-lineage cells are more efficient than IbSSC-lineage cells at recruiting metastases, a phenotype that is due in part to increased secretion of the novel metastatic trophic factor MFGE8. Similarly, genetically targeting loss-of-function to the vSSC lineage results in reduced metastasis rates in the native vertebral environment. Taken together, vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of metastatic seeding of the vertebrae.

[0011] In some embodiments, the disclosure provides isolated vertebral skeletal stem cells (vSSCs), wherein said cells express the transcription factors ZIC1 and PAX1 and wherein said cells, when administered to a subject in need thereof: (1) promote the proliferation of osteogenic cells; or (2) promote the generation of osteoblasts in vertebrae; or (3) promote the generation of a spine fusion mass. In some embodiments, expression of ZIC1 and PAX1 is determined by flow cytometry. In some embodiments, expression of ZIC1 and PAX1 is determined by qPCR.

[0012] In some embodiments, the disclosure provides an isolated population of cells comprising the isolated vSSCs. In some embodiments, at least 50% of the cells in said population are vSSCs. In some embodiments, at least 90% of the cells in said population are vSSCs. In some embodiments, the disclosure provides a pharmaceutical composition comprising the isolated vSCCs. In some embodiments, the disclosure provides a permanent or degradable decellularized or synthetic matrix or scaffold comprising the vSCCs.

[0013] In some embodiments, the disclosure provides methods of improving the outcome of an orthopedic procedure in an individual, comprising administering a population of isolated vSCCs to said individual in an amount and for a time sufficient for detectable improvement of one or more outcomes of said orthopedic procedure. In some embodiments, the orthopedic procedure is a spine fusion procedure.

[0014] In some embodiments, the disclosure provides methods of improving the outcome of an orthopedic procedure in an individual, comprising administering a population of cells comprising isolated vertebral skeletal stem cells (vSSCs) to said individual in an amount and for a time sufficient for detectable improvement of one or more outcomes of said orthopedic procedure. In some embodiments, the orthopedic procedure is a spine fusion procedure. In some embodiments, the vSCCs express the transcription factors ZIC1 and PAX1.

[0015] In some embodiments, the vSCCs, when administered to the individual: (1) promotes the proliferation of osteogenic cells; or (2) promotes the generation of osteoblasts in vertebrae; or (3) promotes the generation of a spine fusion mass.

[0016] In some embodiments, expression of ZIC1 and PAX1 is determined by flow cytometry. In some embodiments, expression of ZIC1 and PAX1 is determined by qPCR. In some embodiments, the population of cells is administered to a surgical site. In some embodiments, the population of cells is administered to a surgical site during a spine fusion procedure. In some embodiments, at least 50% of the cells in the population of cells are vSCCs. In some embodiments, at least 90% of the cells in the population of cells are the cells are vSCCs.

[0017] In some embodiments, the disclosure provides pharmaceutical compositions comprising isolated vSCCs that express the transcription factors ZIC1 and PAX1. In some embodiments, the disclosure provides a permanent or degradable decellularized or synthetic matrix or scaffold comprising isolated vSCCs that express the transcription factors ZIC1 and PAX1.

[0018] In some embodiments, the disclosure provides methods of promoting the proliferation of osteogenic cells in a subject, the subject having undergone a spine fusion procedure, the method comprising administering to the subject a population of cells comprising isolated vSSCs. In some embodiments, one or more of the vSSCs express the transcription factors ZIC1 and PAX1.

[0019] In some embodiments, the disclosure provides methods of promoting the generation of osteoblasts in a vertebrae of a subject, the subject having undergone a spine fusion procedure, the method comprising administering to the subject a population of cells comprising isolated vSSCs. In some embodiments, one or more of the vSSCs express the transcription factors ZIC1 and PAX1.

[0020] In some embodiments, the disclosure provides methods of promoting the generation of a spine fusion mass in a subject, the subject having undergone a spine fusion procedure, the method comprising administering to the subject a population of cells comprising isolated vSSCs. In some embodiments, one or more of the vSSCs express the transcription factors ZIC1 and PAX1.

[0021] In some embodiments, the population of cells is administered to a surgical site. In some embodiments, the population of cells is administered to a surgical site during the spine fusion procedure. In some embodiments, the isolated vSSCs are allogenic to the subject. In some embodiments, the isolated vSSCs are autologous to the subject. In some embodiments, the pharmaceutical composition is administered by injection to the subject.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0023] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0024] DESCRIPTION OF DRAWINGS

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] FIG. 1 is a principal component analysis (PCA) plot of RNA-Seq following FACS of Lin-THYl-6C3-CD200+CD105- populations from the vertebrae (candidate vSSCs) and long bone (IbSSCs) of PIO mice.

[0027] FIG. 2 is a volcano plot highlighting the differentially expressed transcriptional factors in candidate vSSCs and IbSSCs (FDR<0.05).

[0028] FIG. 3 is a plot of flow cytometry analysis of the mGFP+ Zicl -lineage cells within immunophenotypic SSCs (Lin-THYl-6C3-CD200+CD105-EMB- populations) in lumbar vertebrae (L1-L6) and femurs from female Zicl -ere mTmG mice at PIO. n=4.

[0029] FIG. 4 is a scatter plot of flow cytometry analysis of H2B-GFPhighcells (label retaining cells) before and after doxycycline treatment. Representative of 4 biologic replicates.

[0030] FIG. 5 is a series of plots of flow cytometry analysis of vSSC-derived cell populations after the first round and second round of intramuscular transplantation. Plots are representative of 3 independent experiments.

[0031] FIG. 6A is a 3D reconstruction of L5 vertebra pCT from 4- week-old male Osxzlcland Osxf / fmice. Scale bar, 100 pm.

