WO2021097538A1 - Bone formation - Google Patents

Abstract

The present disclosure relates to compositions and methods for use in forming bone and in particular to compositions and methods for producing osteoblasts. Specifically, the present disclosure relates to compositions comprising an insulin-like growth factor-binding protein and methods for use thereof. In one aspect, the present disclosure provides a method of producing an osteoblast the method comprising contacting a first cell with IGFBP7 or a fragment thereof thereby to promote conversion of the first cell to an osteoblast.

Description

BONE FORMATION Field of the disclosure

[0001] The present disclosure relates to compositions and methods for use in forming bone and in particular to compositions and methods for producing osteoblasts.

Background of the disclosure

[0002] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Bone is a dynamic tissue and its homeostasis represents a balance between bone formation and bone resorption. In bone formation, adult stem cells differentiate into bone progenitor cells (ie, osteoprogenitor cells) that have the ability to mature into osteoblasts, osteocytes, and form mature bone and mineralized matrix. In bone resorption, osteoclasts (cells that resorb bone tissue) dissolve the mineralized matrix and create cavities on the bone surface. The balance between bone formation and bone resorption is instrumental in the maintenance of healthy bones.

[0004] Despite the capacity for bone tissue to rejuvenate itself, repairing non-union bone fractures and regenerating bone defects remains a major challenge. Indeed, bone is now second only to blood as the most transplanted tissue.

[0005] Bone grafts, where bone is harvested from a patient (eg, from the hip, leg or calvarial bones) and re-applied to a bone defect, fracture or void, are commonly used to repair bone injuries that cannot be efficiently repaired through natural processes, However, the amount of graft substance available to fill a bone defect is limited by the amount of bone that can be harvested from a patient. Moreover, the bone harvesting procedure has been associated with patient morbidity, surgical blood loss, and is not generally suitable for patients with an underlying bone disease that compromises the quality of the graft. Pathogen transmission also presents a risk, particularly with allografts.

[0006] Artificial biomaterials have shown promise as bone graft alternatives, however, most biomaterials have poor biointegration (do not resorb) and brittleness due to the lack of biological components in the composition. [0007] In this context there is need for compositions and methods for promoting bone formation and/or treating bone disorders.

Summary of the disclosure

[0008] The present disclosure relates to compositions and methods for promoting bone formation and/or treating bone disorders. In work leading to the present disclosure, the inventors have surprisingly found that insulin-like growth factor-binding protein 7 (IGFBP7) can promote conversion of differentiated cells such as fibroblasts to osteoblasts.

[0009] In a first aspect, the present disclosure provides a method of producing an osteoblast the method comprising contacting a first cell with IGFBP7 or a fragment thereof thereby to promote conversion of the first cell to an osteoblast.

[0010] In some examples, the first cell is a somatic cell. For example, the first cell may be a fibroblast. The fibroblast may be a skin fibroblast. The skin fibroblast may be a foreskin fibroblast. The first cell may be a human cell. Preferably, the first cell is not genetically modified.

[0011] In some examples, the IGFBP7 or fragment thereof promotes transdifferentiation of the first cell to an osteoblast.

[0012] In some examples, the conversion is carried out in a medium comprising the IGFBP7 or fragment thereof. The medium may comprise IGFBP7 or fragment thereof at a concentration of between about 250 ng/mL and 1500 ng/ml_.

[0013] In certain examples, the medium is an osteogenic-conditioned medium. In certain examples, the method is performed for a period of between about 5 days and about 30 days. The IGFBP7 or fragment thereof may be isolated, recombinant or synthesised.

[0014] The IGFBP7 or fragment thereof may comprise the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

[0015] In a second aspect, the present disclosure provides an osteoblast produced by the method of the first aspect.

[0016] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the osteoblast of the second aspect and a pharmaceutically acceptable carrier.

[0017] In a fourth aspect, the present disclosure provides a method of treating a bone disorder in a subject the method comprising administering to the subject an osteoblast produced by the method of the first aspect.

[0018] In some examples, the bone disorder is a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer or osteoporosis. For example, the bone disorder may be a bone fracture. The osteoblast may be administered at or adjacent the bone fracture.

[0019] In certain examples, the osteoblast is transdifferentiated from a fibroblast obtained from the subject.

[0020] In a fifth aspect, the present disclosure provides a method of treating a bone disorder in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof.

[0021] In certain examples, the bone disorder is a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer or osteoporosis. For example, the bone disorder may be a bone fracture. The IGFBP7 or fragment thereof may be administered at or adjacent the bone fracture.

[0022] In some examples, the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

[0023] In a sixth aspect, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject an osteoblast produced by the method of the first aspect.

[0024] In some examples, the osteoblast is transdifferentiated from a fibroblast obtained from the subject.

[0025] In some examples, the subject suffers from a bone disorder selected from the group consisting of a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer and osteoporosis. [0026] In a seventh aspect, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof.

[0027] In some examples, the subject suffers from a bone disorder selected from the group consisting of a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer and osteoporosis,

[0028] In some examples, the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

[0029] In an eighth aspect, the present disclosure provides a cell culture medium comprising IGFBP7 or a fragment thereof.

[0030] In certain examples, the medium is suitable for culturing fibroblasts. The medium may be suitable for culturing osteoblasts. In some examples, the medium is suitable for culturing fibroblasts and osteoblasts.

[0031] In some examples, the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

[0032] In some examples, the medium is a conditioned medium. For example, the medium may be an osteogenic-conditioned medium.

[0033] In some examples, the medium comprises isolated, recombinant or synthesised IGFBP7 or a fragment thereof.

[0034] In some examples, the medium comprises the IGFBP7 or fragment thereof at a concentration of between about 250 ng/mL and 1500 ng/ml_.

[0035] The medium may further comprise one or more of ascorbic acid, fetal calf serum, b- glicerophosphate and dexamethasone.

[0036] In a ninth aspect, the present disclosure provides an isolated, recombinant or synthesised IGFBP7 molecule or a fragment thereof.

[0037] In some examples, the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

[0038] In a tenth aspect, the present disclosure provides an isolated or recombinant nucleic acid encoding IGFBP7 or a fragment thereof

[0039] In some examples, the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

[0040] In an eleventh aspect, the present disclosure provides an expression vector comprising the nucleic acid of the tenth aspect.

Brief description of drawings

[0041] Figure 1. A-D: Osteogenic gene expression in cells grown on conditioned medium collected from either osteoblasts (OB-CM) or fibroblasts (FB-CM). E: Alizarin staining of fibroblasts grown on OB-CM.

[0042] Figure 2, Comparative results of protein mass spectrometry performed on FB-CM and OB-CM.

[0043] Figure 3. A-D: Osteogenic gene expression in cells grown in various concentrations of IGFBP7, E: Alizarin staining of fibroblasts grown in various concentrations of IGFBP7.

[0044] Figure 4. IGFBP7-treatment of fibroblasts shows formation of mineralized tissue in vivo. A: IGFBP7-treated fibroblasts versus untreated controls. B-C: 3D reconstructed images from MicroCT analysis. D-E: Images of histological sections stained with Von Kossa.

[0045] Figure 5. Induction of senescence plays a role in IGFBP7 reprograming of fibroblasts to osteoblasts. A-C: IGFBP7 significantly increased expression of key senescence-associated genes ( P16 , P21 and P53). D-F: IGFBP7 significantly increased expression of SASP-associated genes (IL-1a, TNF-o and IL-6). [0046] Figure 6. The senescence inhibitor, rapamycin, antagonizes IGFBP7 reprogramming in fibroblast. A-C: Rapamycin decreased expression of senescence- associated genes ( P16 , P21 and P53). D-F: Rapamycin decreased expression of SASP- associated genes ( IL-1a , TNF-a and IL-6I) GJ : Rapamycin decreased expression of osteogenic genes ( Runx2 , BMP-2, bone sialoprotein and osteocalcin). K: Matrix mineralization and bone nodules formation were also impaired in cells treated with rapamycin.

Detailed description

Definitions

[0047] The articles "a" and "an" are used herein to refer to one or to more than one [i.e. to at least one) of the grammatical object of the article. By way of example, "a cell" means one cell or more than one cell.

