Human oral mucosa stem cell secretome

Abstract

To provide secretome derived from human oral mucosa stem cells (hOMSC), and cell-free compositions comprising hOMSC-derived secretome.SOLUTION: Secretome according to the present invention is a composition comprising soluble and insoluble substances in their various forms that are secreted or released into the culture medium from human oral mucosa derived stem cells. Amongst others, these substances include soluble molecules such as: a. proteins, b. peptides, c. hormones, d. various DNA and RNA species, e. oligomers of nucleic acids, and f. other molecules with a molecular weight higher than 1,000 Daltons.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention is in the field of stem cells and regenerative medicine. Specifically, the present invention provides a composition of human oral mucosa stem cell-derived secretome. Methods for obtaining, manipulating and using stem cell secretome in therapy are also provided. [Background technology]

[0002] Human oral mucosa-derived stem cells (hOMSCs) are a unique stem cell population derived from the lamina propria of the oral mucosa (Marynka-Kalmani et al., 2010). hOMSCs express a unique immunophenotype consisting of markers of embryonic stem cells, neural crest stem cells and mesenchymal stem cells. Comprehensive genetic analysis has revealed that in vivo to in vitro transfer of hOMSCs results in differential expression of genes involved in the development of neural crest lineage during embryonic and fetal development in mammalian organisms.

[0003] The neural crest is a temporary developmental structure that gives rise to various cell lineages of ectodermal and mesenchymal origin, including neural and glial lineages as well as chondroblastic, osteoblastic, adipocyte and fibroblastic lineages, respectively. hOMSCs have also been shown to differentiate into these lineages in vitro and in vivo (Marynka-Kalmani et al., 2010; Treves-Manushevitz et al., 2013; Ganz et al., 2014a, 2014b).

[0004] hOMSCs differentiated into dopaminergic-like neurons or astrocyte-like cells and transplanted in vivo have been shown to have therapeutic effects in animal models (Ganz et al., 2014a, 2014b). Furthermore, naive hOMSCs have been shown to have some therapeutic activity in these animal models, with the therapeutic effect being comparable to that of a placebo.

[0005] Recently, it has been shown that embryonic and adult stem cells are also repositories of extracellular vesicles (EVs) (Desrochers et al., 2016; Konala et al., 2016). Extracellular vesicles (EVs) are vesicles ranging in size from 50 nm to 1.5–2 microns that are released from the cytoplasm of eukaryotic cells.

[0006] EVs are broadly divided into two main types according to their biogenesis and size: microvesicles or shed microvesicles, which range in size from 50 to 1500 nm, and exosomes, which range in size from 30 to 120 nm. Exosomes are lipid bilayer vesicles derived from the luminal membrane of multivesicular bodies, and they are constitutively released by fusion with the plasma membrane. The biogenesis of microvesicles and exosomes is different. Microvesicles are formed by membrane budding and fission at the plasma membrane. Exosomes originate from the endosome / Golgi system and are anchored at least in part to the cell surface by endosomal sorting complexes required for transport, where they undergo exocytosis.

[0007] Extracellular vesicles contain cargo consisting of proteins, lipids and nucleic acids (Desrochers et al., 2016; Xu et al., 2016). The content of EVs is heterogeneous and in a dynamic state depending on the cell of origin, its physiological and pathological state and the cellular site of release. The composition of exosomes can be different from their cell of origin due to the sorting of cargo into exosomes. The cargo of EVs has been shown to serve as a method of cell-cell communication in addition to the classical methods of cell-cell contact and secretion of soluble factors for paracrine and autocrine action. Most of the knowledge on the biological action of EVs comes from studies performed on cancer cells. It has been shown that the cargo of EVs induces the proliferation and survival of cancer cells as well as angiogenesis and the migration, survival and growth of tumor fibroblasts (Antonyak et al., 2015). Exosomes have been revealed to be carriers of genetic material and are considered as biomarkers for cancer diagnosis and prognosis and proposed for monitoring the efficacy of treatment. Exosome-based tumor vaccine and drug delivery are currently being evaluated as a therapeutic strategy for cancer (Gue et al., 2017).

[0008] Conditioned medium obtained from adult stem cells derived from bone marrow and adipose tissue has been shown to have therapeutic potential in cardiac ischemia and wound healing ( Lai et al., 2010 ; Hu et al., 2016 ).

[0009] Accumulating evidence suggests that cargo content differs between microvesicles and exosomes, depending on the cellular origin (Kanada et al., 2015). Furthermore, recent data suggest that cargo properties are cell-specific in EVs released by various types of cancer cells and also by various types of stem cells (Villarroya-Beltri et al., 2014; Lopez-Verrilli MA et al., 2016).

[0010] WO 2008 / 132722 discloses the gastrointestinal mucosa, in particular the lamina propria of the oral mucosa, as a source of multipotent adult stem cells.

[0011] WO2013076726 discloses stem cells derived from the lamina propria of the oral mucosa (OMSCs) as a source for selective differentiation into various neural cell lineages and their use in inducing or maintaining neurogenesis, for the treatment of neurodegenerative and psychiatric disorders and in neural tissue loss due to trauma.

[0012] US Patent Application Publication No. 2016 / 0256496 relates to gingival fibroblast-derived products, such as conditioned media, and their use in methods for the prevention or treatment of orthopedic conditions, such as osteoarthritis and rheumatoid arthritis.

[0013] Med Cell's WO 2017 / 001649, WO 2016 / 082882 and WO 2016 / 083500 disclose specific methods for producing secretomes secreted by mesenchymal stem cells or dendritic cells.

[0014] WO 2014 / 057097 discloses a method for modulating the secretome of adult human mesenchymal stem cells by co-culturing adult human mesenchymal stem cells with adult fully differentiated cardiomyocytes in an appropriate medium to obtain preconditioned adult human mesenchymal stem cells.

[0015] There remains an unmet need for compositions useful for the prevention and treatment of diseases and disorders and for promoting tissue regeneration. Such compositions may advantageously be derived from unique, scalable, and readily available sources. Summary of the Invention

[0016] Conditioned medium or secretome of human stem cells (hOMSC) derived from the lamina propria of the oral mucosa is disclosed herein as a therapeutic composition. The invention is based in part on the discovery that the composition of naive hOMSC secretome is unique and therefore has unique therapeutic potential, which is more advantageous than secretome of naive stem cells derived from other sources. Furthermore, hOMSC stimulation can induce cellular responses reflected in changes in the content of the secretome and thus in its therapeutic potential. It is also conceivable that the secretome generated by the above hOMSC stimulation is unique to the hOMSC stimulated cells, and that the same stimulation applied to adult stem cells derived from other sources will result in a different secretome.

[0017] It is disclosed herein for the first time that the secretome of naive hOMSCs has unique characteristics that differ from the secretomes of other stem cells in the presence, absence or relative amounts of cytokines, chemokines and nucleic acids.

[0018] It is also disclosed herein that acellular compositions comprising hOMSCs and secretomes derived therefrom can promote diabetic wound healing, suggesting that hOMSC secretomes may be used to promote neovascularization, cell proliferation and connective tissue formation. Unexpectedly, the activity of hOMSCs and their secretomes in wound healing is superior to stem cells and secretomes derived from other sources.

[0019] The secretome according to the present invention, derived from an accessible, reproducible and disposable naive hOMSC cell source, is easily accessible and usable without the need for induction of cell differentiation.

[0020] The present invention in one aspect provides a cell-free composition comprising material secreted from human oral mucosal stem cells (hOMSC-derived secretome) together with at least one carrier, excipient or diluent.

[0021] Cell-free compositions comprising hOMSC-derived secretomes according to the present invention are unique in their content and distinct from secretomes of stem cells from other sources.

[0022] In some embodiments, the hOMSCs are naive hOMSCs.

