HUMAN ORAL MUCOSA STEM CELL SECRETOME

Abstract

The present invention provides secretome derived from human oral mucosa stem cells (hOMSC), and cell-free compositions comprising hOMSC-derived secretome. Methods for obtaining, manipulating and using hOMSC-derived secretome in therapy, cosmetics and tissue regeneration are also provided.

Claims

1. A method of treating a disease or disorder comprising administering to a subject in need thereof a cell-free pharmaceutical composition comprising substances secreted from human oral mucosa stem cells (hOMSC-derived secretome), together with at least one carrier, excipient, or diluent, wherein the method involves tissue remodeling, tissue repair or tissue regeneration.

2. The method of claim 1, wherein the cell-free pharmaceutical composition comprises: (i) The proteins: Stromal cell-derived factor 1 (CXCL12/SDF1), Superoxide dismutase [CuZn] (SOD1), Mesencephalic astrocyte-derived neurotrophic factor (MANF), Cystatin-C(CST3), Galectin-1 (LGALS1), Glia-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) The proteins: hepatocyte growth factor (HGF), placental 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) The proteins: 1 SV, 3 SV, ACTG2, ADAM10, ADAMTSL1, ADM, ANXA4, APOD, CALM2, CD109, CD59, CDH6, CFD, COL15A1, COL1A2, COLEC12, CTHRC1, CTSC, CTSL, CXCL12, DCD, DDAH2, DKK1, DSG1, DSP, DSTN, ECH1, EDIL3, EFEMP1, ELN, FLG, GNB2, GREM2, H3F3B, HBA1, HIST1H2AH, HIST1H2BK, HIST1H4A, HMGN2, HNRNPAB, HSP90AA1, HSPA1A, HSPG2, IGFBP5, JUP, KHSRP, LDHA, MNB2, LTBP4, MAN1A1, MFAP4, MMP1, MMP14, MT2A, NBL1, OMD, PFN1, PI16, PSG5, PSMB6, PTGDS, RARRES2, SLIT3, SPOCKI, SPTBN4, STOM, TMSB10, TMSB4X, TNFAIP6, TNXB, TPI1, TUBA1C, UBC, VIT and WNT5A; or (iv) the microRNA (miRNA) molecules: 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-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-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; or combinations thereof.

3. The method of claim 1, wherein the cell-free pharmaceutical composition comprises the substances from (i), (ii), (iii), and (iv).

4. The method of claim 1, wherein the cell-free pharmaceutical composition 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 [CuZn] (SOD1), hepatocyte growth factor (HGF), placental growth factor (PIGF), macrophage colony-stimulating factor (MCSF) and vascular endothelial growth factor (VEGF).

5. The method of claim 1, wherein the cell-free pharmaceutical composition comprises the microRNA molecules: 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-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-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.

6. The method of claim 1, wherein at least one protein selected from the group consisting of: Stromal cell-derived factor 1 (CXCL12/SDF1, P48061), Superoxide dismutase [CuZn] (SOD1, P00441), Mesencephalic astrocyte-derived neurotrophic factor (MANF, P55145), hepatocyte growth factor (HGF), placental growth factor (PIGF), macrophage colony-stimulating factor (MCSF), and vascular endothelial growth factor (VEGF), is present in the cell-free pharmaceutical composition in a significant higher concentration than in secretome derived from skin or bone marrow stem cells.

7. The method of claim 1, wherein at least one protein 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), is present in the cell-free pharmaceutical composition in a significant lower concentration than in secretome derived from skin or bone marrow stem cells.

8. The method of claim 1, wherein the cell-free composition comprises at least one protein involved in the homeostasis of the nervous system, wherein said protein is 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 [CuZn] (SODC).

9. The method of claim 1, wherein the cell-free composition comprises soluble factors, extracellular vesicles (EV), microvesicles and/or exosomes.

10. The method of claim 1, wherein the cell-free pharmaceutical composition comprises soluble factors having a molecular size of 1,000 Daltons or higher.

11. The method of claim 1, comprising at least one process selected from the group consisting of: enhancing wound healing, preventing or reducing scar formation, enhancing scar healing or enhancing cartilage- or bone-formation; enhancing repair or regeneration of the central nervous system or the peripheral nervous system caused by trauma, neurodegenerative disease or vascular diseases of the neural system; and enhancing neo-angiogenesis and neovascularization of an ischemic organ.