[0048] The term "about" is understood to refer to a range of +/- 10%, preferably +/- 5% or +/- 1% or, more preferably, +/- 0.1%.

[0049] The terms "administration concurrently" or "administering concurrently" or "co administering" and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By "simultaneously" is meant that the active agents are administered at substantially the same time, and preferably together in the same formulation.

[0050] As used herein, the term "allogeneic" refers to tissue, cells or stem cells being genetically different, but deriving from the same species.

[0051] As used herein, the term "autologous" refers to tissue, cells or stem cells that are derived from the same subject's body.

[0052] A "cell culture medium" (also referred to herein as a "culture medium" or "medium" in certain contexts) is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art. A "conditioned medium" is one that has previously been used for culturing cells. For example, an osteoblast-conditioned medium is a medium that has previously been used to culture osteoblasts.

[0053] The terms "comprise", "comprises", "comprised" or "comprising", "including" or "having" and the like in the present specification and claims are used in an inclusive sense, ie, to specify the presence of the stated features but not preclude the presence of additional or further features.

[0054] "Cell differentiation" or "differentiation" refers to a process wherein a cell that exhibits a less specialised state of differentiation (eg, a pluripotent stem cell) becomes a cell that exhibits a more specialised state of differentiation (eg, a fibroblast or keratinocyte). "Transdifferentiation" refers to a process wherein a cell that exhibits a more specialised state of differentiation is directly converted to another, different cell having a more specialised state of differentiation. The term "conversion" when used herein in the context of cell type conversion, refers to a process wherein one cell type becomes a different cell type. Conversion may be used to describe the re-programming of a somatic cell to a stem cell. Conversion may also be used to describe cell differentiation or transdifferentiation.

[0055] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (ie, % homology = # of identical positions/total # of positions x 100), with optimal alignment determined taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0056] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program, using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J . Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0057] The term "isolated'' as used herein refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide" as used herein refers to a polynucleotide which has been purified from the sequences which flank it in a naturally-occurring state, eg, a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, ie, it is not associated with in vivo substances.

[0058] A "genetically modified" or "genetically engineered" cell as used herein refers to a cell into which an exogenous nucleic acid has been introduced by a process involving the hand of man (or a descendant of such a cell that has inherited at least a portion of the nucleic acid). The nucleic acid may, for example, contain a sequence that is exogenous to the cell, it may contain native sequences (ie, sequences naturally found in the cells) but in a non-naturally occurring arrangement (eg, a coding region linked to a promoter from a different gene), or altered versions of native sequences, etc.

[0059] The term "pharmaceutically acceptable" as used herein refers to substances that do not cause substantial adverse allergic or immunological reactions when administered to a subject. A "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, coatings, dispersion agents, wetting agents, isotonic and absorption delaying agents and disintegrants.

[0060] "Prevention" includes reduction of risk, incidence and/or severity of a condition or disorder. The terms "treatment" and “treat" include both prophylactic or preventive treatment (that prevent and/or slow the development of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The terms "treatment" and "treat" do not necessarily imply that a subject is treated until total recovery. The terms "treatment" and "treat" also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms "treatment” and "treat" are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As non-limiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional.

[0061] The term "recombinant polynucleotide" as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.

[0062] The term "recombinant polypeptide” as used herein refers to a polypeptide made using recombinant techniques, ie, through the expression of a recombinant polynucleotide.

[0063] "Similarity" refers to the percentage of amino acids that are identical or constitute conservative substitutions as set out in Tables 1 and 2. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0064] "Stem cells" are cells that are capable of self-renewal and of giving rise to more differentiated cells. As known in the art, a "pluripotent" cell has the ability to differentiate into or give rise to cells derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm) and typically has the potential to divide in vitro for a long period of time, eg, greater than one year or more than 30 passages. A "multipotent" cell is a cell that is able to differentiate into some but not all of the cells derived from all three germ layers, Adult stem cells are multipotent cells. Adult stem cells include, for example, hematopoietic stem cells and neural stem cells.

[0065] As used herein, the term "induced pluripotent stem cell" (iPSC) is understood to mean a pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing forced expression of specific genes. The term encompasses pluripotent cells, that, like embryonic stem (ES) cells, can be cultured for a long period of time while maintaining the ability to differentiate into all types of cells in an adult organism, but that, unlike ES cells (which are derived from the inner cell mass of blastocysts), are derived from somatic cells which had a narrower, more defined potential and that in the absence of experimental manipulation could not give rise to all types of cells in the organism.

[0066] Many methods are known in the art that can be used to generate pluripotent stem cells from somatic cells, Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described. Mouse somatic cells can be converted to embryonic stem cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, eg, Takahashi and Yamanaka. 2006. Cell 126(4): 663-76. Human iPSCs can be obtained using similar transduction methods, and the transcription factors OCT4, SOX2 and NANOG have been described as important regulators of pluripotency; see, eg, Budniatzky and Gepstein. Stem Cells Transl. Med. 2014. 3(4):448-57; Barrett et a/. 2014. Stem Cells Trans. Med. 3:1-6 sctm.2014-0121; Focosi et a/. Blood Cancer J ournal. 2014. 4: e211. The production of iPSCs can be achieved by introducing nucleic acid sequences encoding stem cell- associated genes into an adult, somatic cell, eg, using a viral vector. iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell. Reprogramming may be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct- 3/4 or Pouf51), Soxl, Sox2, Sox3, Soxl5, Soxl8, Nanog, Klfl, Klf2, Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert and LIN28. To confirm the induction of pluripotent stem cells, the cells can be tested for the expression of a stem cell marker. Such expression in a cell derived from a somatic cell identifies the cell as an induced pluripotent stem cell. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl and Natl. In one case, for example, a cell that expresses Oct4 or Nanog is identified as pluripotent, The pluripotent stem cell character of isolated cells can also be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers. iPSCs often have a human ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli.

[0067] A "therapeutically effective amount" is at least the minimum concentration or amount required to effect a measurable improvement of a particular condition (eg, a bone disorder). A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the relevant composition (eg, IGFBP7 or a fragment thereof, or an osteoblast of the present disclosure) to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

Insulin-like growth factor-binding proteins

[0068] Insulin-like growth factors (IGFs) are growth-modulating peptides that act as endocrine hormones and autocrine/paracrine growth factors. A family of proteins called the insulin-like growth factor binding proteins (IGFBPs) bind to IGFs, increasing their half-life in circulation and blocking their ability to bind to the insulin receptor. The IGFBP family in humans comprises six members (IGFBP1-6) all of which share a conserved structure including a highly conserved cysteine-rich N-terminal domain, a linker, and a cysteine-rich C- terminal domain that is also highly conserved. The N- and C-terminal domains are both globular structures, stabilised by disulfide bonds between the conserved cysteine residues, and both domains contribute to the IGF-binding site. The central linker domain is unstructured and can include functional motifs.

[0069] Insulin-like growth factor-binding protein 7 (IGFBP7) is not generally considered to be a member of the core IGFBP family, in part, because it lacks the C-terminal domain found in IGFBP1-6, and because its affinity for IGF-1 is at least 100 times lower than IGFBP family members (Hwa et al. 1999. Endocrine Reviews. 20(6): 761-787). It does, however, bind to insulin with high affinity.

[0070] IGFBP7 is a secreted protein and comprises an N-terminal cysteine-rich domain, a heparin binding site, a Kazal-type trypsin inhibitor domain and a C-terminal Ig-like type C repeat (Oh etal. 1996. J . Bioi. Chem. 271: 30322-30325; Collet and Candy. 1998. Mol. Cell. Endocrinol. 139: 1-6). IGFBP7 is sometimes referred to by other names such as Mac25, TAF, PSF, angiomodulin and insulin-like growth factor-binding protein-related protein 1 (IGFBP-rPl), owing to independent isolation and characterisation experiments, as well as different naming conventions.