[0032] FIG. 6B is a series of plots of quantification of bone volume / total volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of L5 vertebra in 4-week-old male Osxzlcland Osxf / fmice. n=6 per group, data are presented as mean±s.d., two-tailed unpaired t test. FIG. 7A is a 3D reconstruction of L5 vertebrae (top) and femur (bottom) pCT from 8-week-old female Stat3zlcland Stat3f / fmice. Scale bar, 100 pm.

[0033] FIG. 7B is a series of plots of quantification of bone volume / total volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of L5 vertebrae (top) and femur (bottom) in 8-week-old female Stat3zlcland Stat3f / fmice. Stat3f / flumbar, n=7; Stat3zlcllumbar, n=9; Stat3f / ffemur, n=6; Stat3zlclfemur, n=7. data are mean±s.d., two-tailed unpaired t test.

[0034] FIG. 8A is a 3D reconstruction of L5 vertebra (top) and femur (bottom) pCT from 8-week-old male Shn3zlcland Shn3f / fmice. Scale bar, 100 pm.

[0035] FIG. 8B is a series of plots of quantification of bone volume / total volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of L5 vertebrae (top) and femur (bottom) in 8-week-old male Shn3zlcl(n=7) and Shn3f / fmice (n=8). Data are mean±s.d., two-tailed unpaired t test.

[0036] FIG. 9A is a plot of quantification of metastasis rates to long bones and vertebrae of 12- week-old female mice 3-4 weeks after caudal artery injection with Py8119 cells (4 weeks, n=32 C57BL6 / J mice). Chi-square test.

[0037] FIG. 9B is a plot of quantification of metastasis rates to long bones and vertebrae of 12-week-old female mice 3-4 weeks after caudal artery injection with 4T1.2 cells (4 weeks, n=30 BALB / c mice). Chi-square test.

[0038] FIG. 9C is a plot of quantification of metastasis rates to long bones and vertebrae of 12-week-old female mice 3-4 weeks after caudal artery injection with EO771 cells (3 weeks, n=25 C57BL6 / J mice). Chi-square test.

[0039] FIG. 10 is a plot of quantification of the metastasis rate of Py8119 cells to IbSSC- derived and vSSC-derived bone organoids 3 weeks after cancer cell injection. n=40. Chi- square test.

[0040] FIG. 11 is a plot of quantification of Py8119 transwell migration in the presence of MFGE8 at 0 ng / ml (n=5), 100 ng / ml (n=4) or 1000 ng / ml (n=4) in the lower chamber. Data are mean ± s.d., one-way ANOVA followed by Tukey’s multiple comparison test. FIG. 12 is a plot of qPCR analysis of PAX1 and ZIC1 in FACS isolated human vSSCs (n=6) and IbSSCs (Lin- THYl-B2M / HLAabc+CD200+CD105-EMB-, n=8). Data are presented as mean ± s.d., two-tailed unpaired t test.

[0041] FIG. 13 A shows pCT imaging of L4-6 fusion in a 16-week-old Zicl-cre mTmG mouse that underwent L4-6 spine fusion without an iliac crest graft or demineralized bone matrix implantation.

[0042] FIG. 13B shows histological imaging of L4-6 fusion in a 16-week-old Zicl-cre mTmG mouse that underwent L4-6 spine fusion without an iliac crest graft or demineralized bone matrix implantation. mGFP+ Zicl vSSC-lineage morphologic osteoblasts and osteocytes are indicated by arrows.

[0043] FIG. 14A shows pCT imaging of L4-6 fusion in a 16-week-old male SlmS^11Zicl-cre (right) or Zicl-cre controls (left) mouse that underwent spinal fusion. Bone mass in the fusion was determined by pCT as shown in the 3D reconstruction.

[0044] FIG. 14B shows a plot of quantitative determination of the volume of bone in the fusion mass shown in FIG. 14A outside of the underlying vertebrae. N = 6 / group, mean + / - SD. p = 0.0025 by an unpaired two-tailed Student’s t-test.

[0045] DETAILED DESCRIPTION

[0046] Provided herein are methods of using isolated vertebral skeletal stem cells (vSSCs) to promote osteogenesis. The compositions and methods disclosed herein can be used to improve outcomes of orthopedic procedures, e.g., during spine fusion procedures to maximize the fusion mass and thereby prevent, e.g., pseudarthrosis. Here we identify a vertebral skeletal stem cell (vSSC), co-expressing the transcription factors ZIC1 and PAX1 together with additional cell surface markers, whose expression profile and function are markedly distinct from those of long bone skeletal stem cells (IbSSCs). vSSCs display formal evidence of sternness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. Lineage tracing of vSSCs confirms that they make a persistent contribution to multiple mature cell lineages in the native vertebrae. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and sternness. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed clinically in breast cancer. Specifically, when an organoid system is used to place both vSSCs and IbSSCs in an identical anatomic context, vSSC- lineage cells are more efficient than IbSSC-lineage cells at recruiting metastases, a phenotype that is due in part to increased secretion of the novel metastatic trophic factor MFGE8. Similarly, genetically targeting loss-of-function to the vSSC lineage results in reduced metastasis rates in the native vertebral environment. Taken together, vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of metastatic seeding of the vertebrae.

[0047] In some embodiments, improvement in outcomes of orthopedic procedures, wherein the individual is administered the vSSCs or therapeutic compositions comprising the vSSCs, can be assessed or demonstrated by detectable improvement in one or more, indicia of osteogenesis, for example, demonstration of increased mineralization of bone, e.g. of vertebrae, or increase bone mass, e.g., of vertebrae, or decrease in the incidence of pseudarthrosis after orthopedic procedures, e.g. after spinal fusion procedures, as compared to the individual prior to administration of vSSCs or as compared to the individual in the absence of administration of vSSCs.

[0048] Success of administration of the vSSCs is not, in certain embodiments, based on survival in the individual of the administered vSSCs. Success is, instead, based on one or more metrics of improvement of outcomes of orthopedic procedures, e.g., outcomes of spine fusion procedures, as noted above.