[0023] In some embodiments, the cell-free composition comprises: (i) at least one protein from the group consisting of interstitial cell-derived factor 1 (CXCL12 / SDF1), superoxide dismutase [Cu-Zn] (SOD1), mesencephalic astrocyte-derived neurotrophic factor (MANF), cystatin C (CST3), galectin 1 (LGALS1), glial-derived nexin (SERPINE2), insulin-like growth factor II (IGF2), latent transforming growth factor beta binding protein 1 (LTBP1), latent transforming growth factor beta binding protein 2 (LTBP2), latent transforming growth factor beta binding protein 3 fragment (LTBP3), latent transforming growth factor beta binding protein 4 (LTBP4), neuroblast differentiation-associated protein (AHNAK) and pigment epithelium-derived factor (SERPINF1 / PEDF); or (ii) at least one protein selected from the group consisting of hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (GCSF), macrophage inflammatory protein 3 (MIP-3a), growth-regulated oncogene alpha (GRO-a or CXCL1), macrophage-derived / CCL22 chemokine (MDC or CCL22), growth-regulated oncogene (GRO), IGFBP-2, neurotrophin 4 (NT-4), monocyte chemoattractant protein 2 (MCP-2 / CCL8), insulin growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 2 (IL-2), and brain-derived neurotrophic factor (BDNF); or (iii) 1SV, 3SV, ACTG2, ADAM10, ADAMTSL1, ADM, ANXA4, APOD, CALM2, CD109, CD59, CDH6, CFD, COL15A1, COL1A2, COLEC12, CTHRC1, CTSC, CTSL, CXC L12, DCD, DDAH2, DKK1, DSG1, DSP, DSTN, ECH1, EDIL3, EFEMP1, ELN, FLG, GNB2, GREM2, H3F3B, HBA1, HIST1H2AH, HIST1H2BK, HIST1H4A, HMGN2, HNRN at least one protein selected from the group consisting of PAB, HSP90AA1, HSPA1A, HSPG2, IGFBP5, JUP, KHSRP, LDHA, MNB2, LTBP4, MAN1A1, MFAP4, MMP1, MMP14, MT2A, NBL1, OMD, PFN1, PI16, PSG5, PSMB6, PTGDS, RARRES2, SLIT3, SPOCK1, SPTBN4, STOM, TMSB10, TMSB4X, TNFAIP6, TNXB, TPI1, TUBA1C, UBC, VIT, and WNT5A; or (iv) hsa-miR-4454+hsa-miR-7975, hsa-miR-23a-3p, hsa-let-7b-5p, hsa-miR-612, hsa-miR-12 5b-5p, hsa-miR-3144-3p, hsa-miR-199a-3p+hsa-miR-199b-3p, hsa-miR-191-5p, hsa-miR-100-5 p, hsa-miR-127-3p, hsa-miR-1260a, hsa-miR-378h, hsa-miR-379-5p, hsa-miR-376a-3p, hsa-let -7i-5p, hsa-miR-526a+hsa-miR-518c-5p+hsa-miR-518d-5p, hsa-miR-212-3p, hsa-miR-520c-3p , hsa-miR-28-5p, hsa-miR-758-3p+hsa-miR-411-3p, hsa-miR-29a-3p, hsa-miR-1206, hsa-miR-1 286, hsa-miR-514a-3p, hsa-miR-548ah-5p, hsa-miR-184, hsa-miR-543, hsa-miR-626, hsa-miR-3 at least one microRNA (miRNA) selected from the group consisting of hsa-miR-39-3p, hsa-miR-1234-3p, hsa-miR-155-5p, hsa-miR-888-5p, hsa-miR-542-3p, hsa-miR-514b-5p, hsa-miR-548m, hsa-miR-30e-5p, and hsa-miR-1290; or The combination Includes.

[0024] In some embodiments, the cell-free composition comprises more than one of substances (i), (ii), (iii), or (iv).

[0025] In yet another embodiment, the cell-free composition comprises at least one protein from (i), at least one protein from (ii), at least one protein from (iii), and optionally at least one miRNA from (iv).

[0026] In some specific embodiments, the cell-free composition of hOMSC-derived secretome comprises at least one factor selected from the group consisting of stromal cell-derived factor 1 (CXCL12 / SDF1), mesencephalic astrocyte-derived neurotrophic factor (MANF), superoxide dismutase [Cu-Zn] (SOD1), hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), and vascular endothelial growth factor (VEGF).

[0027] In another embodiment, the cell-free composition of hOMSC-derived secretome comprises hsa-miR-4454+hsa-miR-7975, hsa-miR-23a-3p, hsa-let-7b-5p, hsa-miR-612, hsa-miR-125b-5p, hsa-miR-3144-3p, hsa-miR-199a-3p+hsa-miR-199b-3p, hsa-miR-2106-3p, hsa-miR-2108 ... -191-5p, hsa-miR-100-5p, hsa-miR-127-3p, hsa-miR-1260a, hsa-miR-378h, hsa-miR-379-5p, hsa- miR-376a-3p, hsa-let-7i-5p, hsa-miR-526a+hsa-miR-518c-5p+hsa-miR-518d-5p, hsa-miR-212-3p , hsa-miR-520c-3p, hsa-miR-28-5p, hsa-miR-758-3p+hsa-miR-411-3p, hsa-miR-29a-3p, hsa-miR- 1206, hsa-miR-1286, hsa-miR-514a-3p, hsa-miR-548ah-5p, hsa-miR-184, hsa-miR-543, hsa-miR-62 6, at least one microRNA molecule selected from the group consisting of hsa-miR-339-3p, hsa-miR-1234-3p, hsa-miR-155-5p, hsa-miR-888-5p, hsa-miR-542-3p, hsa-miR-514b-5p, hsa-miR-548m, hsa-miR-30e-5p and hsa-miR-1290.

[0028] In some specific embodiments, the cell-free composition of hOMSC-derived secretome comprises hsa-miR-4454+hsa-miR-7975, hsa-miR-23a-3p, hsa-let-7b-5p, hsa-miR-612, hsa-miR-125b-5p, hsa-miR-3144-3p, hsa-miR-199a-3p+hsa-miR-199b-3p, hsa-miR-2222-3p, hsa-miR-221 ... a-miR-191-5p, hsa-miR-100-5p, hsa-miR-127-3p, hsa-miR-1260a, hsa-miR-378h, hsa-miR-379-5p, hsa-miR-376a-3p, hsa-let-7i-5p, hsa-miR-526a+hsa-miR-518c-5p+hsa-miR-518d-5p, hsa-miR-212 -3p, hsa-miR-520c-3p, hsa-miR-28-5p, hsa-miR-758-3p+hsa-miR-411-3p, hsa-miR-29a-3p, hsa-mi R-1206, hsa-miR-1286, hsa-miR-514a-3p, hsa-miR-548ah-5p, hsa-miR-184, hsa-miR-543, hsa-miR- 626, hsa-miR-339-3p, hsa-miR-1234-3p, hsa-miR-155-5p, hsa-miR-888-5p, hsa-miR-542-3p, hsa-miR-514b-5p, hsa-miR-548m, hsa-miR-30e-5p and hsa-miR-1290.

[0029] In some embodiments, the cell-free composition comprises at least one protein at a concentration significantly higher than the concentration of said protein in secretomes derived from other stem cell sources. In some particular embodiments, the cell-free composition comprises at least one of (i), (ii) or (iii) proteins at a concentration significantly higher than the concentration in secretomes derived from other stem cell sources.

[0030] In some embodiments, the at least one protein present in a cell-free composition of hOMSC-derived secretomes at a significantly higher concentration than in secretomes derived from other stem cell sources is selected from the group consisting of stromal cell-derived factor 1 (CXCL12 / SDF1, P48061), superoxide dismutase [Cu-Zn] (SOD1, P00441), mesencephalic astrocyte-derived neurotrophic factor (MANF, P55145), hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), and vascular endothelial growth factor (VEGF).

[0031] In certain embodiments, the secretome is a protein that is a part of the cellular cellular phenotype, such as stromal cell-derived factor 1 (CXCL12 / SDF1), mesencephalic astrocyte-derived neurotrophic factor (MANF), superoxide dismutase [Cu-Zn] (SOD1), hepatocyte growth factor (HGF), placental growth factor (PIGF), macrophage colony-stimulating factor (MCSF), vascular endothelial growth factor (VEGF), growth-regulated oncogene (GRO), granulocyte colony-stimulating factor (GCSF), macrophage inflammatory protein 3 (MIP-3a), growth-regulated oncogene alpha (GRO-a or CXCL1), macrophage-derived / CCL22 chemokine (MDC or CCL22), insulin-like growth factor binding protein 2 (IGFBP-2), neurotrophin 4 (NT-4), monocyte chemoattractant protein 2 (MC chemoattractant protein 2), also known as chemokine (CC motif) ligand 8 (CCL8), P-2), insulin growth factor 1 (IGF-1), insulin-like growth factor 2, cystatin C (CST3), galectin 1 (LGALS1), glial-derived nexin (SERPINE2), latent transforming growth factor beta binding protein 1 (LTBP1), latent transforming growth factor beta binding protein 2 (LTBP2), latent transforming growth factor beta binding protein 3 (LTBP3), latent transforming growth factor beta binding protein 4 (LTBP4), neuroblast differentiation-associated protein (AHNAK), pigment epithelium-derived factor (SERPINF1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at a concentration significantly higher than the concentration of the above proteins in secretome derived from skin stem cells or bone marrow stem cells.

[0032] In certain embodiments, the secretome comprises at least one protein selected from the group consisting of hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF) and vascular endothelial growth factor (VEGF) at a concentration significantly higher than the concentration of said proteins in secretomes derived from skin stem cells or bone marrow stem cells.

[0033] In another embodiment, the hOMSC-derived secretome comprises at least one protein at a concentration significantly lower than the concentration of said protein in secretomes derived from other stem cell sources.

[0034] In some embodiments, a cell-free composition of hOMSC-derived secretome comprises at least one protein at a concentration significantly lower than the concentration of said protein in secretomes derived from other stem cell sources.

[0035] In certain embodiments, the at least one protein present at a significantly lower concentration than in secretomes derived from other stem cell sources is selected from the group consisting of monocyte chemoattractant protein 3 (MCP-3 / CCL7), epithelial neutrophil activating peptide or CXC motif chemokine 5 (ENA-78 or CXCL5), leptin, Fms-related tyrosine kinase 3 ligand (Flt-3 ligand), interleukin 6 (IL-6), monokine induced by gamma interferon (MIG or CXCL9) and interleukin 8 (IL-8) at a significantly lower concentration than the concentration of said proteins in secretomes derived from skin stem cells or bone marrow stem cells. Each possibility represents a separate embodiment of the present invention.

[0036] In some embodiments, the protein and nucleic acid content of the hOMSC-derived secretome differs from the protein and nucleic acid content of the secretome of other stem cells.

[0037] In certain embodiments, the secretome comprises at least one protein involved in nervous system homeostasis.