[0071] IGFBP7 plays diverse regulatory roles in humans including the regulation of cell growth, apoptosis and angiogenesis. IGFBP7 expression has also been linked to cancer development such as lung, breast and pancreatic cancer as well as hepatocellular carcinoma, oesophageal adenocarcinoma and head and neck squamous cell carcinomas (Wajapeyee et al. 2008. Cell. 132: 363-374; Chen et al. 2007. J . Pathol. 211: 431-438; Smith et al. 2007. Clin. Cancer Res. 13: 4061-4068; An et al. 2012. Ann. Surg. Oncol. 19: 3971- 3978; Tomimaru et al. 2012. Int. J . Cancer 130: 319-327; Smith et al. 2014. Br. J .

Cancer. 110: 775-782). [0072] In humans, IGFPB7 exists in a number of different forms. It predominantly exists as a 282 amino acid isoform comprising the following sequence:

MERPSLRALLLGAAG LLLLLLPLSSSSSSDTCGPCEPASCPPLPPLGCLLG ETRDA CGCCPMCARGEGEPCGGGGAGRGYCAPGMECVKSRKRRKGKAGAAAGGPGVS

GVCVCKSRYPVCGSDGTTYPSGCQLRAASQRAESRGEKAITQVSKGTCEQGPSIV TPPKDIWNVTGAQVYLSCEVIGIPTPVLIWNKVKRGHYGVQRTELLPGDRDNLAIQT RGGPEKHEVTGWVLVSPLSKEDAGEYECHASNSQGQASASAKITVVDALHEIPVK KGEGAEL (SEQ ID NO. 1).

[0073] A 279 amino acid isoform comprising the following sequence has also been detected:

MERPSLRALLLGAAG LLLLLLPLSSSSSSDTCGPCEPASCPPLPPLGCLLG ETRDA

CGCCPMCARGEGEPCGGGGAGRGYCAPGMECVKSRKRRKGKAGAAAGGPGVS

GVCVCKSRYPVCGSDGTTYPSGCOLRAASQRAESRGEKAITQVSKGTCEQGPSIV

TPPKDIWNVTGAQVYLSCEVIGIPTPVLIWNKVKRGHYGVQRTELLPGDRDNLAIQT

RGGPEKHEVTGWVLVSPLSKEDAGEYECHASNSQGQASASAKITVVDALHEIPVK

KGTQ (SEQ ID NO. 2).

[0074] Posttranscriptional deamination of adenosine to inosine also gives rise to R78G and K95R IGFBP7 isoforms, In each case, the protein is referred to as IGFBP7 and the present disclosure is not limited to any particular isoform. In certain examples, the IGFBP7 or fragment thereof of the present disclosure comprises an amino acid sequence having at least about 50% identity to the sequence set forth in SEQ ID NO. 3. For example, the IGFBP7 or fragment thereof of the present disclosure comprises a sequence having at least about 55% identity to the sequence set forth in SEQ ID NO. 3, or at least about 60% identity to the sequence set forth in SEQ ID NO. 3, or at least about 65% identity to the sequence set forth in SEQ ID NO. 3, or at least about 70% identity to the sequence set forth in SEQ ID NO. 3, or at least about 75% identity to the sequence set forth in SEQ ID NO. 3, or at least about 80% identity to the sequence set forth in SEQ ID NO. 3, or at least about 85% identity to the sequence set forth in SEQ ID NO. 3, or at least about 90% identity to the sequence set forth in SEQ ID NO. 3, or at least about 95% identity to the sequence set forth in SEQ ID NO. 3, or at least about 96% identity to the sequence set forth in SEQ ID NO. 3, or at least about 97% identity to the sequence set forth in SEQ ID NO. 3, or at least about 98% identity to the sequence set forth in SEQ ID NO. 3, or at least about 99% identity to the sequence set forth in SEQ ID NO. 3, or 100% identity to the sequence set forth in SEQ ID NO. 3. [0075] Those skilled in the art will understand that a full-length IGFBP7 polypeptide may be used in the methods and compositions of the present disclosure or a fragment of IGFBP7 may be used. The fragment will have similar osteogenic properties as the full-length IGFBP7, but will be shorter in length. In certain examples, the IGFBP7 fragment may be between about 10 and 282 amino acids in length, such as between about 10 and 279 amino acids, or between about 10 and 257 amino acids, or between about 10 and 250 amino acids, or between about 10 and 240 amino acids, or between about 10 and 230 amino acids, or between about 10 and 220 amino acids, or between about 10 and 210 amino acids, or between about 10 and 200 amino acids, or between about 10 and 190 amino acids, or between about 10 and 180 amino acids, or between about 10 and 170 amino acids, or between about 10 and 160 amino acids, or between about 10 and 150 amino acids, or between about 10 and 140 amino acids, or between about 10 and 130 amino acids, or between about 10 and 120 amino acids, or between about 10 and 110 amino acids, or between about 10 and 100 amino acids, or between about 10 and 90 amino acids, or between about 10 and 80 amino acids, or between about 10 and 70 amino acids, or between about 10 and 60 amino acids, or between about 10 and 50 amino acids, or between about 10 and 40 amino acids, or between about 15 and 40 amino acids, or between about 15 and 35 amino acids, or between about 15 and 30 amino acids, or between about 15 and 25 amino acids. In certain examples, the IGFBP7 fragment is less than 282 amino acids in length, such as less than about 279 amino acids, or less than about 257 amino acids, or less than about 250 amino acids, or less than about 240 amino acids, or less than about 230 amino acids, or less than about 220 amino acids, or less than about 210 amino acids, or less than about 200 amino acids, or less than about 190 amino acids, or less than about 180 amino acids, or less than about 170 amino acids, or less than about 160 amino acids, or less than about 150 amino acids, or less than about 140 amino acids, or less than about 130 amino acids, or less than about 120 amino acids, or less than about 110 amino acids, or less than about 100 amino acids, or less than about 90 amino acids, or less than about 80 amino acids, or less than about 70 amino acids, or less than about 60 amino acids, or less than about 50 amino acids, or less than about 40 amino acids, or less than about 30 amino acids, or less than about 20 amino acids in length.

[0076] Variant IGFBP7 and fragments thereof may contain conservative amino acid substitutions at various locations relative to a naturally occurring IGFBP7. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Those skilled in the art will understand that different amino acids can be grouped based on the properties of their side chains. Such groupings are set out below.

[0077] Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

[0078] Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (eg, histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

[0079] Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (ie, glutamic acid, aspartic acid, arginine, lysine and histidine).

[0080] Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

[0081] Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

[0082] Certain amino acids may also be characterized as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally- occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the a- carbon. For the purposes of the present disclosure, however, proline is considered to be a "small” amino acid.

[0083] Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub- classification according to this scheme is presented in Table 1.

Table 1

[0084] Conservative amino acid substitutions are also grouped based on amino acid side chains, For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change alters the activity of a variant of IGFBP7 or a fragment thereof can readily be determined using, eg, the methods described herein. Conservative substitutions are shown in Table 2 under the heading of exemplary and preferred substitutions.

Table 2

[0085] Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G. Biochemistry, third edition, Wm.C. Brown Publishers (1993).

[0086] Accordingly, the present disclosure also contemplates as variants of naturally occurring IGFBP7 polypeptide sequences or fragments thereof, wherein the variants are distinguished from the naturally occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. Preferably, variants will display at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99% similarity to a parent or reference IGFBP7 peptide or polypeptide sequence as, for example, set forth in SEQ ID NO: 1, 2 or 3, as determined by sequence alignment programs described elsewhere herein using default parameters. Variants of a naturally occurring IGFBP7 polypeptide or fragment thereof which fall within the scope of a variant polypeptide may differ from the naturally occurring molecule generally by as much 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 amino acid residues or suitably by as few as 10, 9, 8, 7, 6, 5 4, 3, 2 or 1 amino acid residue(s). In some examples, a variant polypeptide differs from a corresponding sequence set forth in any one of SEQ ID NOs: 1 to 3 by at least 1 but by less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues. In other examples, it differs from the corresponding sequence in any one of SEQ ID NOs: 1 to 3 by at least one 1% but less than or equal to 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% of the residues. If the sequence comparison requires alignment, the sequences are typically aligned for maximum similarity or identity. "Looped" out sequences from deletions or insertions, or mismatches, are generally considered differences. The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution, as discussed in more detail below.