[0049] In certain embodiments, the methods of treatment provided herein comprise inducing the vSSCs to differentiate along an osteogenic lineage.

[0050] Administration of vSSCs, or therapeutic compositions comprising such cells, to an individual in need thereof, can be accomplished, e.g., by transplantation, implantation (e.g., of the cells themselves or the cells as part of a matrix-cell combination), injection (e.g., directly to the site of the orthopedic procedure, for example, directly to one or more vertebrae of an individual who is undergoing a spinal fusion procedure), infusion, delivery via catheter, or any other means known in the art for providing cell therapy.

[0051] In some embodiments, the therapeutic cell compositions are provided to an individual in need thereof, for example, by injection into one or more sites in the individual. In a specific embodiment, the therapeutic cell compositions are provided by injection into the spine during a spine fusion procedure, e.g., to one or more vertebrae. In other specific embodiments, vSSCs are introduced by a device or as part of a spinal implant.

[0052] Also provided herein are kits for use in the improvement of outcomes of orthopedic procedures, e.g., outcomes of spine fusion procedures. The kits provide a therapeutic cell composition comprising vSSCs, which can be prepared in a pharmaceutically acceptable form, for example by mixing with a pharmaceutically acceptable carrier, and an applicator, along with instructions for use. Ideally the kit can be used in the field, for example in a physician's office, or in a surgical suite. In a specific embodiment, the methods of treatment provided herein comprise administering vSSCs, e.g., a therapeutic composition comprising the cells, to a patient undergoing an orthopedic procedure, e.g., a spine fusion procedure; and evaluating the patient for improvements in outcomes of the orthopedic procedure, wherein the therapeutic cell composition is administered as a matrix-cell complex. In certain embodiments, the matrix is a scaffold, preferably bioabsorbable, comprising at least the cells.

[0053] To this end, further provided herein are populations of vSSCs contacted with, e.g., incubated or cultured in the presence of, one or more factors that stimulate stem or progenitor cell differentiation along an osteogenic pathway. Such factors include, but are not limited to factors, such as growth factors, chemokines, cytokines, cellular products, demethylating agents, and other factors which are now known or later determined to stimulate differentiation, for example of stem cells, along osteogenic pathways or lineages. In certain embodiments herein, vSSCs contact with one or more factors as identified above may become osteogenic cells, or progenitors. Preferably at least some of the cells integrate at least partially into a recipient's bone tissue, including but not limited to vertebrae at the site of a spinal fusion procedure. In certain embodiments, the differentiated vSCCs differentiate into cells exhibiting two or more characteristics of osteogenic cells or their progenitors and are able to partially or fully integrate into a recipient's bone tissue. In certain embodiments, the vSSCs act to promote the differentiation of stem cells naturally present in the patient's bone tissue to themselves differentiate into, for example, bone tissue comprising a fusion mass. vSSCs, and populations of such cells, can be provided therapeutically to an individual, e.g., an individual undergoing an orthopedic procedure, e.g., a spinal fusion procedure.

[0054] In certain embodiments, the individual is administered a therapeutically effective amount of vSSCs, e.g., in a population of cells that comprise the vSSCs. In a specific embodiment, the population comprises about 50% vSSCs. In another specific embodiment, the population is a substantially homogeneous population of vSSCs. In other embodiments the population comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, 95%, 98% or 99% vSSCs.

[0055] In certain embodiments, the isolated stem cells are isolated vSSCs. In certain other embodiments, the isolated stem cells are isolated vertebral multipotent cells. In some embodiments, the isolated stem cells, e.g, vSSCs, express the transcription factor ZIC1. In some embodiments, the isolated stem cells, e.g, vSSCs, express the transcription factor PAX1. In some embodiments, the isolated vSSCs co-express the transcription factors ZIC1 and PAX1 as detected by flow cytometry. In another specific embodiment, the isolated vSSCs have the potential to differentiate into cells of a osteogenic phenotype, and / or cells of a chondrogenic phenotype.

[0056] Gene profiling confirms that isolated vSSCs, and populations of isolated vSSCs, are distinguishable from other cells, e.g., mesenchymal stem cells, e.g., bone marrow- derived mesenchymal stem cells. The isolated vSSCs described herein can be distinguished from, e.g., mesenchymal stem cells on the basis of the expression of one or more genes, the expression of which is significantly higher in the isolated vSSCs, in comparison to other stem cells. In particular, the isolated vSSCs, useful in the methods provided herein, can be distinguished from other stem cells on the basis of the expression of one or more genes, the expression of which is significantly higher (for example, at least twofold higher) in the isolated vSSCs than in an equivalent number of other stem cells, wherein the one or more genes are ZIC1 and PAX1.

[0057] Expression of the above-referenced genes can be assessed by standard techniques. For example, probes based on the sequence of the gene(s) can be individually selected and constructed by conventional techniques. Expression of the genes can be assessed, e.g., on a microarray comprising probes to one or more of the genes, e.g., an Affymetrix GENECHIP® Human Genome U133 A 2.0 array, or an Affymetrix GENECHIP® Human Genome U133 Plus 2.0 (Santa Clara, California). Expression of these genes can be assessed even if the sequence for a particular GenBank accession number is amended because probes specific for the amended sequence can readily be generated using well- known standard techniques.

[0058] The level of expression of these genes can be used to confirm the identity of a population of isolated vSSCs, to identify a population of cells as comprising at least a plurality of isolated vSSCs, or the like. Populations of isolated vSSCs, the identity of which is confirmed, can be clonal, e.g., populations of isolated vSSCs expanded from a single isolated vSSCs, or a mixed population of stem cells, e.g., a population of cells comprising solely vSSCs that are expanded from multiple isolated vSSCs, or a population of cells comprising isolated vSSCs, as described herein, and at least one other type of cell.