[0038] In some embodiments, the at least one protein involved in nervous system homeostasis may be selected from cystatin C, galectin 1, glial-derived nexin, insulin-like growth factor II, (IGF2), latent transforming growth factor beta binding protein 1 (LTBP1), latent transforming growth factor beta binding protein 2 (LTBP2), latent transforming growth factor beta binding protein 3 (LTBP3), latent transforming growth factor beta binding protein 4 (LTBP4), mesencephalic astrocyte-derived neurotrophic factor (MANF), neuroblast differentiation-associated protein (AHANK), pigment epithelium-derived factor (PEDF), stromal cell-derived factor 1 (SDF1) and superoxide dismutase [Cu-Zn] (SODC). Each possibility represents an individual embodiment of the invention.

[0039] The hOMSC-derived secretome according to the present invention comprises substances secreted or released into the medium in which they are grown or maintained, such medium being referred to herein as conditioned medium.

[0040] In some embodiments, the hOMSC-derived secretome and cell-free compositions comprise extracellular vesicles (EVs).

[0041] In some embodiments, the hOMSC-derived secretome and the cell-free composition comprise microvesicles.

[0042] In another embodiment, the hOMSC-derived secretome and cell-free composition comprises exosomes.

[0043] In some embodiments, the hOMSC-derived secretome and cell-free compositions comprise microvesicles and exosomes.

[0044] In some embodiments, the composition hOMSC-derived secretome and the cell-free composition comprise soluble factors.

[0045] In some embodiments, the soluble factors are selected from the group consisting of proteins, peptides, hormones, DNA and RNA species, oligonucleotides and polynucleotides, and combinations thereof.

[0046] In some embodiments, the soluble factor is a molecule with a molecular size of 1,000 Daltons (Da) or greater.

[0047] In some specific embodiments, the soluble factors are molecules having molecular sizes of, for example, 1,000-10,000 Da; 1,000-3000 Da; 1,000-5,000 Da; 2,000-6,000 Da; 5,000-10,000 Da; 7,000-10,000 Da, 10,000-30,000 Da; 10,000-50,000 Da, etc., or even larger, with each possibility representing a separate embodiment of the present invention.

[0048] In some embodiments, the conditioned medium of hOMSCs is concentrated using methods known in the art to obtain an hOMSC secretome that contains components at higher concentrations than those in the conditioned medium.

[0049] In some embodiments, the secretome is derived from hOMSCs that have been exposed to a stimulus or condition that affects the content of the secretome.

[0050] In some embodiments, the stimulus or condition includes, but is not limited to, a chemical stimulus, a physical stimulus, a substrate stimulus, and / or a biological stimulus.

[0051] In some embodiments, the cell-free composition is a pharmaceutical composition comprising the hOMSC-derived secretome together with a pharma- ceutically acceptable carrier, excipient or diluent.

[0052] In some embodiments, the cell-free composition is a cosmetic composition comprising the hOMSC-derived secretome together with an acceptable carrier, excipient or diluent suitable for cosmetic use.

[0053] In some embodiments, the acellular composition comprising an hOMSC-derived secretome is for use in tissue remodeling or tissue regeneration.

[0054] In some embodiments there is provided a pharmaceutical composition according to the invention for use in promoting wound healing, preventing or reducing scar formation, promoting scar healing or promoting cartilage or bone formation.

[0055] In some embodiments, the cell-free composition of hOMSC-derived secretome is for use in promoting or accelerating diabetic wound healing.

[0056] In another aspect, the present invention provides a method for producing a cell-free secretome from hOMSCs, the method comprising: i. isolating hOMSCs by explantation or enzymatic digestion; ii. expanding the hOMSCs in culture medium; iii. replacing the medium with basal medium; iv. culturing the hOMSCs in basal medium for a period of time ranging from 1 hour to 120 hours; v. Recovering the medium from the culture; and vi. Optionally, concentrating the medium 1.1 to 10,000 times. Includes.

[0057] In some embodiments, the hOMSCs isolated are naive cells.

[0058] In some embodiments, at or after step (iv), the hOMSCs are exposed to a stimulus or condition that affects the content of the secretome. In some embodiments, the stimulus is selected from the group consisting of a chemical stimulus, a physical stimulus, a substrate stimulus, or a biological stimulus.

[0059] Naïve or stimulated hOMSCs used according to the present invention for the generation of secretome and cell-free compositions are maintained and expanded in tissue culture in an undifferentiated state.

[0060] The pharmaceutical and cosmetic cell-free compositions comprising hOMSC-derived secretomes can be used according to the present invention for the repair and regeneration of organs and tissues destroyed in whole or in part by mechanical trauma, chemical injury, radiation and heat or any other type of iatrogenic injury. Examples of such injuries include, but are not limited to, contusions of the central nervous system, spinal cord injuries, spinal cord transections, crushed or severed peripheral nerves, burns, chemotherapy-induced neuropathy and heart disease, bone fractures, ruptured tendons and ligaments. Each possibility represents an individual embodiment of the present invention.

[0061] In some embodiments, the compositions of the invention comprise a secretome derived from autologous hOMSCs, ie, the individual receiving treatment serves as the donor of hOMSCs for the generation of the secretome.

[0062] In another embodiment, the compositions of the invention comprise a secretome derived from allogeneic hOMSCs, ie, a donor unrelated to the patient serves as the donor of hOMSCs for secretome generation.

[0063] In yet another aspect, the present invention provides a method of preventing or treating a disease or disorder comprising administering to a subject in need thereof a cell-free composition comprising a hOMSC-derived secretome.

[0064] Any disease or disorder suitable for prevention or treatment with stem cells may be treated or prevented with the compositions according to the present invention.

[0065] In some embodiments, diseases or disorders suitable for prevention or treatment with the compositions of the invention include: i. Inflammatory diseases (e.g., osteoarthritis); ii. Autoimmune diseases (e.g., rheumatoid arthritis, scleroderma); iii. Vascular diseases (e.g., arteritis / Buerger's disease); iv. Heart disease (e.g., myocardial infarction, chronic heart failure); v. Respiratory diseases (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis); vi. Skeletal system diseases (e.g. bone regeneration, avascular necrosis, osteomyelitis, cartilage repair, tendon repair, muscular dystrophies); vii. Gastrointestinal disorders (e.g., fistulas, ulcers, esophageal strictures, liver cirrhosis, incontinence, Crohn's disease); viii. Renal disease (e.g., kidney failure); ix. Urinary tract (e.g., incontinence); x. Skin diseases (e.g., foot ulcers, epidermolysis bullosa, pemphigus, diabetic ulcers, static venous ulcers, chronic pressure ulcers); xi.Aging-related diseases; xii. Peripheral nerve and skeletal muscle diseases (e.g., chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barré syndrome, muscular dystrophies); xiii. Diseases of the central nervous system (e.g. neurodegenerative diseases, e.g. demyelinating diseases (multiple sclerosis), Alzheimer's disease, Parkinson's disease, bulbospinal atrophy, etc., diseases of the autonomic nervous system such as stroke, spinal cord ischemia, multiple system atrophy); xiv. Eye diseases (e.g., retinopathy [age-related macular degeneration, diabetic retinopathy, arteriosclerotic retinopathy], optic neuritis); xv. Endocrine system diseases (e.g. diabetes mellitus and its complications: [vascular disorders, neuropathy, chronic ulcers, renal disorders]); and xvi. Dental and oral diseases (e.g., dental pulp-related disease, periodontal disease, alveolar bone loss, oral mucosal ulcers caused by immune diseases) is selected from the group consisting of:

[0066] Each possibility represents a separate embodiment of the present invention.

[0067] In certain embodiments, the disorder is a diabetic wound.

[0068] In some embodiments, the disorder is a cosmetic disorder.

[0069] In some embodiments, the method of treatment includes tissue remodeling, tissue repair or tissue regeneration and comprises administering to a subject in need thereof a cell-free composition comprising a hOMSC-derived secretome, which may be a pharmaceutical or cosmetic composition.

[0070] In some embodiments, a method is provided for promoting or accelerating diabetic wound healing comprising administering to a subject in need thereof a cell-free composition comprising an hOMSC-derived secretome.

[0071] In yet another aspect, the present invention provides a method for tissue repair and regeneration comprising administering at least one acellular composition comprising a substance secreted from human oral mucosal stem cells (hOMSCs) according to the present invention.

[0072] In some embodiments, the repair or regeneration method is for organs and tissues destroyed in whole or in part by mechanical trauma, chemical injury, radiation and heat or any other type of iatrogenic injury. Examples of such injuries include, but are not limited to, contusions of the central nervous system, spinal cord injuries, spinal cord transections, crushed or severed peripheral nerves, burns, chemotherapy-induced neuropathy and heart disease, bone fractures, ruptured tendons and ligaments. Each possibility represents a separate embodiment of the present invention.

[0073] In some embodiments, the tissue repair or regeneration is associated with a condition, disease or disorder selected from the group consisting of wound healing, degenerative diseases, congenital defects, age-related defects and iatrogenic defects.

[0074] In certain embodiments, the stem cells are allogeneic or autologous stem cells.

[0075] The composition of the present invention can be administered to a subject in need thereof by any suitable administration route, including but not limited to topical, subcutaneous, intramuscular, intravenous, intraarterial, intraarticular, intralesional, intratumoral or parenteral.In some embodiments, topical administration can be used for wound healing.Therefore, the pharmaceutical and cosmetic compositions according to the present invention are formulated to suit the specific administration route used.For example, for topical administration, the composition can be formulated as cream, foam, gel, lotion and ointment using methods known in the art.

[0076] In some embodiments, the composition is administered locally to the damaged tissue.