12. The method of claim 11, comprising repair or regeneration of organs and tissues that were totally or partially destroyed by at least one iatrogenic injury.

13. The method of claim 12, wherein the at least one iatrogenic injury is caused by mechanical trauma, chemical injury, chemotherapy, radiation or heat.

14. The method of claim 12, wherein the at least one injury is selected from the group consisting of: contusion of the central nervous system, spinal injuries, section of the spinal cord, peripheral nerve crush or section, burns, neuropathy, cardiopathy, bone fractures, tendon and ligament rupture.

15. The method of claim 12 wherein tissue remodeling, tissue repair or tissue regeneration comprises at least one process selected from the group consisting of: enhancing wound healing, preventing or reducing scar formation, enhancing scar healing or enhancing cartilage- or bone-formation; enhancing repair or regeneration of the central nervous system or the peripheral nervous system caused by trauma, neurodegenerative disease or vascular diseases of the neural system; and enhancing neo-angiogenesis and neovascularization of an ischemic organ.

16. The method of claim 15 wherein the ischemic organ is selected from the group consisting of heart, brain, peripheral nerves, and kidney.

17. The method of claim 1 wherein the treatment results in protection or repair of the nervous central nervous system or the peripheral nervous system, or in enhancement of diabetic wound healing.

18. The method of claim 1 wherein the cell-free pharmaceutical composition is administered to a subject in need thereof via a route selected from the group consisting of: topically, subcutaneously, intramuscularly, intraarterial, intraperitoneal, intrathecal, intravenously or directly injected in any tissue at the site in need.

19. The method of claim 1 wherein the cell-free pharmaceutical composition comprises secretome derived from autologous or allogeneic hOMSC.

20. The method of claim 1, wherein the hOMSC-derived secretome is derived from nave cells.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0108] FIG. 1. Comparison between the stem cell marker profile of hOMSC and foreskin stem cells (hSkin). The results are represented as Ct (cycle threshold) values relative to the house-keeping gene GAPDH. A higher negative values means a lower level of expression.

[0109] FIG. 2 Relative differences in protein expression between hOMSC and hSkin SC secretomes from immunofluorescent staining with antibodies against pluripotency- and neural crest-associated stem cell markers.

[0110] FIG. 3. Ratio of expression of selected makers in hOMSC and hSkin SC.

[0111] FIG. 4. Protein expression of the secretome of mesenchymal stem (stromal) cells obtained from young human bone marrow. Data taken from Park et al., International Journal of Stem Cells, 2009.

[0112] FIG. 5. The upper left panel shows the donut-shaped ring sutured to the dorsal skin of a diabetic db/db mouse before wounding. The upper right panel depicts the site immediately after wounding. The lower left panel illustrates the site of intradermal injections. The lower right panel shows the histology or the excised skin.

[0113] FIGS. 6A and 6B. Quantitative (6A) and representative qualitative photographs (6B) that illustrate the rate of diabetic wound healing in groups of db/db diabetic mice treated with either hOMSC or hSkin SC or with PBS vehicle (Untreated).

[0114] FIG. 7. Quantitative illustration of the rate of diabetic wound healing in groups of: db/db diabetic mice treated with hOMSC, db/db diabetic untreated mice, or wild-type (WT)-untreated mice.

[0115] FIG. 8. Average time required for complete wound closure in WT-untreated mice, WT-hOMSC-treated mice, db/db-untreated mice, db/db-hOMSC treated mice and db/db hADSC (human adipose tissue-derived stem cells) treated mice. The 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 treated=0.00044; db-untreated vs. db-hADSC treated=0.48983; WT-untreated vs. db-hOMSC treated=1.9933E-5; WT-untreated vs. db-untreated=2.262E-5.

[0116] FIG. 9. Quantitative illustration of the rate of diabetic wound healing in groups of db/db diabetic mice treated with: hOMSC, hSkin SC, hADCS or with PBS vehicle (Untreated).

[0117] FIG. 10. Quantitative illustration of the rate of diabetic wound healing in groups of db/db diabetic mice treated with:hOMSC, hOMSC-derived cell-free secretome, hSkin stem cells or with PBS vehicle (Untreated).