[0059] Isolated vSSCs can be selected on the basis of the level of expression of one or more such genes as compared to the level of expression in said one or more genes in, e.g., a mesenchymal stem cell control, for example, the level of expression in said one or more genes in an equivalent number of bone marrow- derived mesenchymal stem cells. In one embodiment, the level of expression of said one or more genes, e.g., ZIC1 and / or PAX1, in a sample comprising an equivalent number of mesenchymal stem cells is used as a control. In another embodiment, the control, for vSSCs tested under certain conditions, is a numeric value representing the level of expression of said one or more genes in a different population of other stem cells under said conditions.

[0060] Populations of isolated vSSCs, or populations of cells comprising the isolated vSSCs, can be formulated into pharmaceutical compositions for use in vivo, e.g., in the methods provided herein. Such pharmaceutical compositions comprise a population of isolated vSSCs, or a population of cells comprising isolated vSSCs, in a pharmaceutically-acceptable carrier, e.g., a saline solution or other accepted physiologically-acceptable solution for in vivo administration.

[0061] Pharmaceutical compositions comprising the isolated vSSCs described herein can comprise any, or any combination, of the isolated vSSC populations, or isolated vSSCs, described elsewhere herein. The pharmaceutical compositions can comprise fetal, maternal, or both fetal and maternal isolated placental cells. The pharmaceutical compositions provided herein can further comprise isolated vSSCs obtained from a single individual, or from a plurality of individuals. In some embodiments, the pharmaceutical compositions provided herein can comprise isolated vSSCs obtained from the patient undergoing the orthopedic procedure, including subsequence administration the patient undergoing the orthopedic procedure.

[0062] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0063] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manners using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0064] The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or procedure, the general medical condition of each patient, and the resulting method of administration.

[0065] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. Such penetrants are generally known in the art. As discussed above, in some embodiments, the pharmaceutical composition is formulated in an injection vehicle comprising sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, HSA, and water.

[0066] Compositions are also provided that include a scaffold, such as a polymeric carrier and / or an extracellular matrix, and an effective amount of the vSSCs generated by the methods disclosed herein. The matrix material is generally physiologically acceptable and suitable for use in in vivo applications. For example, the physiologically acceptable materials include, but are not limited to, solid matrix materials that are absorbable and / or nonabsorbable, such as small intestine submucosa (SIS), crosslinked or non-crosslinked alginate, hydrocolloid, foams, collagen gel, collagen sponge, polyglycolic acid (PGA) mesh, fleeces and bioadhesives. Suitable polymeric carriers also include porous meshes or sponges formed of synthetic or natural polymers, as well as polymer solutions. For example, the matrix is a polymeric mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; and polysaccharides such as alginate and polymers of hyaluronic acid. Synthetic polymers include both biodegradable and nonbiodegradable polymers. For example, biodegradable polymers include polymers of hydroxyl acids such as polyactic acid (PLA), polyglycolic acid (PGA) and polylactic acid-glycolic acid (PGLA), polyorthoesters, polycaprolactone (PCL), polyanhydrides, polyphosphazenes, and combinations thereof. Nonbiodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohols.

[0067] The pharmaceutical compositions provided herein can comprise any number of isolated vSSCs. For example, a single unit dose of isolated vSSCs can comprise, in various embodiments, about, at least, or no more than 1 x 105, 5 x 105, 1 x 106, 5 X 106, 1 X 107, 5 X 107, 1 X 108, 5 X 108, 1 X 109, 5 X 109, 1 X IO10, 5 X IO10, 1 X 1011or more isolated vSSCs.

[0068] In some embodiments, the subject is a human being and the vSSCs cells are from a human origin. In some embodiments, the cells are non-syngeneic with the subject. According to one embodiment, the cells are allogeneic with the subject. According to one embodiment, the cells are xenogeneic with the subject. According to one embodiment, the cells are syngeneic with the subject (e.g. autologous).

[0069] The cell suspension of vSSCs may be obtained by any mechanical or chemical (e.g. enzymatic) means. Several methods exist for dissociating cell clusters to form cell suspensions (e.g. single cell suspension) from primary tissues, attached cells in culture, and aggregates, e.g., physical forces (mechanical dissociation such as cell scraper, trituration through a narrow bore pipette, fine needle aspiration, vortex disaggregation and forced filtration through a fine nylon or stainless steel mesh), enzymes (enzymatic dissociation such as trypsin, collagenase, Accutase and the like) or a combination of both.

[0070] According to the present invention, the cell suspension of differentiated cells comprises viable cells. Cell viability may be monitored using any method known in the art, as for example, using a cell viability assay (e.g. MultiTox Multiplex Assay available from Promega), Flow cytometry, Trypan blue, etc.

[0071] Typically, the vSSCs are immediately used in a surgical procedure. However, in situations in which the cells are to be maintained in suspension prior to the surgical procedure, e.g., for 1-72 hours, the cells may be placed in a culture medium, suspension medium, or vehicle that is capable of supporting their viability and / or efficacy. Such a medium can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which can benefit maintaining the isolated population of cells in a viable state. For example, a suspension medium according to this aspect of the disclosure can be a salt solution such as Balanced Salt Solution Combination 2 or a synthetic tissue culture medium such as RPMI-1640 (Life Technologies, Israel), Ko-DMEM (Gibco-Invitrogen Corporation products, Grand Island, NY, USA), DMEM / F12 (Biological Industries, Beit Haemek, Israel), Mab ADCB medium (Hy Clone, Utah, USA) or DMEM / F12 (Biological Industries, Beit Haemek, Israel) supplemented with the necessary additives.