[0077] The compositions according to the invention comprising hOMSC-derived secretomes may be administered to damaged tissue according to any treatment regimen, for example, the compositions may be administered once or multiple times at the same or different sites.

[0078] In some embodiments, the cell-free compositions of the invention comprising hOMSC-derived secretomes are administered to a subject in need thereof as part of a treatment regimen that includes at least one additional pharmaceutical or cosmetic agent or treatment.

[0079] Virtually any use known or conceivable in the art for stem cells can be implemented with the secretomes of the invention derived from hOMSCs, including prophylactic and therapeutic techniques.

[0080] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description set forth hereinafter, however, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0081] [Figure 1]FIG. 1 shows a comparison between stem cell marker profiles of hOMSCs and foreskin stem cells (hSkin). Results are expressed as -ΔCt (cycle threshold) values ​​relative to the housekeeping gene GAPDH. Highly negative values ​​indicate low expression levels. [Diagram 2] FIG. 1 shows the relative differences in protein expression between hOMSC and hDermal SC secretomes from immunofluorescence staining with antibodies against pluripotent stem cell markers and neural crest-associated stem cell markers. [Diagram 3] FIG. 1 shows the expression ratios of selected markers in hOMSCs and hSkin SCs. [Figure 4] Figure 1 shows protein expression in the secretome of mesenchymal stem (stromal) cells from young human bone marrow. Data taken from Park et al., International Journal of Stem Cells, 2009. [Diagram 5] The top left panel shows a donut-shaped ring sutured onto the dorsal skin of a diabetic db / db mouse prior to wounding. The top right panel shows the site immediately after wounding. The bottom left panel shows the site of intradermal injection. The bottom right panel shows histology or excised skin. [Figure 6A] FIG. 6A shows quantitative (6A) and representative qualitative (6B) photographs showing the rate of diabetic wound healing in groups of db / db diabetic mice treated with hOMSCs or hSkin SCs or PBS vehicle (untreated). [Figure 6B] FIG. 6A shows quantitative (6A) and representative qualitative (6B) photographs showing the rate of diabetic wound healing in groups of db / db diabetic mice treated with hOMSCs or hSkin SCs or PBS vehicle (untreated). [Figure 7] FIG. 13 Quantitative depiction of the rate of diabetic wound healing in groups of db / db diabetic mice treated with hOMSCs, db / db diabetic untreated mice or wild type (WT) untreated mice. [Figure 8]1 shows the average time required for complete wound closure in WT untreated, WT-hOMSC-treated, db / db untreated, db / db-hOMSC-treated and db / db hADSC (human adipose tissue derived stem cell)-treated mice. Calculated t-test p-values ​​are WT untreated vs. db-hOMSC=0.19533; db-hOMSC-treated vs. db untreated=5.34E-6; db-hOMSC-treated vs. db-hADSC=0.00044; db untreated vs. db-hADSC=0.48983; WT untreated vs. db-hOMSC=1.9933E-5; WT untreated vs. db untreated=2.262E-5. [Figure 9] FIG. 13 Quantitative depiction of the rate of diabetic wound healing in groups of db / db diabetic mice treated with hOMSCs, hSkin SCs, hADCS or PBS vehicle (untreated). [Figure 10] FIG. 13 Quantitative depiction of the rate of diabetic wound healing in groups of db / db diabetic mice treated with hOMSCs, hOMSC-derived acellular secretomes, hskin stem cells or PBS vehicle (untreated). [Figure 11] FIG. 1 shows 369 hOMSC secretome proteins identified by mass spectrometry and their average relative abundance (intensity). [Figure 12] FIG. 294 hOMSC secretome proteins common to 1534 proteins listed in secretomes derived from either BMSCs, ASCs or DPSCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] Detailed Description of the Invention The present invention provides the secretome of human oral mucosa-derived adult stem cells for the treatment and prevention of diseases and disorders.

[0083] hOMSCs are a type of neural crest (NC)-derived stem cells that co-express pluripotency markers Oct4, Nanog, and Sox2 and NC-SC markers Snail, Slug, Sox10, Twist, and Notch1 in developing colonies (Marynka-Kalmani et al., 2010; Widera et al., 2009). The NC is a transient neuroectodermal structure in vertebrate embryos. During the NC's existence in the embryo, it gives rise to migratory multipotent stem cells that colonize various primitive tissues, where these cells differentiate into neural cell lineages and / or cell lineages with mesenchymal phenotypes, termed ectomesenchymal tissues or mesoectoderm. A portion of these NC-SCs remain in a relatively undifferentiated state in adults and show a propensity for neural differentiation even in tissues of mesenchymal origin, such as the dermis and bone marrow.

[0084] Typical total adult populations contain small numbers of stem cells, which makes stem cell expansion and isolation laborious, time-consuming, and usually inefficient. Primary and expanded total cell populations derived from the lamina propria of the oral mucosa were found to consist primarily (>80%) of naive stem cells. The majority (80-90%) of cell populations obtained from oral mucosa of three different donors were shown to express mesenchymal stem cell markers. A study by Marynka-Kalmani et al., 2010 (ibid.) demonstrated that trillions of hOMSCs could be obtained in a cost-effective and reproducible manner from biopsy samples of 3-4 x 2 x 1 mm taken with negligible morbidity.

[0085] Typical methods for isolating stem cells from solid tissues for clinical use include releasing the cells from the extracellular matrix by enzymatic digestion or explantation; expanding the primary whole population to obtain a sufficiently large population; and isolating stem cells from the whole population.

[0086] The quality and quantity of the isolated stem cell population derived from the oral lamina propria mucosa is not significantly affected by aging, can be expanded in vitro without losing its multipotency, and is therefore a safe and reliable source of secretome to be re-administered to subjects in need thereof to effectively effect tissue regeneration and other therapeutic processes.

[0087] definition The oral mucosa is the mucosa that covers the oral cavity, i.e. the oral parts of the cheeks and alveolar ridges, including the gums and palate, the tongue, the floor of the mouth, and the lips. The oral mucosa consists of epithelial tissue of ectodermal origin and the lamina propria (LP), a connective tissue of ectomesenchymal origin. Just as the connective tissues in the oral cavity are of ectomesenchymal origin, the cells of the oral mucosa lamina propria (OMLP) originate from the embryonic ectodermal neural crest. Wounds in the human oral mucosa heal primarily by regeneration. The healing rate is faster than that of the skin or other connective tissues, and appears to be only slightly affected by age and sex (Szpaderska, AM et al., J Dent Res, 2003, 82, 621-626).

[0088] "Stem cells" (SCs) are undifferentiated cells that can give rise to a range of mature functional cells.

[0089] "Embryonic stem (ES) cells" are cells that are derived from the inner cell mass of the embryonic blastocyst and are pluripotent and therefore have the ability to develop into any organ or tissue type, or at least potentially, into an entire embryo.

[0090] "Adult stem cells" are postnatal stem cells derived from the tissues, organs or blood of a postnatal organism.

[0091] "Pluripotent stem cells" are stem cells that can give rise to the three germinal cell layers and their derivative cell lines and tissues.

[0092] A "multipotent stem cell" is a stem cell that is capable of giving rise to the multiple cell lineages that make up an entire tissue or organ.

[0093] The secretome according to the present invention is a composition comprising soluble and insoluble substances in their various forms secreted or released into the medium by human oral mucosa-derived stem cells. These substances include, inter alia: 2. A protein B peptide C. hormones D various DNA and RNA species e. Nucleic acid oligomers f. Other molecules with a molecular weight greater than 1,000 daltons Soluble molecules such as 3. Proteins: growth factors, cytokines, hormones, cell surface receptors, cytoplasmic and nuclear proteins, metabolic enzymes, receptor ligands, adhesion proteins, endosome-associated proteins, tetraspanins, lipid raft-associated proteins, antigens, etc. b. RNA species: mRNA, miRNA, tRNA, rRNA, siRNA and lncRNA and possible other RNA species c.DNA: mitochondrial DNA (mtDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) d. Lipids: cholesterol, sphingomyelin, hexosylcermide and others e. Lectins, glycans, proteoglycans, glycoproteins Extracellular vesicles containing

[0094] Extracellular vesicles are membrane-bound particles that carry a cargo of soluble and insoluble substances as mentioned above. The term "extracellular vesicles" refers to a group of secretory or shed vesicles of various species. They are divided into the following subtypes (Xu et al., JIC 2016): 1. Microvesicles or shed microvesicles; size range - 50~1500nm 2. Exosomes; size range - 30~120nm 3. Vesicles; size range <500nm.

[0095] The culture medium or expansion medium is the medium in which the hOMSCs are cultured and expanded. The culture / expansion medium according to some embodiments of the present invention comprises at least one of the following components: low glucose Dulbecco's Modified Eagle Medium (LGDMEM), streptomycin, penicillin, gentamicin, amphotericin B, glutamine and serum, such as fetal calf serum (FCS).

[0096] In some embodiments, culture expansion medium comprises reduced LGDMEM supplemented with 100 μg / ml streptomycin, 100 U / ml penicillin (Biological Industries, Bete Haemek, Israel), 2 mM glutamine (Invitrogen) and 10% fetal calf serum (FCS, Gibco).

[0097] The basal medium is a serum-free medium.

[0098] Conditioned medium according to the present invention refers to medium collected from hOMSC cultures that contains hOMSC-derived substances secreted or released into the medium in which the hOMSCs are grown or maintained.