[0087] The IGFBP7 polypeptides and fragments thereof described herein may also comprise other modifications such as amino acids with modified side chains, incorporation of unnatural amino acid residues and/or their derivatives during peptide, polypeptide or protein synthesis and the use of cross-linkers and other methods which impose conformational constraints. Examples of side chain modifications include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBhU; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ; and trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS). The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatization, by way of example, to a corresponding amide. The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4- nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N-bromosuccinimide. Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. The imidazole ring of a histidine residue may be modified by N-carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

[0088] Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t- butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated by the present disclosure is shown in Table 3.

Table 3

[0089] IGFBP7 polypeptides and fragments thereof may be prepared by any suitable procedure known to those of skill in the art. For example, the IGFBP7 or fragment thereof may be produced by purifying the IGFBP7 or fragment from a naturally-occurring reservoir such as a cell population or tissue (eg, a mammalian cell population or tissue). Methods of purification include size exclusion, affinity or ion exchange chromatography/separation. Alternatively, the IGFBP7 or fragment thereof may be synthesised by chemical synthesis, eg, using solution synthesis or solid phase synthesis as described, eg, in Chapter 9 of Atherton and Shepard (Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989) and in Roberge et al. 1995. Science. 269: 202. Alternatively, the IGFBP7 or fragment thereof may be produced recombinantly, eg, by expressing a recombinant polynucleotide encoding the IGFBP7 or fragment in a suitable host cell.

Cells and methods for producing osteoblasts

[0090] The present disclosure provides methods and compositions for converting a first cell to an osteoblast. The first cell is preferably a somatic cell, and may be obtained by well- known methods from bodily fluids (eg, blood or urine) or various organs, eg, skin such as foreskin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc., generally from any organ or tissue containing live somatic cells. For example, skin cells may be collected from the border of a surgical incision, eg, during an accompanying surgical procedure, or using a traditional skin punch as a stand-alone procedure. Skin could be collected from any area, including, but not limited to, collection from the scalp, forearm or foreskin. Mammalian somatic cells that may be useful in the compositions and methods of the present disclosure include, for example, fibroblasts, sertoli cells, granulosa cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, endothelial cells, hepatocytes, adipocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle cells, etc., generally any living somatic cells. Preferably, the first cell is a fibroblast, such as a skin fibroblast (eg, a foreskin fibroblast). Fibroblasts are a group of extracellular matrix- and collagen-producing cell which are abundant in the connective tissue of animals including humans. They play important roles in wound healing. Within the skin, dermal fibroblasts produce and organise the extracellular matrix of the dermis and communicate with other cell types, playing an important role in regulating skin physiology.

[0091] As described herein, the first cell (eg, the somatic cell) can be converted to an osteoblast by contacting the first cell with IGFBP7 or a fragment thereof. Those skilled in the art will be aware of several ways by which osteoblast development can be detected and measured. For example, calcium deposition can be assayed by alizarin red staining (see, eg, Example 1). Calcium deposition can also be assayed by von Kossa staining. In one non limiting example of von Kossa staining, cells may be fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with water. 5% silver nitrate can then be added and the cells exposed to UV radiation for about 30 minutes. 5% sodium thiosulfate may be used to reduce non-specific staining.

[0092] Alkaline phosphatase assays and/or osteocalcin assays can also be used to measure osteoblast development. In one non-limiting example of an alkaline phosphatase assay, cells may be lysed in a suitable buffer, and the lysate (eg, 10 pl_) is then incubated with p-nitrophenyl phosphate substrate solution at 37°C for about 30 minutes, The reaction can be stopped by adding 0.5 N NaOH and the absorbance can be measured at 405 nm on a microplate reader. For alkaline phosphatase staining, cells can be fixed, for example, with 4% paraformaldehyde for about 30 minutes, and washed with PBS. Fixed cells can then be stained with an alkaline phosphatase staining solution, which may be obtained from a commercial supplier.

[0093] The expression of certain genes can also be indicative of osteoblast development. Such genes may include, for example, RUNX2, SP7, BSP, COL1A1, BMP-2, bone sialoprotein and osteocalcin (OCN).

[0094] It will be appreciated that the compositions and methods of the present disclosure enable conversion of a first cell such as a somatic cell to an osteoblast without the need for genetic engineering of the first cell. Accordingly, the first cell of the present disclosure is preferably not genetically modified. It will further be appreciated that the compositions and methods described herein enable transdifferention of a first cell (eg, a fibroblast) to an osteoblast without the formation of a pluripotent stem cell.

[0095] In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote conversion of the fibroblast to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast, wherein the fibroblast is not genetically modified. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast, wherein the fibroblast is not genetically modified, and wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3. Although the present disclosure describes methods for producing an osteoblast, it will be understood that the disclosure, including the claims, encompasses methods of producing a plurality of osteoblasts. A plurality of osteoblasts may be produced using one first cell or a plurality of first cells.

[0096] In other examples, the first cell may be a stem cell such as a pluripotent stem cell or a multipotent stem cell. Accordingly, in some examples, the present disclosure provides a method of producing an osteoblast the method comprising contacting a stem cell with IGFBP7 or a fragment thereof thereby to promote conversion of the stem cell to an osteoblast. The stem cell may be a bone marrow-derived stem cell (eg, a bone marrow- derived mesenchymal stem cell) or an iPSC.

[0097] The present disclosure also provides cell culture media comprising IGFBP7 or a fragment thereof. The media may be used to induce conversion (eg, transdifferentiation) of a first cell (eg, a fibroblast) to an osteoblast. Alternatively, or in addition, the media may be used to culture cells such as osteoblasts. The media of the present disclosure preferably comprises IGFBP7 or a fragment thereof at a concentration of at least about 5 ng/mL such as at least about 20 ng/mL, or at least about 40 ng/mL, or at least about 50 ng/mL, or at least about 60 ng/mL, or at least about 70 ng/mL, or at least about 80 ng/mL, or at least about 90 ng/mL, or at least about 100 ng/mL, or at least about 125 ng/mL, or at least about 150 ng/mL, or at least about 175 ng/mL, or at least about 200 ng/mL, or at least about 225 ng/mL, or at least about 250 ng/mL, or at least about 275 ng/mL, or at least about 300 ng/mL, or at least about 325 ng/mL, or at least about 350 ng/mL, or at least about 375 ng/mL, or at least about 400 ng/mL, or at least about 425 ng/mL, or at least about 450 ng/mL, or at least about 475 ng/mL, or at least about 500 ng/mL, or at least about 525 ng/mL, or at least about 550 ng/mL, or at least about 575 ng/mL, or at least about 600 ng/mL, or at least about 625 ng/mL, or at least about 650 ng/mL, or at least about 675 ng/mL, or at least about 700 ng/mL, or at least about 725 ng/mL, or at least about 750 ng/mL, or at least about 775 ng/mL, or at least about 800 ng/mL, or at least about 825 ng/mL, or at least about 850 ng/mL, or at least about 875 ng/mL, or at least about 900 ng/mL, or at least about 925 ng/mL, or at least about 950 ng/mL, or at least about 975 ng/mL, or at least about 1000 ng/mL, or at least about 1250 ng/mL, or at least about 1500 ng/mL, or at least about 1750 ng/mL, or at least about 2000 ng/mL, or at least about 2500 ng/mL, or more. Preferably, the media of the present disclosure comprises IGFBP7 or a fragment thereof at a concentration of more than about 500 ng/mL. In some examples, the media may comprise IGFBP7 or a fragment thereof at a concentration of between about 5 ng/mL and 5000 ng/mL, such as between about 10 ng/mL and 3000 ng/mL, or between about 15 ng/mL and 2500 ng/mL, or between about 20 ng/mL and 2000 ng/mL, or between about 20 ng/mL and 2000 ng/mL, or between about 40 ng/mL and 2000 ng/mL, or between about 50 ng/mL and 1750 ng/mL, or between about 60 ng/mL and 1750 ng/mL, or between about 75 ng/mL and 1750 ng/mL, or between about 100 ng/mL and 1750 ng/mL, or between about 150 ng/mL and 1750 ng/mL, or between about 200 ng/mL and 1750 ng/mL, or between about 250 ng/mL and 1750 ng/mL, or between about 250 ng/mL and 1500 ng/mL, or between about 300 ng/mL and 1500 ng/mL, or between about 500 ng/mL and 1500 ng/mL, or between about 750 ng/mL and 1500 ng/mL, or between about 750 ng/mL and 1000 ng/mL. The IGFBP7 or fragment thereof is preferably isolated, recombinant or synthetic. The cell culture medium may also comprise any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. In certain examples, the cell culture medium of the present disclosure comprises IGFBP7 or a fragment thereof and foetal calf serum (FCS). In certain examples, the cell culture medium of the present disclosure comprises IGFBP7 or a fragment thereof and one or more of ascorbic acid (eg, l-ascorbic acid phosphate), FCS (or fetal bovine serum), b- glicerophosphate and dexamethasone.