[0072] EXAMPLES

[0073] Example 1 - Identifying Vertebral Skeletal Stem Cell (vSSC) Markers

[0074] To identify specific markers of vertebral stem cells (vSSC), it was noted that skeletal stem cells (SSCs) at multiple anatomic sites, including calvarium, long bone periosteum, and endosteum share a core set of surface markers, all being Lineage- CD200+CD105-THY1-6C3-CD51+ cells. To identify candidate genes that would, in combination with this immunotype, uniquely mark candidate vSSCs, cells bearing this immunophenotype were sorted from vertebrae and long bones (long bone SSCs, IbSSCs) and subjected to transcriptional profiling. As shown in FIG. 1, this transcriptional profiling revealed that candidate vSSCs and IbSSCs displayed broadly different transcriptional profiles, supporting that candidate vSSCs represent a distinct cell type.

[0075] Example 2 - Defining vSSC Immunophenotype

[0076] Transcriptome analysis was also used to refine the SSC immunophenotypic definition, identifying EMBIGIN (EMB) as a marker of non-stem osteoblast and chondrocyte-lineage cells contaminating current SSC definitions.

[0077] We sought additional markers that could be used to identify and exclude non-stem populations present in current SSC definitions. Cell surface markers expressed in candidate vSSCs and IbSSCs were screened, ultimately finding that EMB marked a distinct subset of both populations. Transcriptome analysis shows the EMB+ fraction of SSCs expresses much higher levels of osteoblast lineage markers including Alpl, Sppl, Runx2, and Sp7 / Osterix, and hypertrophic chondrocyte markers including CollOal and Ihh, than the EMB- fraction. In line with this, immunostaining validated that both osteocalcin+ committed osteoblast lineage cells and hypertrophic chondrocytes express EMB. Additionally, FACS of Osteocalcin-cre (Ocn-cre) mTmG mice identifies a very high degree (mGFP+EMB+ / Total mGFP+ >90%) of concordance between the EMB+ fraction of vSSCs and IbSSCs and the Ocn-cre mGFP+ fraction of cells. In transplantation studies, the EMB-fraction of candidate vSSCs was able to generate the EMB+ fraction of vSSCs, but the EMB+ fraction was not able to generate the EMB- fraction of vSSCs, demonstrating that the EMB+ fraction of SSCs does not sit at the apex of their differentiation hierarchy. Thus, EMB identifies non-stem cell types contaminating current stem cell immunophenotypic definitions and thereby refines the definition of SSCs. Hereafter candidate vSSCs were defined as Lin-CD200+CD105- THY1-6C3-EMB cells that are further fractionated based on genetic lineage reporters.

[0078] Example 3 - Transcription Profiling of vSSCs

[0079] Transcriptional profiling identified a set of genes that showed selective or differential expression in postnatal candidate vSSCs but not IbSSCs. Notably, this included a number of genes reflecting the somitic origins of the vertebrae such as Paxl, Zicl, Pax9 and Prdm69-11, postnatally.

[0080] Next, genes comprising a specific transcriptional “signature” for vSSCs that can be followed as a mark of vSSC identity were established, with Dpt and Ramp2 showing robust differential expression in vSSCs versus IbSSCs. Next, among the genes selectively expressed in vSSCs, we additionally sought genes that could be used to construct ere lines selectively targeting vSSCs, focusing on transcription factors (FIG. 2). These transcription factors were screened for their ability to drive expression of vSSC transcripts when their expression was enforced in IbSSCs. Through this, we found that Zicl (Zinc finger in cerebellum 1), which is best known as a transcription factor regulating cerebellar development, displayed highly specific expression in candidate vSSCs but not IbSSCs. Zicl was able to enforce expression of the vSSC-specific markers, including Dpt and Ramp2. The transcription factor Paxl was also prioritized for further evaluation based on both displaying highly selective expression in vSSCs and not IbSSCs and longstanding observations that “undulated” mice with spontaneous mutations in Paxl display vertebral defects.

[0081] Example 4 - ZIC1 and PAX1 Expression in vSSCs

[0082] Based on these findings, a Zicl -ere and a Paxl-creERT2 were constructed, finding that both provide selective labeling of vertebrae when bred to the mTmG reporter allele. Within the skeleton, Zicl -ere provided absolute specificity for vertebrae versus long bones, with no detectable long bone signal (FIG. 3). Consistent with vSSCs being Zicl -positive, Zicl -ere labeled multiple skeletal lineages, including endplate cartilage, osteoblasts, marrow adipocytes, marrow LepR+ cells, Nestin+ cells and the annulus fibrosis surrounding the intervertebral disc. Similar labeling was observed throughout the spinal column, from cervical to sacral vertebrae. Flow cytometry showed a corresponding diversity in Zicl -lineage cells, with Zicl -ere labeling candidate vSSCs in addition to other populations similar to reported non-stem populations in long bones, such as CD 105+ or THY1+ cells.

[0083] For Paxl, a creERT2 strategy was selected to avoid any labeling observed being due to the known expression of Paxl in the developmental precursors of the vertebrae. Pulse-chase labeling in Paxl-creERT2 mice demonstrated that sparse initial labeling was seen in the vertebral endplate cartilage, with subsequent spread of the labeled clones through the proliferating cartilage columns over the subsequent month, ultimately leading to labeling of subchondral osteoblasts. This pattern of labeling indicates that the most immature Paxl -lineage cells localize to the resting zone of the endplate cartilage and progress through proliferative and hypertrophic chondrocytes before generating osteoblasts. This physical progression of Paxl-creERT2 labeled cells from the resting zone of the endplate, through proliferating and hypertrophic chondrocytes to osteoblasts also paralleled a differentiation sequence observed by flow cytometry, with initial labeling of EMB- vSSC populations that over time progressed to labeling of EMB+CD200+ mature cells. Similar to observations in Zicl-cre mice, Paxl-creERT2 mice displayed no detectable labeling of skeletal cells in long bones, and Paxl- creERT2 labeled cells displayed the ability to generate adipocytes in bone organoids. Immunofluorescence staining of Zicl-cre- labeled populations for PAX1 demonstrated that the majority of endplate Zicl -lineage cells at the resting zone are also PAX1 positive. Similarly, FACS-isolated Zicl -lineage vSSCs, but not vertebral hematopoietic lineage cells nor IbSSCs, displayed robust co-expression of Zicl and Paxl. In line with the restriction of Paxl expression to the endplate region housing vSSCs, Paxl mRNA expression is lost upon in vitro differentiation of vSSCs into osteoblasts, whereas Zicl expression is maintained upon vSSC differentiation into osteoblasts. Thus, multiple lines of evidence indicate that, with reference to immunophenotypic vSSCs, Zicl and Paxl label highly overlapping populations, therefore these two mouse strains offer complementary approaches to examine a Paxl+Zicl+ candidate vSSC.