[0099] The conditioned medium containing the hOMSC secretome can optionally be concentrated using methods known in the art to increase the concentration of the secretome components and then stored, for example, frozen. Alternatively, the conditioned medium can be lyophilized and the secretome can be stored as a frozen powder and reconstituted for injection with water for injection or saline or other solutions known in the art.

[0100] Concentrated conditioned medium containing secretome or lyophilized secretome may be supplemented with any additives or preservatives known in the art and then stored at conditions and temperatures according to some embodiments to maintain the substances in their original and effective form.

[0101] The secretome of the present invention may be mixed with at least one excipient or carrier that is pharma- ceutically acceptable and compatible with the components of the secretome, as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), Plasma Lyte, dextrose, glycerol, ethanol, polyethylene glycol, mineralized excipients, such as hydroxyapatite particles and tricalcium phosphate putty or particles, and combinations thereof. Excipients and carriers may also include extracellular matrix components, such as proteins (collagen, elastin, adhesion proteins, such as fibronectin, vitronectin, albumin, etc.); glycoproteins (osteopontin, bone sialoprotein, thrombonspondin, tenascin, etc.); proteoglycans and glycoseaminoglycans (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, etc.). Other suitable excipients and carriers are well known to those skilled in the art.

[0102] In addition, if desired the composition may contain minor amounts of auxiliary substances such as emulsifying agents, pH buffering agents and the like.

[0103] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0104] The term "administering" a composition to a subject or "administration" of a composition to a subject may be performed using one of a variety of methods known to those of skill in the art. For example, the composition may be administered enterally or parenterally. Enterally refers to administration via the digestive tract, including orally, sublingually, or rectally. Parenteral administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, intraocular, sublingual, intranasal, by inhalation, intraspinal, intracerebral, and transdermal administration (e.g., by absorption through the skin tract). The composition may be conveniently introduced by rechargeable or biodegradable polymeric or other devices, such as patches and pumps, or formulations that provide sustained, sustained, or controlled release of the compound or agent. Administration may be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a patient is administered a drug by a physician who instructs the patient to self-administer the drug or to have another person administer the drug and / or who provides the patient with a prescription for the drug.

[0105] The following examples are intended to illustrate how to make and use the compounds and methods of the present invention, and should not be construed as limiting in any way. Although the present invention will now be described with specific embodiments thereof, it is apparent that numerous modifications and variations will become apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the appended claims.

[0106] (Example) The results described below are obtained in part from the secretome of a cell population derived from the lamina propria (but not the epithelial portion) of human gingiva, an integral part of the oral mucosa lining the oral cavity. hOMSCs isolated from palatal and alveolar mucosa show the same properties.

[0107] Isolation and culture of hOMSCs Specifically, hOMSCs were obtained from oral mucosal biopsy samples of gingival origin from donors aged 25-80 years, as described above and in WO 2008 / 132722.

[0108] Briefly, gingival or alveolar mucosa biopsies measuring 3–4 × 2 × 1 mm were cut into pieces and cut into 25 cm pieces as described by Marinka-Kalmani et al. 2010 (ibid.). 2 Explants were cultured in low glucose Dulbecco's modified Eagle's medium (LGDMEM) supplemented with 100 μg / ml streptomycin, 100 U / ml penicillin (Biological Industries, Bete Haemek, Israel), 2 mM glutamine (Invitrogen), and 10% fetal calf serum (FCS) (Gibco) in tissue culture flasks. This medium is referred to as growth medium or expansion medium. In some cases, streptomycin and penicillin are replaced with gentamicin, and amphotericin B is also included in the expansion medium.

[0109] Secretome generation Cultures of hOMSCs expanded in Expansion Medium and with cumulative population doublings between 5 and 80 are used for generation of hOMSC secretomes. Expansion Medium is removed and cultures are washed extensively with PBS, then basal medium or LGDMEM is added. After 24-120 hours, the medium is collected and centrifuged to remove any dead cells. The supernatant contains the secretome.

[0110] The concentration of the secretome components can be increased by concentrating the supernatant using devices and methods known in the art. As shown herein, in some embodiments, concentration ratios ranging from 1.1 to 10,000 times are believed to be useful and effective to achieve the desired therapeutic effect.

[0111] Array analysis at the protein and molecular levels indicates that the hOMSC secretome has unique compositional features not previously seen in stem cell secretomes derived from other sources, including skin-derived stem cells and bone marrow-derived stem cells.

[0112] The composition of the secretome can be altered by subjecting hOMSCs to various culture conditions and stimuli. Examples of such culture stimuli and conditions include, but are not limited to, the following: Chemical: e.g. hypoxia, hyperoxia, chemicals, various types of chemical stimulants or inhibitors or various pathways, e.g. Ca ++ high or low ionic concentrations such as and / or various chemicals such as glucose, statins, bisphosphonates, etc.; Physical: e.g. ultrasound, mechanical vibration, electrical stimulation, continuous or intermittent strain, light, radiation; Substrates: e.g. adhesion proteins, three-dimensional matrices, beads for suspension cell culture; Biological: e.g. growth factors, cytokines, hormonal stimuli, differentiation factors, DNA and RNA species, genetic manipulation.

[0113] Example 1. Comparison of stem cell markers of hOMSCs with their or other sources hOMSCs were obtained as previously described (Marynka-Kalmani et al., 2010 and WO 2008 / 132722). Foreskin SCs (hSkin SCs) were isolated by enzymatic digestion from the foreskins of 8-day-old infants. Both cell types were grown in T-75 cell flasks in low glucose DMEM supplemented with essential amino acids, antibiotics and fetal bovine serum.

[0114] Stem cell markers of hOMSCs and foreskin stem cells (hSkin SCs) were assessed by RT-PCR and immunochemistry (Marinka-Kalmani et al., 2010, ibid. and unpublished data).

[0115] As shown in Figure 1 , hOMSCs have higher expression of pluripotency- and neural crest-associated markers than hdermal SCs.

[0116] The markers OCT4, SOX2 and NANOG are distinctive pluripotency-associated markers, c-MYC and KLF4 are both pluripotency-associated and early neural crest markers, and SNAIL is a distinctive neural crest stem cell marker.

[0117] Molecular data were confirmed at the protein level by immunofluorescence using antibodies against pluripotency-associated stem cell markers and neural crest-associated stem cell markers, which revealed higher abundance of the markers NANOG, SOX2, C-MYC, KLF4 and SNAIL in hOMSCs compared to hdermal SCs. We also found that staining in hOMSCs was restricted to the nucleus, suggesting functional activity of these transcription factors. Therefore, we conclude that expression of the above markers is significantly higher in hOMSCs compared to hdermal SCs.

[0118] Example 2. Comprehensive analysis of hOMSC secretome We identify unique features of the hOMSC secretome by characterizing its protein and nucleic acid content. Three different methods, namely protein array, mass spectrometry (MS) and microRNA (miRNA) characterization, are used to obtain a comprehensive range of hOMSC secretome components.

[0119] hOMSCs are generated as described above and expanded in expansion medium.The protein profile of conditioned medium is evaluated by mass spectrometry and commercial protein array kit.Using methods known in the art, the sequence of RNA contained in hOMSCs secretome is identified to determine the gene cargo of hOMSCs secretome.

[0120] Protein Profile The protein content of the hOMSC secretome was analyzed by MS and protein arrays.

[0121] MS analysis: Four secretomes of four different hOMSC cultures, each derived from a separate donor, are prepared as described above. 0.1 ml samples are digested with trypsin and analyzed by LC-MS / MS on a Q exactive plus (Thermo Fisher) and analyzed with Discoverer software version 1.4 against human and bovine uniprot databases (for fetal bovine serum). Identified proteins are filtered at peptide and protein level with a false discovery rate (FDR) < 0.01 using a target-decoy strategy.

[0122] Proteins were filtered to eliminate common contaminants and single peptide identification. Semi-quantification was performed by calculating the peak area of ​​each peptide. Protein areas are the average of the three most intense peptides from each protein.

[0123] A total of 369 proteins were identified in the hOMSC secretome (Figure 11), including extracellular matrix proteins, glycoproteins, protein receptors, proteases and protease inhibitors for extracellular matrix proteins, proteins involved in metabolism, proteins involved in stress response such as heat shock proteins, nuclear proteins, proteins involved in tissue development and repair, integral membrane proteins such as integrins and cluster of differentiation (CD) proteins including the exosome marker CD63, immunomodulatory proteins and other protein clusters.

[0124] Of note is a cluster of 13 proteins involved in nervous system homeostasis, protection and repair, detailed in Table 1: [Table 1]

[0125] Of particular interest is the presence of SOD1 and mesencephalic astrocyte-derived neurotrophic factor (MANF) in the hOMSC secretome. These proteins suppress intracellular stress, a marker of neurodegenerative diseases and a cause of cell death. Furthermore, MANF has been shown to be effective in treating Parkinson's disease in animal models (Voutilainen et al., 2015).

[0126] The protein composition of the hOMSC secretome determined by MS is unique: when comparing the hOMSC secretome with those of mesenchymal stem cells derived from human bone marrow (BMSC), adipose tissue (ASC) or dental pulp (DPSC) (Tachida et al., 2015), the hOMSC secretome is found to contain 75 proteins not detected in any of the secretomes.

[0127] The 75 unique proteins identified are listed in Table 2: [Table 2]

[0128] The protein CXCL12, unique to the hOMSC secretome, is known to aid in stem cell recruitment to injured organs and promote proliferation and migration of neural progenitor cells (Wu et al., 2009). This protein is abundant in the hOMSC secretome, ranking 58th out of 369 proteins, i.e., in the top 20% of proteins detected.