[0098] The media may be used to produce an osteoblast from, eg, a fibroblast. In that regard, the present disclosure provides methods for producing an osteoblast the method comprising contacting a first cell such as a fibroblast with IGFBP7 or a fragment thereof so as to promote conversion of the first cell to an osteoblast. The method may be performed by incubating the first cell (eg, the fibroblast) in media comprising IGFBP7 or a fragment thereof for a period of between about 2 hours and 60 days, for example, between about 12 hours and 60 days, or between about 1 day and 60 days, or between about 1 day and 50 days, or between about 2 days and 50 days, or between about 5 days and 50 days, or between about 5 days and 40 days, or between about 10 days and 40 days, or between about 15 days and 40 days, or between about 20 days and 40 days, or between about 20 days and 35 days, or between about 20 days and 30 days, or between about 25 days and 30 days.

[0099] In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a first cell in a cell culture medium comprising IGFBP7 or a fragment thereof thereby to promote conversion of the first cell to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast in a cell culture medium comprising IGFBP7 or a fragment thereof thereby to promote conversion of the fibroblast to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast in a cell culture medium comprising IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast in a cell culture medium comprising IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. wherein the medium comprises the IGFBP7 or fragment thereof at a concentration of between about 250 ng/mL and 1500 ng/mL. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast for a period of between about 5 days and 30 days in a cell culture medium comprising between about 250 ng/mL and 1500 ng/mL IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast in an osteoblast-conditioned medium thereby to promote transdifferentiation of the fibroblast to an osteoblast. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast in an osteoblast-conditioned medium for a period of between about 10 days and 20 days thereby to promote transdifferentiation of the fibroblast to an osteoblast, The osteoblast-conditioned medium may be supplemented with isolated, recombinant or synthesised IGFBP7. In certain examples, the present disclosure provides a method of producing an osteoblast the method comprising culturing a fibroblast in an osteoblast- conditioned medium comprising between about 250 ng/mL and 1500 ng/mL IGFBP7 or a fragment thereof for a period of between about 10 days and 20 days (eg, about 14 days) thereby to promote transdifferentiation of the fibroblast to an osteoblast.

Compositions and treatments

[0100] The present disclosure provides compositions and methods for promoting bone formation and treating bone disorders, Bone disorders that may be treated in accordance with the present disclosure include, eg, a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer and osteoporosis. Bone fractures that may be treated using the compositions and methods of the present disclosure include non-union fractures, simple fractures, greenstick fractures, compound fractures, comminuted (multifragmentary) fractures, impacted fractures, complicated fractures, hairline fractures, compression fractures, fatigue fractures and/or pathological fractures. Examples of bone fractures that may be advantageously treated by the method of the disclosure include, but are not limited to, fractures of the spine, leg and arm. A further example of a fracture that may be advantageously treated in accordance with the present disclosure is a vertebral compression fracture. Such fracture occurs when one or more of the bones of the vertebral column fractures or collapses, typically when the vertebrae are already weakened for instance as a result of ageing or a disease that weakens bone, such as osteoporosis, Paget's disease or bone cancer.

[0101] Osteoblasts also give rise to alveolar bone around the roots of teeth. Accordingly, in some examples, the IGFBP7 or fragment thereof, or the osteoblasts of the present disclosure may by useful in the treatment of a dental or maxillofacial disorder.

[0102] The compositions and methods of the present disclosure may be used to treat a subject suffering from an imbalance in bone formation and resorption. Imbalance of bone formation and resorption usually causes loss of bone mass and can lead to bone related diseases, such as osteoporosis, rickets and osteomalacia. These bone diseases are associated with increased risk of bone fractures, increased severity of fractures and protracted time periods for healing. Additionally, with age or injury the incidence of disc degenerative disease or deformity of the spine is increased, leading to spondylolisthesis.

[0103] The present disclosure also provides a method of treating a bone disorder and/or promoting bone formation in a subject the method comprising administering to the subject an osteoblast produced using the methods described herein. The osteoblast is preferably transdifferentiated from a fibroblast obtained from the subject. In other words, the subject's own fibroblasts are transdifferentiated using the methods described herein to produce osteoblasts which are then administered to the subject.

[0104] In certain examples, the present disclosure provides a method of treating a bone disorder in a subject the method comprising administering to the subject an osteoblast produced by a method described herein. For example, the present disclosure provides a method of treating a bone disorder in a subject the method comprising administering to the subject an osteoblast, wherein the osteoblast has been produced by contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. For example, the present disclosure provides a method of treating a bone fracture in a subject the method comprising administering to the subject an osteoblast, wherein the osteoblast has been produced by contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. In other examples, the present disclosure provides a method of treating a bone fracture in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof. In some examples, the present disclosure provides a method of treating a bone disorder (eg, a bone fracture) in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof, wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3. In some examples, the present disclosure provides a method of treating a bone disorder such as a bone fracture in a subject the method comprising administering to the subject: an osteoblast, wherein the osteoblast has been produced by contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast; and isolated, recombinant or synthesised IGFBP7 or a fragment thereof. In some examples, the present disclosure provides a method of treating a bone disorder such as a bone fracture in a subject the method comprising administering to the subject an osteoblast, wherein the osteoblast has been produced by culturing a fibroblast in an osteoblast-conditioned medium thereby to promote transdifferentiation of the fibroblast to an osteoblast.

[0105] In certain examples, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject an osteoblast produced by a method described herein. For example, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject an osteoblast, wherein the osteoblast has been produced by contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. For example, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject an osteoblast, wherein the osteoblast has been produced by contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast. In other examples, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof. In some examples, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof, wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3. In some examples, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject: an osteoblast, wherein the osteoblast has been produced by contacting a fibroblast with IGFBP7 or a fragment thereof thereby to promote transdifferentiation of the fibroblast to an osteoblast; and isolated, recombinant or synthesised IGFBP7 or a fragment thereof. In some examples, the present disclosure provides a method of promoting bone formation in a subject the method comprising administering to the subject an osteoblast, wherein the osteoblast has been produced by culturing a fibroblast in an osteoblast-conditioned medium thereby to promote transdifferentiation of the fibroblast to an osteoblast.

[0106] Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, transcutaneous, intradermal, intramedullary delivery (eg, injection), as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular delivery (eg, injection). For injection, the IGFBP7 or fragment thereof, or the osteoblast, may be formulated in an aqueous solution, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art.

[0107] The IGFBP7 or fragment thereof of the present disclosure or the osteoblasts of the present disclosure may be administered in combination with additional compounds that are useful for promoting bone formation. For example, suitable compounds may include Alendronate (Foxamax), Risedronate (Actonel), Ibandronate (Boniva) or Zoledronic acid (Reclast or Aclasta). Alternatively, or in addition, the other compound may be a corticosteroid, eg, prednisone or cortisone. Alternatively, or in addition, the other compound may be denosumab (Prolia). Alternatively, or in addition, the other compound may be strontium ranelate (Protos). Alternatively, or in addition, the other compound may be a selective oestrogen receptor modulator (SERMS), such as raloxifene (Evista). Alternatively, or in addition, the other compound may be a drug used in hormone replacement therapy (HRT), such as oestrogen or progesterone. Alternatively, or in addition, the other compound may be teriparatide (Forteo). Alternatively, or in addition, the other compound may be a nonsteroidal anti-inflammatory agent or analgesic. Forexample, a suitable non-steroidal antiinflammatory agent may be ibuprofen, naproxen or a COX-1 and/or COX-2 inhibitor selected from ketoprofen, indomethacin (Indocin orTivorbex), fenoprofen (Nalfon).