[0084] Example 5 - Evaluating Sternness of vSSCs

[0085] Given this evidence, the sternness of these candidate vSSC was next interrogated, finding that serial transplantation, in vivo clonality assays, and label retention studies all converge on vSSCs representing true stem cells. First, the clonality of the contribution of Paxl -lineage vSSCs to vertebral cellularity was confirmed using Paxl-creERT2; Rosa26confetti mice, observing a clonal contribution, which was particularly evident in proliferating chondrocytes. Label retention studies using both “tet-off ’ (R26-M2rtTA; TetOP-H2B-GFP) and “tet-on” (R26-tTA; TetOP-H2B-GFP) pulse chase systems revealed preferential label retention within the vertebrae after a 6-month chase period in a Paxl+ population within the cartilage endplate. Flow cytometry showed that these label retaining cells are vSSCs (FIG. 4). This simultaneous flow cytometry and imaging analysis further supports the localization of vSSCs to the resting zone of the cartilage endplate. Due to the lower efficiency of labeling with the inducible Paxl-creERT2 system, Zicl-cre was used for experiments requiring isolation of vSSCs. First, FACS isolated Zicl -lineage vSSCs formed bone organoids after transplantation, a functional signature of SSCs. Both vSSCs and IbSSCs robustly retain their respective signature distinguishing transcriptional features after co-transplantation to form a mixed organoid containing both cell types prior to re-isolation. Thus, vSSCs and IbSSCs retained their respective transcriptional features and lineage identity despite being placed together into an identical environmental context. Similar to IbSSCs, vSSC-derived organoids included bone matrix-forming osteoblasts, an internal marrow space including both host-derived hematopoietic cells and graft-derived marrow adipocytes, and cartilage, demonstrating the in vivo multipotency of Zicl -lineage vSSCs. While EMB-vSSC-derived organoids included durable cartilage tissue that persisted at late timepoints, cartilage in Zicl -lineage EMB+CD200+ cell-derived organoids were only transiently present. Additionally, Zicl -lineage vSSCs, but not Zicl -lineage EMB+CD200+ cells were uniquely able to both self-renew and reconstitute their entire lineage after transplantation (FIG. 5). Moreover, Zicl -lineage vSSCs reisolated after the first round of transplantation were capable of again reconstituting the entire Zicl -lineage, demonstrating that the Zicl- lineage vSSCs present after the first round of transplantation do not merely retain their immunophenotype, but moreover retained their original differentiation potential (FIG. 5). Taken together, vSSCs display all the major sternness features demonstrated for IbSSCs.

[0086] Example 6 - Functional Disruption of Zicl -Lineage vSSCs

[0087] Ablating the contribution of Zicl -lineage vSSCs to the pool of bone forming osteoblasts demonstrates their physiologic importance for vertebral bone formation. Conditional deletion of Osterix (Sp7), a transcription factor absolutely required for osteoblast differentiation, with Zicl-cre selectively blocks the contribution of Zicl lineage vSSCs to bone formation. The resulting Zicl-cre Osxfl / flmice display severe vertebral defects, including paraplegia with 100% penetrance and accompanying limb unloading effects, starting from 2 weeks of age due to spine instability. This corresponded to severe kyphoscoliosis and an absence of the majority of the dorsal neural arch. Additionally, the vertebral body was substantially impacted, with an approximately 50% reduction in bone mass (FIGs. 6A-6B). While this phenotype was severe, the residual presence of vertebral bone in Zicl-cre Osxfl / flmice raised the possibility that an additional, second vertebral stem cell may exist. As the paresis associated with the severe spinal instability phenotype of Zicl-cre Osxfl / flmice could potentially confound assessment of bone mass, we also sought to generate mice with a less severe attenuation of Zicl -lineage vSSC osteogenic capacity. STAT3 is a positive regulator of osteoblast differentiation. Zicl-cre Stat3fl / flmice displayed decreased bone mass in vertebral body but not in long bones, and only a low rate of paraplegia (10%) (FIGs. 7A-7B). Zicl-cre Stat3fl / flmice also exhibited a reduction in vertebral bone formation rate and mineral apposition rate, while vertebral osteoclast numbers and endothelial cell numbers were unchanged. Conversely, deletion of Schnurri3 (Shn3; Hivep3), a cell-intrinsic inhibitor of osteoblast function and bone formation, with Zicl-cre led to an increase in bone mass within the vertebral body but no significant change in long bone parameters (FIGs. 8A- 8B). Thus, Zicl -lineage vSSCs play a specific role in producing the osteoblasts that form the vertebrae.