[0129] Peripheral tissues under stress due to disease or injury secrete CXCL12 to recruit endothelial progenitor cells and mesenchymal stem (stromal) cells from the bone marrow. In injured diabetic tissues, this process is significantly suppressed (Rodrigues et al., 2015; Tepper et al., 2010). Administration of hOMSC secretome, which contains CXCL12 as a major trophic factor, in injured tissues promotes wound healing in general and in diabetic individuals in particular.

[0130] As shown in Figure 12, a total of 294 hOMSC secretome proteins are common to the 1534 proteins listed in the secretomes derived from BMSCs, ASCs or DPSCs. Meanwhile, the unique features of a secretome are determined not only by its components but also by the relative abundance of these components. For example, insulin growth factor 2 (IGF2), which belongs to the insulin family of growth factors and has pleiotropic functions in tissue homeostasis and repair, is one of the major components of the hOMSC secretome, but is barely detected in BMSCs and completely absent in ASCs and DPSCs.

[0131] Protein Array: The protein components of the secretome of hOMSCs were further analyzed and compared with that of hskin SCs by a protein array kit of 80 proteins (RayBio® G-Series Cytokine Array, RayBiotech, USA). Protein analysis revealed differential secretion of at least 21 proteins that were over- or under-expressed in the hOMSC secretome compared with that of hskin SCs (Figures 2 and 3). Of note are the proteins PlGF, MSCF, VEGF and HGF, which are cytokines with higher abundance compared with hskin SCs, and the proteins leptin, ENA-78 and MCP-3, which are lower abundance.

[0132] The secretomes of hOMSCs and hskin SCs were further compared with those published for human mesenchymal stem cells derived from young human bone marrow using the same antibody array kit used to reveal the secretomes of hOMSCs and hskin SCs above (Park et al., 2009). Taking into account a lower limit of detection value of 50, a comparison of Figures 2 and 4 shows that the growth factors and neurotrophic agents PlGF, EGF, SDF-1, BDNF, GDNF, IGF1, angiogenin as well as many others are not detected in the secretome of bone marrow derived mesenchymal stem cells but are expressed in hOMSCs.

[0133] Some of the factors and agents, such as HGF, are highly expressed in hOMSCs. The differences between the hOMSC secretome and the skin SC secretome are shown in Figure 3, which shows the ratio of the amounts of various chemokines in the hOMSC secretome and the skin SC secretome. The data shown in Figures 1-4 clearly show that the three stem cell types have different secretion profiles for the tested proteins.

[0134] MicroRNA Analysis: Conditioned media was collected from three different hOMSC cultures, each derived from a different donor, as described above for secretome preparation, except that no enrichment step was performed. The conditioned media was centrifuged at 12000G for 4 minutes at 4°C, and total miRNA was extracted according to the following procedure: 1. Add 7 ml of Trizol™ reagent to 10 ml of sample and vortex. Then leave the sample at room temperature for 5 minutes. 2. Add 1.4 ml of chloroform and shake the sample vigorously for 15 seconds. 3. Centrifuge the sample at 15,300G for 15 minutes at 4°C. 4. Transfer the upper aqueous phase to a new tube, carefully avoiding the interphase. 5. Isolate miRNA using the mirVana™ PARIS miRNA Isolation Kit (Ambion®) according to the manufacturer's protocol. The mirVana™ Kit uses two GFFs in series. Elute miRNA with 50 μl of RNase-free water.

[0135] The concentration and purity of miRNA was assessed using a NanoDrop™ optical spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The wavelength-dependent extinction coefficients represent the traces of total RNA in solution, as shown in Table 3: [Table 3]

[0136] Characterization of microRNA expression miRNA expression profiling was performed using the nCounter miRNA Expression Assay (described at https: / / www.nanostring.com), a method that detects 800 miRNAs by using molecular barcodes called nCounter Reporter Probes without reverse transcription or amplification. All data analysis and normalization was performed using nSolver™ Software Analysis (free download from NanoString Technologies), which normalizes specific miRNA counts to selected stably expressed miRNAs based on CVS statistics calculated across all experimental samples, or using Spike-in controls.

[0137] The normalized data shown in Table 4 shows 39 miRNAs expressed in three hOMSC secretomes, each from a different donor, each with a relative expression value above 20, the lower miRNA detection threshold allowed using the methodology described above.

[0138] [Table 4]

[0139] The profile of these 39 miRNAs was compared with published profiles for bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ASCs) (Baglio et al., 2015). The results show that the miRNA profile of the hOMSC secretome is unique. Only five and three miRNAs are shared between the miRNAs of the hOMSC secretome and those detected in the BMSC and ASC secretomes, respectively. Of the five miRNAs shared by the hOMSC and BMSC secretomes, three are also shared with the ASC secretome. Thus, the 34 miRNAs are not included in the miRNA composition of adult BMSC and adult ASC secretomes, which are considered to have high therapeutic potential.

[0140] Example 3. Therapeutic potential of hOMSCs in wound healing In diabetes, wound healing is delayed due to abnormalities in local and systemic signaling and inappropriate tissue responses to wound healing cues. This multifactorial defect in the wound healing process leads to delayed cell migration and reduced formation of new blood vessels and connective tissue. Stem cells have previously been proposed as a state-of-the-art treatment for diabetic wounds due to their multifactorial secretome.

[0141] Transgenic mice lacking the leptin receptor (db / db mice) were used to increase food intake, resulting in obesity and the development of type 2 diabetes, with a pathogenesis similar to that of human type 2 diabetes. Diabetic db / db mice have the slowest rate of skin wound closure among all known mouse diabetes models (Michaels J et al., 2007).

[0142] A full-thickness skin wound of 6 mm in diameter was performed on the back of diabetic (blood glucose >300 mg / dcl) db / db mice. To prevent wound contraction, a silicone donut-shaped ring with an inner diameter of 8 mm was sutured around the wound to serve as a standard reference for calculating the rate of wound closure at the macroscopic level. Mice were divided into three groups of 5–10 individuals each: i) a negative control group injected with PBS, which served as a vehicle for cell delivery; ii) a hOMSC-treated group; and iii) a hDSC-treated group. 5 × 10 cells were injected intradermally at four equidistant sites. 5 100 cells / site were injected. Mice were photographed every 2-4 days to determine the rate of wound closure. To do this, the wound area at each time point for each animal was determined by image analysis of the photographs, and the wound area was normalized by determining the area outlined by the inner circumference of a donut-shaped ring seen on the same photograph (Figure 5).

[0143] The results shown in Figure 6A (quantitative results) and 6B (representative qualitative photographs) indicate that hOMSC-treated animals had a statistically significant (p<0.05) faster wound healing rate to wound closure than untreated animals (PBS vehicle) or hDermal SC-treated animals. Wound closure in all animals treated with hOMSC was observed 16 days after wounding, whereas complete wound closure in all animals in the untreated group was observed 26 days after wounding. Furthermore, no statistically significant differences were observed between the untreated and hDermal SC-treated groups.

[0144] In a separate study, we also investigated the natural wound healing kinetics in healthy untreated wild-type (WT) individuals. For this purpose, we used the same experimental setup as above. Mice were age-matched to their db / db counterparts.

[0145] As shown in Figure 7, the wound healing rate of hOMSC-treated mice was similar to that of WT untreated mice. The mean time to complete wound closure in db / db diabetic mice treated with hOMSC was 14.3 ± 1.4 days, the mean time in WT untreated mice was 15.14 ± 1.06 days, and the mean time in db / db diabetic untreated mice was 22.75 ± 2.16 days. These results indicate that hOMSCs have the ability to overcome the deleterious effects of the diabetic state on wound healing and restore the rate of diabetic wound healing to normal.

[0146] The lack of stimulatory effect of hDermal SCs on diabetic wound healing was surprising, so the potential of another SC population, i.e., adipose tissue-derived stem cells (hADSCs), was examined in the same experimental setting. The results shown in Figures 8 and 9 show that the wound healing rate of hADSC-treated mice was statistically significantly lower than that of hOMSC-treated mice, but higher than that of untreated or hDermal SC-treated mice (p<0.05). Nevertheless, complete wound healing was observed in all hADSC-treated mice 24 days after wounding, similar to that of hDermal SC-treated mice.

[0147] Therefore, it was concluded that naïve hOMSCs are superior to other stem cells in promoting diabetic wound healing.

[0148] Example 4. Therapeutic potential of hOMSC secretome To investigate whether the therapeutic effects of hOMSCs are attributable, at least in part, to their unique secretome, we 6 The hOMSCs were maintained in serum-free medium for 24 h.

[0149] The medium was then collected and concentrated with a 1,000 Dalton cutoff concentration filter (Amicon). The concentrated medium contains components of the naive hOMSC secretome as analyzed by MS. This hOMSC-derived concentrated conditioned medium was injected into a diabetic wound healing model as described in Example 2 and Figure 5. Each diabetic db / db mouse was injected with 2x10 cells / mL of conditioned medium at each site indicated by the arrow in Figure 5 for 24 hours. 5 Each animal was injected with 50 μl of conditioned medium, which indicates secretion of hOMSCs. Thus, a total of 200 μl of concentrated conditioned medium was injected around the wound. Conditioned medium was administered only at the beginning of the experimental period. Mice were followed macroscopically as described above and sacrificed at the time of closure. The wound area including the silicone ring was read, processed, and subjected to histological analysis.