[0108] Components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). The release system material can be selected so that components having different molecular weights are released by diffusion or through degradation of the material. Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as polyflactic acid), polyfglycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as polyethylene oxide), polyethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Poly(lactide-co-glycolide) microspheres can also be used.

[0109] In examples where cells (eg, osteoblasts) are administered to a subject as a composition, the composition may also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilisation of the cells. Suitable ingredients include matrix proteins or gel polymers that support or promote adhesion of the cells or complementary cell types.

[0110] The IGFBP7 or fragment thereof of the present disclosure or the osteoblasts of the present disclosure may be administered to a subject in association with a scaffold. The scaffold material may be as described in U.S. Patent Nos. 5,681,872; 5,914,356; 5,939,039; 6,325,987; 6,383,519; 6,521,246; 6,736,799; 6,800,245; 6,969,501; 6,991,803; 7,052,517; 7,189,263; 7,534,451; 8,303,967; 8,460,686; or 8,647,614. Other suitable scaffold materials may include VITOSS®, CORTOSS®, biopolymers, bone, decellularized bone, extracellular matrix or components thereof, fibronectin, laminin collagen, chitosan, alginate, calcium phosphate, calcium sulfate, poly(alpha-hydroxy acids) such as poly(lactic- co-glycolic acid) and polyglycolic acid, CUPE polymer, polyethylene glycol, or any combinations thereof. The scaffold material may be porous. The scaffold material may be a natural material, synthetic material, or a combination thereof. The scaffold material may be biocompatible, nontoxic and/or non-inflammatory. The scaffold material may support cell attachment, cell proliferation, extracellular and/or bone matrix production, and/or cell conversion. The scaffold material may be biodegradable. The scaffold material may be sterilized. Other scaffold materials and attributes will be appreciated by those of skill in the art.

[0111] Suitable synthetic material for a cell transplantation scaffold should be able to support extensive cell growth and cell function. Such scaffolds may also be resorbable. Suitable scaffolds may include polyglycolic acid scaffolds, eg, as described by Vacanti, et al. 1988. J . Ped. Surg. 23:3-9; Cima, et al. 1991. Biotechnol. Bioeng. 38:145; Vacanti, et al. 1991. Plast. Reconstr. Surg. 88:753-9; or synthetic polymers such as polyanhydrides, polyorthoesters and polylactic acid. In another example, the cells may be administered in a gel scaffold (such as Gelfoam from Upjohn Company).

[0112] The compositions described herein may be administered alone or as admixtures with other cells. Cells that may be administered in conjunction with the compositions of the present disclosure include, but are not limited to, multipotent or pluripotent stem cells, or bone marrow cells. The cells of different types may be admixed with a composition of the disclosure immediately or shortly prior to administration, or they may be co-cultured together for a period of time prior to administration.

[0113] The present disclosure also provides a scaffold for supporting bone development or bone repair, wherein the scaffold comprises IGFBP7 or a fragment thereof. The IGFBP7 or a fragment thereof may, for example, be conjugated to the scaffold.

[0114] The exact amount of osteoblasts and/or IGFBP7 or fragment thereof to be administered will depend upon a variety of factors, including the age, weight, and sex of the patient, and the extent and severity of the condition to be treated.

[0115] In some instances it may be desirable or appropriate to pharmacologically immunosuppress a subject prior to initiating cell therapy and/or reduce an immune response of a subject against the cellular composition. Means for reducing or eliminating an immune response to transplanted cells are known in the art.

[0116] The present disclosure also provides medical devices for use in delivering osteoblasts or promoting the growth or development of osteoblasts in a subject. For example, the present disclosure provides a syringe or catheter or other suitable delivery device comprising osteoblasts of the present disclosure, and/or comprising IGFBP7 or a fragment thereof.

[0117] The osteoblasts of the present disclosure may be surgically implanted, injected, delivered (eg, by way of a catheter or syringe), or otherwise administered directly or indirectly to the site in need of repair or augmentation. Exemplary routes of parenteral administration include intravenous, intra-arterial, intramuscular, intraperitoneal, or intrathecal, and infusion techniques.

[0118] Dosages may vary with the type and severity of the condition to be treated, and may include single or multiple dosses. Specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the practitioner administering the composition. When administered to a human subject, the dosage regimen may vary depending on a variety of factors including the type and severity of the condition, the age, sex, weight or medical condition of the subject and the route of administration. In that regard, precise amounts of the composition to be administered will depend on the judgement of the practitioner.

[0119] The compositions described herein may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the condition being treated, whether a recurrence is considered likely, etc. The administration may be constant, eg, constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, eg, once per day over a period of days, once per hour over a period of hours, or any other such schedule as deemed suitable.

[0120] In other examples, the osteoblasts of the present disclosure may be useful for research or drug screening. For example, the osteoblasts may be exposed to a test compound and the effect of the test compound on the cells may be observed or measured.

Examples

[0121] Data were obtained from four independent experiments and represented as mean

± SE. Tukey HSD Post Hoc test ANOVA was used for the statistical analysis of data among three groups. SPSS 24.0 program was employed for all statistical analysis and differences were considered significant if p < 0.05.

Example 1

Cell culture

[0122] Human trabecular bone was chopped into 1 mm³ pieces and washed several times in phosphate buffered saline (PBS), followed by digestion for 90 min at 37 °C with 0.02% (w/v) trypsin (Sigma-Aldrich, USA) in PBS. Digested cells were cultured in complete media containing a-Minimal Essential Medium (a-MEM, Gibco Laboratories, USA), supplemented with 10% (v/v) fetal calf serum (Life Technology), 30 mg/mL penicillin, 100 mg/mL streptomycin (Life Technology) and ImM l-ascorbic acid phosphate magnesium salt (Wako Pure Chemicals, Osaka, J apan). The cells were cultured at 37 °C with 5% CO2, and the medium was refreshed every three days until 80-90% confluence when cells were passaged. All human osteoblasts (HOBs) were at passage 2.

[0123] Foreskin fibroblast cells were cultured in complete media containing DMEM (Gibco Laboratories, USA), supplemented with 10% (v/v) fetal calf serum (FCS, Life Technology), 30 mg/mL penicillin, 100 mg/mL streptomycin (Life Technology). The cells were cultured at 37 °C with 5% CO2, and the medium was refreshed every three days until 80-90% confluence when cells were passaged. All fibroblasts were at passage 10.

Collection of conditioned media

[0124] HOBs (at passage 2) and fibroblasts (at passage 10) were grown in flasks (5% CO2 atmosphere) in alpha-MEM + 10% fetal calf serum (FCS) and Dulbecco's modified eagle medium (DMEM) + 10% FCS, respectively, up to a confluence of 80-90%. The medium and detached cells were removed, and the cell layer was washed three times with PBS, and then two times with serum-free DMEM. Serum-free DMEM was then added (typically 15 mL for a 75 cm² flask), and the cells were incubated at 37 °C for 48 hours, and the cell culture media were collected as osteoblast-conditioned medium (OB-CM) or fibroblast-conditioned medium (FB-CM). The CM was then filtered at 0.2 pm to remove detached cells and large debris. The CM was used immediately or otherwise stored at -80 °C.

Conversion of fibroblasts using conditioned medium

[0125] Fibroblasts at passage 10 were seeded on 12 well plates with 80,000 cells/well and cultured in DMEM medium supplemented with 10% FCS, 30 mg/mL penicillin and 100 mg/mL streptomycin overnight. The fibroblasts were then refreshed with one of four media types: i) FB-CM supplemented with 2% FCS as a control; ii) osteogenic medium (FB-CM supplemented with 2% FCS, ImM l-ascorbic acid phosphate and 10 mM b- glicerophosphate); iii) OB-CM supplemented with 2% FCS; iv) OB-CM supplemented with 2% FCS, ImM l-ascorbic acid phosphate and 10 mM b-glicerophosphate. The cells were cultured at 37 °C with 5% C02, and the medium was refreshed every three days. The cells were harvested for gene expression analysis at day 3 and 7, and for alizarin red staining at day 28.