[0088] To evaluate the role of vSSCs in vertebral metastatic tropism, we first confirmed that preferential vertebral versus long bone tropism is conserved in mice using several metastasis models, including caudal artery injection, intracardiac injection, and spontaneous metastasis models with several syngeneic breast cancer cell lines, including EO771, 4T1.2 and Py8119 cells (FIGs. 9A-9C). This vertebral tropism is also preserved in the ovariectomy model of post-menopausal osteoporosis. In line with the increased ultimate outgrowth of vertebral metastases seen, a greater number of initially seeding tumor cells were present at vertebrae than long bones. Interestingly, the site of initial seeding was predominantly in the marrow space in the primary spongiosum adjacent to the growth plate in long bones and the endplate in vertebrae, placing the initially seeding tumor cells in relative physical proximity to vSSCs and their immediate derivates. We next investigated whether passive alterations in blood flow contribute to the observed differences in tumor cell early seeding. Measurement of relative blood flow rates using fluorescent microspheres identified increased blood flow in long bones versus vertebrae. Thus, the increased early tumor seeding of vertebrae cannot be explained by passive blood flow distribution. Similarly, transmission electron microscopy showed no detectable differences in fenestrations or other ultrastructural features between vertebral and long bone vascular endothelial cells, and differences in the total vertebral versus long bone marrow volume available for metastatic seeding cannot account for the vertebral metastatic preference observed here.

[0089] To identify whether vSSCs provide a basis for the high rates of vertebral versus long bone metastases, we examined the ability of bone organoids derived from Zicl- lineage vSSCs versus IbSSCs to recruit tumor cells in an in vivo competitive seeding assay. vSSCs and IbSSCs were isolated by FACS and transplanted into the thigh musculature in contralateral legs in the same host. After allowing these bone organoids of defined cellular composition to mature and mineralize for 4 weeks, host mice were challenged with Py8119 or 4T1.2 tumor cells by caudal artery injection. Histology demonstrated that tumor cells infiltrated into these bone organoids and were physically adjacent to graft-derived skeletal cells. The numbers of stem and non-stem populations generated in vSSC-derived vs IbSSC-derived organoids were comparable. Even after normalizing the surrounding anatomic context using this organoid system, Zicl -lineage vSSC-derived bone organoids displayed a higher rate of metastatic recruitment than that of IbSSCs (FIG. 10). In addition to showing that vSSC-lineage cells are sufficient to drive increased metastatic seeding using organoids, we sought to establish that vSSC-lineage cells are necessary for the high rates of metastatic seeding in the native vertebral microenvironment. Zicl -ere Stat3fl / flmice were used to attenuate the generation of Zicl- lineage vSSC derived osteoblasts. Zicl -ere Stat3fl / flmice showed a decreased vertebral metastasis rate after caudal artery challenge with Py8119 cells, indicating that vSSC- lineage cells contribute to metastasis in the native vertebral microenvironment. Example 7 - vSSC-derived Mediator of Vertebral Metastatic Tropism

[0090] Based on these findings, we searched for a vSSC-derived mediator of vertebral metastatic tropism, ultimately identifying milk fat globule epidermal growth factor 8 (Mfge8). Mfge8 encodes a secreted protein with higher levels of expression in vSSC- lineage cells than IbSSC-lineage cells. However, no difference in Mfge8 expression was detected between a variety of other vertebral and long bone cell types, including osteoclasts, endothelial cells, and a number of leukocyte populations. MFGE8 was sufficient to induce tumor cell migration in vitro, with Py8119 cells showing a dose dependent migration in response to recombinant MFGE8 in a transwell assay (FIG. 11). Mfge8 was also necessary for tumor cell migration in a co-culture assay. Vertebral bone marrow stromal cells or vSSC-derived osteoblasts induced a greater degree of migration of Py8119 cells in a transwell assay than their long bone counterparts, and this effect was Mfge8 dependent, as the same vertebral and long bone populations from Mfge8- / - mice no longer displayed a differential ability to induce migration. In addition, MFGE8 was able to similarly induce migration of prostate and lung cancer cells. MFGE8 also contributed to vertebral metastasis in vivo, as Mfge8' / _mice displayed markedly reduced rates of vertebral metastasis after caudal artery challenge with Py8119 cells (Fig. 5o, p). In line with Mfge8 being also expressed in IbSSCs, albeit at a lower level than in vSSCs, a more modest decrease in long bone metastases was observed, and Mfge8-deficiency ablated the difference between vertebral and long bone metastasis rates.

[0091] In line with this, the number of early seeding tumor cells in vertebrae also significantly dropped in Mfge8' / _. Mfge8' / _mice displayed no baseline bone phenotype at 8 weeks of age. To demonstrate that the reduced vertebral metastasis phenotype of Mfge8' / _mice reflected vSSC metastatic function, bone organoids were generated from vSSCs derived cells from Mfge8' / _and WT control mice in contralateral thigh musculature within the same hosts before caudal artery challenge with Py8119 cells. In this setting, the Mfge8 deficient vSSC derived organoids displayed a markedly decreased rate of metastatic recruitment. MFGE8 additionally regulated the post-seeding outgrowth of metastatic tumor cells, as Py8119 cells displayed reduced outgrowth after direct injection into the L6 vertebrae of Mfge8' / _hosts. Thus, MFGE8 regulates both early metastatic early seeding and subsequent outgrowth.

[0092] Example 8 - Isolating Human vSSCs

[0093] Next, we identified the human counterparts of murine vSSCs. First, we identified a population in the human endplate co-expressing ZIC1 and PAX1. Flow cytometry also identified a population expressing a similar panel of markers as murine vSSCs (Lin- THY1-CD105-CD200+EMB-, hereafter human vSSCs). Human vSSCs display selective expression of PAX1 and ZIC1 relative to human IbSSCs (FIG. 12), and human vSSCs show similar sternness features as murine vSSCs, having the ability to form bone organoids after xenotransplantation into highly immunodeficient NOD-scid gamma (NSG) mice, including giving rise to chondrocytes and bone matrix forming osteoblasts. After transplantation, human vSSCs maintained themselves and concomitantly differentiated to other populations corresponding to the other cell types observed in the murine Zicl vSSC-lineage. Thus, humans and mice share an analogous vSSC population.