[0150] The rate of wound closure is shown in Figure 10. The results indicate that a single administration of hOMSC-secretome entrapped in enriched hOMSC-conditioned medium was as effective as hOMSC in promoting diabetic wound healing. Histomorphometric analysis of blood vessel number and collagen content in the center of the healed wound revealed i) no statistically significant difference between the amount of collagenous connective tissue in hOMSC-treated animals and that in hOMSC-secretome-treated animals; and ii) increased blood vessel number in hOMSC-secretome-treated animals compared to untreated or hDSC-treated animals, and decreased blood vessel number compared to hOMSC-treated animals.

[0151] Without wishing to be bound by any theory or mechanism of action, it is suggested that continuous or multiple treatments with secretomes may be required compared to treatments with stem cells that continue to secrete substances, yet secretome compositions are safer and easier to manipulate, characterize, maintain, and administer than cells.

[0152] The results collectively suggest that hOMSC-secretome retains the efficiency of hOMSCs to promote diabetic foot ulcer healing and therefore they may be used as an independent therapeutic tool or an adjunct to cell therapy, especially whenever de novo angiogenesis is required.

[0153] The above description of the specific embodiments fully reveals the general nature of the present invention, so that others, applying current knowledge, can easily modify and / or adapt such specific embodiments to various uses without undue experimentation and without departing from the general concept, and therefore such adaptations and modifications should be understood and are intended to be within the meaning and scope of equivalents of the embodiments of the present disclosure. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. The means, materials and steps for carrying out the various functions disclosed may take various alternative forms without departing from the present invention.

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[0155] This application may include the following inventions. (1) A cell-free composition comprising material secreted from human oral mucosal stem cells (hOMSC-derived secretome) together with at least one carrier, excipient or diluent. (2) (i) at least one protein from the group consisting of interstitial cell-derived factor 1 (CXCL12 / SDF1), superoxide dismutase [Cu-Zn] (SOD1), mesencephalic astrocyte-derived neurotrophic factor (MANF), cystatin C (CST3), galectin 1 (LGALS1), glial-derived nexin (SERPINE2), insulin-like growth factor II (IGF2), latent transforming growth factor beta binding protein 1 (LTBP1), latent transforming growth factor beta binding protein 2 (LTBP2), latent transforming growth factor beta binding protein 3 fragment (LTBP3), latent transforming growth factor beta binding protein 4 (LTBP4), neuroblast differentiation-associated protein (AHNAK) and pigment epithelium-derived factor (SERPINF1 / PEDF); or (ii) at least one protein selected from the group consisting of hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (GCSF), macrophage inflammatory protein 3 (MIP-3a), growth-regulated oncogene alpha (GRO-a or CXCL1), macrophage-derived / CCL22 chemokine (MDC or CCL22), growth-regulated oncogene (GRO), IGFBP-2, neurotrophin 4 (NT-4), monocyte chemoattractant protein 2 (MCP-2 / CCL8), insulin growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 2 (IL-2), and brain-derived neurotrophic factor (BDNF); or (iii) 1SV, 3SV, ACTG2, ADAM10, ADAMTSL1, ADM, ANXA4, APOD, CALM2, CD109, CD59, CDH6, CFD, COL15A1, COL1A2, COLEC12, CTHRC1, CTSC, CTSL, CXC L12, DCD, DDAH2, DKK1, DSG1, DSP, DSTN, ECH1, EDIL3, EFEMP1, ELN, FLG, GNB2, GREM2, H3F3B, HBA1, HIST1H2AH, HIST1H2BK, HIST1H4A, HMGN2, HNRN At least one protein selected from the group consisting of PAB, HSP90AA1, HSPA1A, HSPG2, IGFBP5, JUP, KHSRP, LDHA, MNB2, LTBP4, MAN1A1, MFAP4, MMP1, MMP14, MT2A, NBL1, OMD, PFN1, PI16, PSG5, PSMB6, PTGDS, RARRES2, SLIT3, SPOCK1, SPTBN4, STOM, TMSB10, TMSB4X, TNFAIP6, TNXB, TPI1, TUBA1C, UBC, VIT, and WNT5A, or (iv)hsa-miR-4454+hsa-miR-7975, hsa-miR-23a-3p, hsa-let-7b-5p, hsa-miR-612, hsa-miR-12 5b-5p, hsa-miR-3144-3p, hsa-miR-199a-3p+hsa-miR-199b-3p, hsa-miR-191-5p, hsa-miR-100-5 p, hsa-miR-127-3p, hsa-miR-1260a, hsa-miR-378h, hsa-miR-379-5p, hsa-miR-376a-3p, hsa-let -7i-5p, hsa-miR-526a+hsa-miR-518c-5p+hsa-miR-518d-5p, hsa-miR-212-3p, hsa-miR-520c-3p , hsa-miR-28-5p, hsa-miR-758-3p+hsa-miR-411-3p, hsa-miR-29a-3p, hsa-miR-1206, hsa-miR-1 286, hsa-miR-514a-3p, hsa-miR-548ah-5p, hsa-miR-184, hsa-miR-543, hsa-miR-626, hsa-miR-3 at least one microRNA (miRNA) selected from the group consisting of hsa-miR-39-3p, hsa-miR-1234-3p, hsa-miR-155-5p, hsa-miR-888-5p, hsa-miR-542-3p, hsa-miR-514b-5p, hsa-miR-548m, hsa-miR-30e-5p and hsa-miR-1290; or The combination The cell-free composition according to (1), (3) The cell-free composition of (2), comprising a plurality of substances from (i), (ii), (iii) or (iv). (4) The cell-free composition of (2), comprising at least one protein from (i), at least one protein from (ii), at least one protein from (iii), and optionally at least one miRNA from (iv). (5) The cell-free composition according to any one of (1) to (4), comprising at least one factor selected from the group consisting of interstitial cell-derived factor 1 (CXCL12 / SDF1), mesencephalic astrocyte-derived neurotrophic factor (MANF), superoxide dismutase [Cu-Zn] (SOD1), hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), and vascular endothelial growth factor (VEGF). (6) hsa-miR-4454+hsa-miR-7975, hsa-miR-23a-3p, hsa-let-7b-5p, hsa-miR-612, hsa-miR-125b-5p, hsa-miR-3144-3p, hsa-miR-199a-3p+hsa-miR-199b-3p, hsa-miR-191-5p, hsa-miR-100-5p, hsa-mi R-127-3p, hsa-miR-1260a, hsa-miR-378h, hsa-miR-379-5p, hsa-miR-376a-3p, hsa-let-7i-5p, hsa -miR-526a+hsa-miR-518c-5p+hsa-miR-518d-5p, hsa-miR-212-3p, hsa-miR-520c-3p, hsa-miR-28- 5p, hsa-miR-758-3p+hsa-miR-411-3p, hsa-miR-29a-3p, hsa-miR-1206, hsa-miR-1286, hsa-miR-5 14a-3p, hsa-miR-548ah-5p, hsa-miR-184, hsa-miR-543, hsa-miR-626, hsa-miR-339-3p, hsa-miR-1 The cell-free composition according to any one of (1) to (4), comprising at least six microRNA molecules selected from the group consisting of miR-234-3p, hsa-miR-155-5p, hsa-miR-888-5p, hsa-miR-542-3p, hsa-miR-514b-5p, hsa-miR-548m, hsa-miR-30e-5p and hsa-miR-1290. (7) The cell-free composition according to any one of (2) to (5), wherein the at least one protein (i), (ii) or (iii) is present at a significantly higher concentration than in secretomes derived from other stem cell sources. (8) 8. The cell-free composition of claim 7, wherein the at least one protein present at a significantly higher concentration than in secretomes derived from other stem cell sources is selected from the group consisting of stromal cell-derived factor 1 (CXCL12 / SDF1, P48061), superoxide dismutase [Cu-Zn] (SOD1, P00441), mesencephalic astrocyte-derived neurotrophic factor (MANF, P55145), hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), and vascular endothelial growth factor (VEGF). (9) The cell-free composition according to any one of (1) to (8), comprising at least one protein at a concentration significantly lower than in secretomes derived from other stem cell sources. (10) 10. The cell-free composition of claim 9, wherein the at least one protein is selected from the group consisting of interleukin-8 (IL-8), monokine induced by gamma interferon (MIG / CXCL9), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 ligand (Flt-3 ligand), leptin, epithelial-derived neutrophil-activating peptide 78 (ENA-78 / CXCL5), and monocyte chemotactic protein 3 (MCP-3 / CCL7). (11) The cell-free composition according to any one of (1) to (10), comprising at least one protein involved in homeostasis of the nervous system, the protein being selected from the group consisting of cystatin C, galectin 1, glia-derived nexin, insulin-like growth factor II, (IGF2), latent transforming growth factor beta binding protein 1 (LTBP1), latent transforming growth factor beta binding protein 2 (LTBP2); latent transforming growth factor beta binding protein 3 (LTBP3); latent transforming growth factor beta binding protein 4 (LTBP4); mesencephalic astrocyte-derived neurotrophic factor (MANF), neuroblast differentiation-associated protein (AHANK), pigment epithelium-derived factor (PEDF), stromal cell-derived factor 1 (SDF1), and superoxide dismutase [Cu-Zn] (SODC). (12) The cell-free composition according to any one of (1) to (11), comprising extracellular vesicles (EVs). (13) The cell-free composition according to any one of (1) to (12), comprising microvesicles. (14) The cell-free composition according to any one of (1) to (13), comprising an exosome. (15) The cell-free composition according to any one of (1) to (14), comprising a soluble factor having a molecular size of 1,000 daltons or more. (16) The cell-free composition according to (15), wherein the soluble factors are selected from the group consisting of proteins, peptides, hormones, DNA and RNA species, oligonucleotides and polynucleotides, and combinations thereof. (17) The cell-free composition according to any one of (1) to (16), comprising a soluble factor, a microvesicle, and an exosome. (18) The composition according to any one of (1) to (17), which is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, excipient, or diluent. (19) The composition according to any one of (1) to (17), which is a cosmetic composition. (20) The composition according to any one of (1) to (19) for use in preventing or treating a disease or disorder. (twenty one) The composition for use according to (20), wherein the disease or disorder is selected from the group consisting of inflammatory diseases; autoimmune diseases; vascular diseases; cardiac diseases; respiratory system diseases; skeletal system diseases; gastrointestinal diseases; renal diseases; urinary tract diseases; skin diseases; aging-related diseases; peripheral nerve diseases, skeletal muscle diseases, central nervous system diseases; eye diseases; endocrine system diseases; and dental and oral diseases. (twenty two) The composition according to any one of (1) to (19) above, for use in tissue remodeling, tissue repair or tissue regeneration. (twenty three) The composition for use according to (22), wherein the tissue remodeling, tissue repair or tissue regeneration comprises at least one process selected from the group consisting of promoting wound healing, preventing or reducing scar formation; promoting scar healing, promoting cartilage or bone formation; promoting the repair or regeneration of damage in the central or peripheral nervous system; and promoting neovascularization and angiogenesis of ischemic organs. (twenty four) The composition for use according to (23), wherein the damage is caused by trauma to the nervous system, a neurodegenerative disease or a vascular disease. (twenty five) 13. The cell-free composition of (11) in combination with a pharma- ceutically acceptable carrier, excipient or diluent for use in promoting repair or regeneration of damage in the central or peripheral nervous system. (26) The cell-free composition according to any one of (1) to (25), wherein the secretome is derived from autologous hOMSCs. (27) The cell-free composition according to any one of (1) to (25), wherein the secretome is derived from allogeneic hOMSCs. (28) 1. A method for generating acellular secretome from hOMSCs, comprising: i. isolating hOMSCs by explantation or enzymatic digestion; ii. Expanding the hOMSCs in culture medium; iii. replacing the medium with basal medium; iv. culturing the hOMSCs in the basal medium for a period of time ranging from 1 hour to 120 hours; v. harvesting said medium from the culture; vi. Optionally, concentrating the medium 1.1 to 10,000 times. A method comprising: (29) The method of (28), wherein during or after step (iv) the hOMSCs are subjected to a stimulus or condition that affects the content of the secretome. (30) A method for preventing or treating a disease or disorder comprising administering to a subject in need thereof the cell-free composition of (1). (31) The method according to (30), wherein the disease or disorder is selected from the group consisting of inflammatory diseases; autoimmune diseases; vascular diseases; cardiac diseases; respiratory system diseases; skeletal system diseases; gastrointestinal diseases; renal diseases; urinary tract diseases; skin diseases; aging-related diseases; peripheral nerve and skeletal muscle diseases; diseases of the central nervous system; eye diseases; diseases of the endocrine system; and dental and oral diseases. (32) The method according to (30) or (31), wherein the method of treatment comprises tissue remodeling, tissue repair or tissue regeneration. (33) The method according to (30), wherein the method of treatment comprises the repair or regeneration of organs and tissues destroyed in whole or in part by at least one iatrogenic injury. (34) 33. The method of claim 32, wherein the at least one iatrogenic injury is caused by mechanical trauma, chemical injury, chemotherapy, radiation, or heat. (35) 33. The method of claim 32, wherein the at least one injury is selected from the group consisting of a contusion of the central nervous system, a spinal cord injury, a spinal cord transection, a crush or severance of a peripheral nerve, a burn, a neuropathy, a heart disease, a bone fracture, and a ruptured tendon or ligament. (36) The method according to (32), wherein the tissue remodeling, tissue repair or tissue regeneration comprises at least one process selected from the group consisting of promoting wound healing, preventing or reducing scar formation, promoting scar healing or promoting cartilage or bone formation; promoting repair or regeneration of the central or peripheral nervous system caused by trauma, neurodegenerative diseases or vascular diseases of the nervous system; and promoting neovascularization and angiogenesis of ischemic organs. (37) The method according to (36), wherein the ischemic organ is selected from the group consisting of the heart, the brain, the peripheral nerves, and the kidneys. (38) The method according to (30), wherein the composition is a pharmaceutical composition. (39) The method according to (30), wherein the composition is a cosmetic composition. (40) The method according to (30), wherein the lesion is a diabetic wound. (41) The method according to (39), wherein the disorder is a cosmetic disorder. (42) The method according to any one of (30) to (41), wherein the cell-free composition comprises a secretome derived from autologous hOMSCs. (43) The method according to any one of (30) to (41), wherein the cell-free composition comprises a secretome derived from allogeneic hOMSCs. (44) The method according to any one of (30) to (43), wherein the composition is administered to a subject in need thereof by a route selected from the group consisting of topical, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, and intravenous, or is directly injected into any tissue at a site in need. (45) The method according to any one of (30) to (43), wherein the composition is administered locally to damaged tissue. (46) The method according to any one of (30) to (43), wherein the composition is administered systemically. (47) The cell-free composition according to any one of (1) to (19), wherein the hOMSCs are naive cells.