[0126] For gene expression analysis, total RNA was isolated from cells by adding RNeasy Mini Kit (Qiagen) after the medium was removed according to the manufacturer's instructions. First strand cDNA was synthesized from 0.7 pg total RNA using the Tetro cDNA Synthesis Kit (Bioline) according to the manufacturer's instructions. Real-time PCR was performed in Rotor-Gene 6000 (Corbett Life Science) using Immomix (Bioline) according to manufacturer's instructions, and the relative gene expression levels for Runx2, BMP-2, bone sialoprotein, and osteocalcin were obtained by normalizing them to a house- keeping gene (18S). Referring to Figure 1, the conditioned medium collected from osteoblasts (OB-CM) significantly increased osteogenic gene expression levels (Runx2, BMP2, bone sialoprotein and osteocalcin) compared with the control, ie, the conditioned medium collected from fibroblasts (FB-CM).

[0127] Alizarin red staining was used to assess mineralisation. The cells were cultured for 28 days in different media at which time point alizarin red staining was carried out to assess calcium deposition. In brief, after 28 days of culturing, the medium from each well was carefully aspirated and cells were fixed by incubating in iced cold 70% ethanol for 1 hour at room temperature followed by rinsing the cells twice (5-10 minutes each) with water. The water was then aspirated and 1 mL alizarin red solution was added to cover each well of the 24-well plate and incubated at room temperature for 30 minutes. After 30 minutes, the alizarin red solution was removed and the cells were washed four times with 1 mL water. 1- 1.5 mL water was added to each well prior to visual inspection and image acquisition. Image J was used to quantify the staining area. Referring to Figure 1, the conditioned medium collected from osteoblasts (OB-CM) promoted bone nodule formation in the cells, particularly in the presence of osteogenic medium.

Example 2

Protein identification

[0128] The OB-CM and FB-CM were dialyzed using a 3.5-kDa-molecular mass cutoff membrane (Thermo Scientific, USA). The CM were dialyzed overnight at 4 °C in 5 litres of 1 mM ammonium bicarbonate solution with two buffer changes. The dialyzed CM were frozen and lyophilized to dryness. Lyophilised conditioned media OB-CM and FB-CM were reconstituted in 25 mM ammonium bicarbonate buffer, denatured with final 1% (w/v) SDS and reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) (pH 7.0) at 65 °C for 15 min. The sample was cooled on ice and alkylated with a final concentration of 55 mM of iodoacetamide at room temperature for 1 h in the dark. Proteins were precipitated using a final concentration of 10% (v/v) of trichloroacetic acid at 4 °C overnight and centrifuged at 10,000 g for 10 min at 4 °C. The protein pellet was washed three times with ice-cold 100% acetone, dried and resolubilised in 70 mI_ of buffer containing 100 mM Tris-HCI pH 8, 1 mM CaC and 8 M urea buffer. Proteins were subsequently diluted in buffer containing 1 M urea buffer (560 mI_) and protein concentrations were determined by absorbance at 280 nm. Proteins were digested overnight with trypsin and buffer components were removed from peptides using solid phase extraction.

[0129] Peptides (1 pg) were injected into LC-MS for analysis. Each sample was analysed three times by mass spectrometry. Differentially expressed peptide ions were analysed by Progenesis Ql for proteomics. The peptide ions across samples were compared and aligned to compensate for variability in the liquid chromatography. Peptide ion peaks with a retention time of between 30 min and 96 min were selected for further analysis since the solvent gradient for elution was set at between 31-90 min. The abundance of peptide ions was adjusted using the normalisation factor and the in-built algorithm "normalise to all proteins". Ions with a charge equal to +1 are generally non-peptide ions and were thus removed from further analysis. The remaining peptide ions were filtered based on their p- value (<0.01) and maximum fold change (>5) for principal component analysis and correlation analysis. Peptide ions (54772 tandem mass spectra) were subsequently used to search the Swissprot database using Mascot search engine for protein identification. To refine protein identification and quantification, peptide ions that scored below 20 with hits value less than 2, and non-human were removed. The remaining peptides were used for protein identification.

[0130] A total of 1088 proteins were identified. The number of unique and common proteins in the two conditioned media is shown in Figure 2. Surprisingly, IGFBP7 was present in the OB-CM at a concentration 46-fold higher compared to the FB-CM (Table 4).

Table 4

Example 3

IGFBP7 treatment of fibroblasts [0131] Fibroblasts at passage 10 were seeded on 12 well plates with 80,000 cells/well and cultured in DMEM medium supplemented with 10% FCS, 30 mg/mL penicillin and 100 mg/mL streptomycin overnight. The media were then removed, and the cell layer was washed with PBS and refreshed with DMEM control medium (supplemented with 2% FCS and 30 mg/mL penicillin, 100 mg/mL streptomycin) as control group, and DMEM control medium added with different concentrations (125 ng/mL, 250 ng/mL, 500 ng/mL and 1000 ng/mL, Sigma) of IGFBP7. The sequence of the IGFBP7 used in the present example is set forth below:

MSSSDTCGPCEPASCPPLPPLGCLLGETRDACGCCPMCARGEGEPCGGGGAGR GYCAPGMECVKSRKRRKGKAGAAAGGPGVSGVCVCKSRYPVCGSDGTTYPSGC QLRAASQRAESRGEKAITQVSKGTCEQGPSIVTPPKDIWNVTGAQVYLSCEVIGIPT PVLIWNKVKRGHYGVQRTELLPGDRDNLAIQTRGGPEKHEVTGWVLVSPLSKEDA GEYECHASNSQGQASASAKITVVDALHEIPVKKGEGAEL (SEQ ID NO. 3)

[0132] The cells were cultured at 37 °C with 5% CO2, and the medium was refreshed every three days. The cells were harvested for gene expression analysis at day 4 and 14, and for alizarin red staining at day 28. As shown in Figure 3, IGFBP7 significantly increased osteogenic gene expression levels (Runx2, BMP2, bone sialoprotein and osteocalcin) and promoted bone nodule formation in a dose-dependent manner.

Example 4

I GFBP7 -treated fibroblasts form mineralized tissue in vivo

[0133] Fibroblasts were incubated in the absence or presence of 1 pg/mL IGFBP7 and capacity for forming mineralized tissue was examined in a mice model. This model is adapted from Fedorovich et aiy Biomed Mater Res A. 2012; 100:2412-2420 where cultured cells are introduced into the hind limb of a nude mouse in Matrigel and bioceramic granules. Growth factor reduced Matrigel (BD Biosciences, San J ose CA) and biphasic calcium phosphonate (BCP) microparticles 100-200 pm (Berkeley Advanced Biomaterials) were used with an optimal time of 4 weeks. Cultured human cells were harvested from tissue culture plates using trypsin, and resuspended in culture media and combined with Matrigel/BCP in a syringe with a 23-gauge needle. Two hundred microliters containing ~1 x 10⁶ cells were injected subcutaneously into the hind limbs of 8-10 week old nude (BALB/c-Foxlnu/Ausb) mice (Australian BioResources). Animals were sedated using inhaled isoflurane and given 0.1 mg/kg buprenorphine as an analgesic after the procedure. Animals were injected bilaterally.

[0134] Mineralized tissue was analysed using a Skyscan 1272 microCT scanner (Bruker, Billerica, Massachusetts) at a magnification of 5 m, with a 0.25 mm aluminum filter, 2016 x 1344 camera, 2050 ms exposure time, 60 kV X-ray tube voltage and 166 mA current. Scan files were reconstructed in NRecon (Bruker) using a 0.0-0 1 gray scale, and analysis was performed using CTan and BatchMan (Bruker). A volume of interest (VOI) of the tissue that encompassed the injection site was analyzed for total volume (TV) and bone volume (BV) using a cutoff for mineralized tissue of 0.3 g/cm³ calcium calibrated to phantoms (0.25 and 0.75 g/cm³).