[0094] Example 9 - Involvement of Human vSSCs in Metastases

[0095] To investigate whether human vSSCs display conserved mechanisms of involvement in vertebral metastases as demonstrated in murine studies, we first confirmed that human MDA231- BoM-1833 breast cancer cells are MFGE8 responsive in vitro. Next, FACS isolated human vSSCs were transduced with lentivirus encoding either a shRNA targeting MFGE8 or a GFP control. These transduced cells were then implanted and allowed to form bone organoids before caudal artery challenge with MDA231-BoM-1833 cells. MFGE8-deficient human vSSC-derived organoids displayed reduced metastasis rates, confirming that vSSC-derived MFGE8 is a relevant driver of metastasis in a human xenograft system.

[0096] These data indicate that the initial formation of vertebral bone and its subsequent postnatal maintenance is achieved by a novel stem cell type that is distinct from all previously studied long bone stem cells in terms of function, location and transcriptional program in both mice and humans. Further, a new marker (EMBIGIN) marks non- stem populations within current- definition SSCs within long bones and vertebrae, thereby removing these contaminating mature cells from stem cell preparations and enhancing future skeletal stem cell studies in all anatomic sites.

[0097] Example 10 - Zicl-Expressing vSSCs Mediate Spine Fusion

[0098] We further investigated the role of Zicl+ vSSCs to the development and clinical management of spine disorders, e.g., improving the outcome of an orthopedic procedure in an individual, including promoting the proliferation of osteogenic cells, promoting the generation of osteoblasts in vertebrae, and promoting the generation of a spine fusion mass.

[0099] We developed mouse models of lumbar spine fusion (pCT of L4-6 fusion shown in FIG. 13 A). In this model, the L4-L6 facets were removed with a rongeur, and the lamina, facets and transverse processes were decorticated with a motorized burr. Robust fusion is observed even without additional bone graft implants by 3 weeks post-surgery. Conducting this model in Zicl-cre mTmG mice reveals that the majority of fusion bone mass skeletal cells, including morphologic osteoblasts and osteocytes, are Zicl -lineage mGFP+ cells (histological imaging shown in FIG. 13B). Flow cytometry similarly found that both Zicl+vSSCs as well as downstream Zicl+vSSC derived cell types were abundantly present in the fusion mass.

[0100] Next, we evaluated the functional contribution of Zicl+ vSSCs to spine fusion. Specifically, we deleted Schnurri 3 (SHN3), an intracellular adapter protein in Zicl+ vSSCs by breeding Shn311 / 11Zicl -ere mice, where Zicl+ vSSC-lineage osteoblasts, but not other osteoblasts not derived from Zicl+ vSSCs, will display an increased bone formation capacity. When Shn3f, / flmice undergo spine fusion, they display a marked >3- fold increase in the volume of the fusion bone mass (pCT shown in FIG. 10A, quantitative determination of bone volume shown in FIG. 10B). These results indicate that the Zicl+ vSSC mediate spine fusion and are useful for improving the outcome of an orthopedic procedure in an individual, including promoting the proliferation of osteogenic cells, promoting the generation of osteoblasts in vertebrae, and promoting the generation of a spine fusion mass.

[0101] OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of improving the outcome of an orthopedic procedure in an individual, comprising administering a population of cells comprising isolated vertebral skeletal stem cells (vSSCs) to said individual in an amount and for a time sufficient for detectable improvement of one or more outcomes of said orthopedic procedure.

2. The method of claim 1, wherein the orthopedic procedure is a spine fusion procedure.

3. The method of claim 1 or claim 2, wherein the vSCCs express the transcription factors ZIC1 and PAX1.

4. The method of any one of claims 1-3, wherein the vSCCs, when administered to the individual:(1) promotes the proliferation of osteogenic cells; or(2) promotes the generation of osteoblasts in vertebrae; or(3) promotes the generation of a spine fusion mass.

5. The method of claim 3 or 4, wherein expression of ZIC1 and PAX1 is determined by flow cytometry.

6. The method of claim 3 or 4, wherein expression of ZIC1 and PAX1 is determined by qPCR.

7. The method of any one of claim 1-6, wherein the population of cells is administered to a surgical site.

8. The method of any one of claims 1-7, wherein the population of cells is administered to a surgical site during a spine fusion procedure.

9. The method of any one of claims 1-8, wherein at least 50% of the cells in the population of cells are vSCCs.

10. The method of claim 9, wherein at least 90% of the cells in the population of cells are the cells are vSCCs.

11. A pharmaceutical composition comprising isolated vSCCs that express the transcription factors ZIC1 and PAX1 and a pharmaceutically acceptable carrier.

12. A permanent or degradable decellularized or synthetic matrix or scaffold comprising isolated vSCCs that express the transcription factors ZIC1 and PAX1.

13. A method of promoting the proliferation of osteogenic cells in a subject, the subject having undergone a spine fusion procedure, the method comprising administering to the subject the pharmaceutical composition of claim 11.

14. A method of promoting the generation of osteoblasts in a vertebrae of a subject, the subject having undergone a spine fusion procedure, the method comprising administering to the subject the pharmaceutical composition of claim 11.

15. A method of promoting the generation of a spine fusion mass in a subject, the subject having undergone a spine fusion procedure, the method comprising administering to the subject the pharmaceutical composition of claim 11.

16. The method of any one of claims 13-15, wherein the population of cells is administered to a surgical site.

17. The method of any one of claims 13-16, wherein the population of cells is administered to a surgical site during the spine fusion procedure.

18. The method of any one of claims 13-17, wherein the isolated vSSCs are allogenic to the subject.

19. The method of any one of claims 13-17, wherein the isolated vSSCs are autologous to the subject.

20. The method of any one of claims 13-19, wherein the pharmaceutical composition is administered by injection to the subject.

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