Claims

1. A cell-free composition comprising a substance secreted from human oral mucosal stem cells (hOMSC-derived secretome) together with at least one carrier, excipient or diluent.

2. (i) at least one protein from the group consisting of stromal cell-derived factor 1 (CXCL12 / SDF1), superoxide dismutase [Cu-Zn] (SOD1), mesencephalic astrocyte-derived neurotrophic factor (MANF), cystatin C (CST3), galectin 1 (LGALS1), glial-derived nexin (SERPINE2), insulin-like growth factor II (IGF2), latent transforming growth factor beta binding protein 1 (LTBP1), latent transforming growth factor beta binding protein 2 (LTBP2), latent transforming growth factor beta binding protein 3 fragment (LTBP3), latent transforming growth factor beta binding protein 4 (LTBP4), neuroblast differentiation-associated protein (AHNAK) and pigment epithelium-derived factor (SERPINF1 / PEDF); or (ii) at least one protein selected from the group consisting of hepatocyte growth factor (HGF), placenta growth factor (PIGF), macrophage colony-stimulating factor (MCSF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (GCSF), macrophage inflammatory protein 3 (MIP-3a), growth regulated oncogene alpha (GRO-a or CXCL1), macrophage-derived / CCL22 chemokine (MDC or CCL22), growth regulated oncogene (GRO), IGFBP-2, neurotrophin 4 (NT-4), monocyte chemoattractant protein 2 (MCP-2 / CCL8), insulin growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 2 (IL-2), and brain-derived neurotrophic factor (BDNF); or (iii) 1SV, 3SV, ACTG2, ADAM10, ADAMTSL1, ADM, ANXA4, APOD, CALM2, CD1 09, CD59, CDH6, CFD, COL15A1, COL1A2, COLEC12, CTHRC1, CTSC, CTSL, CXC L12, DCD, DDAH2, DKK1, DSG1, DSP, DSTN, ECH1, EDIL3, EFEMP1, ELN, FLG, G NB2, GREM2, H3F3B, HBA1, HIST1H2AH, HIST1H2BK, HIST1H4A, HMGN2, HNRN At least one protein selected from the group consisting of PAB, HSP90AA1, HSPA1A, HSPG2, IGFBP5, JUP, KHSRP, LDHA, MNB2, LTBP4, MAN1A1, MFAP4, MMP1, MMP14, MT2A, NBL1, OMD, PFN1, PI16, PSG5, PSMB6, PTGDS, RARRES2, SLIT3, SPOCK1, SPTBN4, STOM, TMSB10, TMSB4X, TNFAIP6, TNXB, TPI1, TUBA1C, UBC, VIT, and WNT5A, or (iv) hsa-miR-4454+hsa-miR-7975, hsa-miR-23a-3p, hsa-let-7b-5p, hsa-miR-612, hsa-miR-12 5b-5p, hsa-miR-3144-3p, hsa-miR-199a-3p+hsa-miR-199b-3p, hsa-miR-191-5p, hsa-miR-100-5 p, hsa-miR-127-3p, hsa-miR-1260a, hsa-miR-378h, hsa-miR-379-5p, hsa-miR-376a-3p, hsa-let -7i-5p, hsa-miR-526a+hsa-miR-518c-5p+hsa-miR-518d-5p, hsa-miR-212-3p, hsa-miR-520c-3p , hsa-miR-28-5p, hsa-miR-758-3p+hsa-miR-411-3p, hsa-miR-29a-3p, hsa-miR-1206, hsa-miR-1 286, hsa-miR-514a-3p, hsa-miR-548ah-5p, hsa-miR-184, hsa-miR-543, hsa-miR-626, hsa-miR-3 at least one microRNA (miRNA) selected from the group consisting of hsa-miR-39-3p, hsa-miR-1234-3p, hsa-miR-155-5p, hsa-miR-888-5p, hsa-miR-542-3p, hsa-miR-514b-5p, hsa-miR-548m, hsa-miR-30e-5p and hsa-miR-1290; or The combination The cell-free composition of claim 1 .

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