[0135] All samples were harvested, fixed in 10% formalin for 24 hours and then stored in 70% ethanol. Bone nodules from the muscle pouch study were embedded in TissueTek OCT compound (Sakura, Japan) and cryosectioned to 5 pm slides using Type 2C cryofilm (Section-Lab, Hiroshima, J apan). Samples were stained for mineralized bone using previously published von Kossa staining methods (McDonald MM, et al J Orthop Res. 2018;36:1106-1113).

[0136] As shown in Figure 4, control fibroblasts showed negligible bone formation, while several specimens from the IGFBP7 treatment group showed large amounts of bone and their mean bone volume values were significantly higher. Specifically, MicroCT analysis of mineralized bone (0.2 g/cm³ threshold) showed a significant increase for IGFBP7-treated fibroblasts (1 pg/L) * p < 0.05 versus untreated controls (Fig. 4A). 3D reconstructed images from MicroCT analysis (Fig. 4B-C), and images of histological sections stained with Von Kossa (Fig. 4D-E) are shown. Scale bars shown in D and E indicate 300 pm. Von kossa staining confirmed the presence of mineralised tissue in the IGFBP7 treatment group and nodules were observed in reconstructed images from microCT analysis.

Example 5

Induction of senescence plays a role in IGFBP7 reprograming of fibroblasts to osteoblasts

[0137] IGFBP7-induced cell senescence in the fibroblast to osteoblast transition was examined. Fibroblasts were treated without or with IGFBP7 (1 pg/mL) and gene expression was measured by qPCR (Lu Z et al, Tissue Eng Part A. 2017; 23:1212-1220). Figure 5 shows that IGFBP7 significantly increased expression of key gene regulators of cell senescence such as P16, P21 and P53 at day 4 and day 14 of culture *P< 0.05 when compared to a control at day 14 using a Student's t test, (Fig, 5A-C). Additionally, the gene expression levels of I L-a, TNF-a and IL-6 was also measured, which are among a plethora of proteins collectively termed senescence associated secretory profiles (SASP) secreted by metabolically active senescent cells. It was found that the expression of these genes were also significantly elevated by IGFBP7 treatment (Fig. 5D-F).

[0138] To further investigate if senescence induction plays a role in IGFBP7 reprograming of fibroblasts to osteoblasts, the cell senescence inhibitor, rapamycin, was employed. Fibroblasts were treated with IGFBP7 (1 pg/mL) alone or in combination with rapamycin (500 mM) and gene expression was measured by qPCR as above. Figure 6 shows that rapamycin reduced expression of cell senescence regulator genes (P16, P21 and P53) and SASP expression (IL-a, TNF-a and IL-6) (Fig. 6A-F). Moreover, rapamycin inhibited transdifferentiation of fibroblasts by decreasing the expression of osteogenic genes (Runx2, BMP-2, bone sialoprotein and osteocalcin, Fig. 6G-J ) and thus diminished the formation of mineralized nodules (Fig. 6K).

[0139] Note that, for immunofluorescence staining of Runx2 expression, anti-Runx2 antibody (Abeam, ab76956, 1:100, Cambridge, MA) followed by Alexa Fluor 488 conjugated anti-mouse antibody (ThermoFisher Scientific, A28175, 1:100, Waltham MA) were used for detection. Fluorescence was analyzed using Olympus Cell Imaging System. For quantification Runx2 positive cells in the population, fluorescent cells in a total of 150 cells were determined from 10 images acquired under x20 magnifications at the same exposure setting from three independent experiments.

[0140] Although the present disclosure has been described with reference to specific embodiments, it will be appreciated by those skilled in the art that it may be embodied in many other forms.

Claims

Claims

1. A method of producing an osteoblast the method comprising contacting a first cell with insulin growth factor binding protein 7 (IGFBP7) or a fragment thereof thereby to promote conversion of the first cell to an osteoblast.

2. The method of claim 1 wherein the first cell is a somatic cell.

3. The method of claim 1 or claim 2 wherein the first cell is a fibroblast.

4. The method of claim 3 wherein the fibroblast is a skin fibroblast.

5. The method of any one of claims 1 to 4 wherein the first cell is a human cell.

6. The method of any one of claims 1 to 5 wherein the first cell is not genetically modified.

7. The method of any one of claims 1 to 6 wherein the IGFBP7 or fragment thereof promotes transdifferentiation of the first cell to an osteoblast.

8. The method of any one of claims 1 to 7 wherein the conversion is carried out in a medium comprising the IGFBP7 or fragment thereof.

9. The method of claim 8 wherein the medium comprises IGFBP7 or fragment thereof at a concentration of between about 250 ng/mL and 1500 ng/mL

10. The method of claim 8 or claim 9 wherein the medium is an osteogenic- conditioned medium.

11. The method of any one of claims 1 to 10 wherein the method is performed for a period of between about 5 days and about 30 days.

12. The method of any one of claims 1 to 11 wherein the IGFBP7 or fragment thereof is isolated, recombinant or synthesised.

13. The method of any one of claims 1 to 12 wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

14. An osteoblast produced by the method of any one of claims 1 to 13.

15. A pharmaceutical composition comprising the osteoblast of claim 14 and a pharmaceutically acceptable carrier.

16. A method of treating a bone disorder in a subject the method comprising administering to the subject an osteoblast produced by the method of any one of claims 1 to 13.

17. The method of claim 16 wherein the bone disorder is a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer or osteoporosis.

18. The method of claim 17 wherein the bone disorder is a bone fracture.

19. The method of claim 18 wherein the osteoblast is administered at or adjacent the bone fracture.

20. The method of any one of claims 16 to 19 wherein the osteoblast is transdifferentiated from a fibroblast obtained from the subject.

21. A method of treating a bone disorder in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof.

22. The method of claim 21 wherein the bone disorder is a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer or osteoporosis.

23. The method of claim 22 wherein the bone disorder is a bone fracture.

24. The method of claim 23 wherein the IGFBP7 or fragment thereof is administered at or adjacent the bone fracture.

25. The method of any one of claims 21 to 24 wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

26. A method of promoting bone formation in a subject the method comprising administering to the subject an osteoblast produced by the method of any one of claims 1 to 13.

27. The method of claim 26 wherein the osteoblast is transdifferentiated from a fibroblast obtained from the subject.

28. The method of claim 26 or claim 27 wherein the subject suffers from a bone disorder selected from the group consisting of a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer and osteoporosis.

29. A method of promoting bone formation in a subject the method comprising administering to the subject isolated, recombinant or synthesised IGFBP7 or a fragment thereof.

30. The method of claim 29 wherein the subject suffers from a bone disorder selected from the group consisting of a bone fracture, a spinal chord injury, spinal disc degeneration, Paget's disease, bone cancer and osteoporosis.

31. The method of claim 29 or claim 30 wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

32. A cell culture medium comprising IGFBP7 or a fragment thereof.

33. The cell culture medium of claim 32 wherein the medium is suitable for culturing fibroblasts.

34. The cell culture medium of claim 32 or claim 33 wherein the medium is suitable for culturing osteoblasts.

35. The cell culture medium of any one of claims 32 to 34 wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

36. The cell culture medium of any one of claims 32 to 35 wherein the medium is a conditioned medium.

37. The cell culture medium of claim 36 wherein the conditioned medium is an osteogenic-conditioned medium.

38. The cell culture medium of any one of claims 32 to 37 wherein the medium comprises isolated, recombinant or synthesised IGFBP7 or a fragment thereof.

39. The cell culture medium of any one of claims 32 to 38 wherein the medium comprises the IGFBP7 or fragment thereof at a concentration of between about 250 ng/mL and 1500 ng/ml_.

40. The cell culture medium of any one of claims 32 to 39 further comprising one or more of ascorbic acid, fetal calf serum, b-glicerophosphate and dexamethasone.

41. An isolated, recombinant or synthesised IGFBP7 molecule or a fragment thereof.

42. The IGFBP7 or fragment thereof of claim 41 wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

43. An isolated or recombinant nucleic acid encoding IGFBP7 or a fragmentthereof.

44. The IGFBP7 or fragment thereof of claim 43 wherein the IGFBP7 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 3, or a sequence having about 90% identity to SEQ ID NO. 3.

45. An expression vector comprising the nucleic acid of claim 43 or claim 44.