[0118] FIG. 11. 369 hOMSC secretome proteins identified by mass spectrophotometry, and their average relative abundance (intensity).

[0119] FIG. 12. 294 hOMSC secretome proteins that are common to the 1534 proteins listed for the secretomes derived from either BMSC. ASC or DPSC.

DETAILED DESCRIPTION OF THE INVENTION

[0120] The present invention provides secretomes of adult stem cells from human oral mucosa for treatment and prevention of diseases and disorders.

[0121] hOMSC are a neural crest (NC)-derived stem cell type, which co-express the pluripotency markers Oct4, Nanog and Sox2 as well as the NCSC markers, Snail, Slug, Sox10, Twist and Notch 1 in developing colonies (Marynka-Kalmani et al. 2010; Widera et al. 2009). The NC is a transient neuroectodermal structure of the vertebrate embryo. During its embryonic existence it gives rise to migratory multipotent stem cells that populate various primordial tissues where they differentiate into neural lineages and or lineages with a mesenchymal phenotype termed ectomesenchyme or mesectoderm. Some of these NCSC remain in a relative undifferentiated state in the adult, with a predisposition for neural differentiation even in tissues of mesenchymal origin such as dermis and bone marrow.

[0122] Typical whole adult populations contain low amount of stem cells and therefore expansion and isolation of stem cells are laborious, long and usually not efficient. It was demonstrated that primary whole population and expanded whole cell population derived from the lamina propria of the oral mucosa consists mainly (more than 80%) of nave stem cells. High proportions (80-90%) of cell populations obtained from the oral mucosa of three different donors were shown to express mesenchymal stem cells markers. The study of Marynka-Kalmani et al. 2010 (ibid) has proved that trillions of hOMSC are cost-effectively and reproducibly generated from a biopsy of 3-421 mm that is obtained with negligible morbidity.

[0123] A typical isolation method of stem cells from a solid tissue for clinical utilization comprises releasing the cells from the extracellular matrix by enzymatic digestion or by explantation; expanding primary whole population in order to obtain sufficiently large populations; and isolation of stem cells from the whole populations.

[0124] The quality and quantity of the isolated stem cells population form the lamina propria oral mucosa is largely unaffected by aging and can be expanded in vitro without losing its pluripotency and is therefore a safe and reliable source for secretomes to be re-administered to a subject in need thereof to effectively achieve tissue regeneration and other therapeutic processes.

Definitions

[0125] Oral Mucosa is the mucosal lining the oral cavity, namely: the cheeks and the alveolar ridge including the gingiva and the palate, the tongue, the floor of the mouth and the oral part of the lips. Oral mucosa consists of an epithelial tissue of ectodermal origin and the lamina propria (LP) which is a connective tissue of ectomesenchymal origin. Similar to the ectomesenchymal origin of connective tissues in the oral cavity, cells of the oral mucosa lamina propria (OMLP) originate from the embryonic ectodermal neural crest. Wounds in human oral mucosa heal mainly by regeneration. The rate of healing is faster than that in the skin or other connective tissues and seems to be affected negligibly by age and gender (Szpaderska, A. M., et al., J Dent Res, 2003, 82, 621-626).

[0126] Stem cells (SC) are undifferentiated cells, which can give rise to a succession of mature functional cells.

[0127] Embryonic stem (ES) cells are cells derived from the inner cell mass of the embryonic blastocysts that are pluripotent, thus possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo.

[0128] Adult stem cells are post-natal stem cells derived from tissues, organs or blood of an organism after its birth.

[0129] Pluripotent stem cells are stem cells capable of generating the three embryonic cell layers and their derivatives cell lineages and tissues;

[0130] Multipotent stem cells are stem cells capable of forming multiple cell lineages that constitutes an entire tissue or organ;

[0131] 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: [0132] 2. Soluble molecules as: [0133] a. Proteins [0134] b. Peptides [0135] c. Hormones [0136] d. various DNA and RNA species [0137] e. oligomers of nucleic acids [0138] f. other molecules with a molecular weight higher than 1,000 Daltons [0139] 3. Extracellular vesicles that contain: [0140] a. Proteins: growth factors, cytokines, hormones, cell surface receptors, cytosolic and nuclear proteins, metabolic enzymes, receptor ligands, adhesion proteins, endosome associated proteins, tetraspanins, lipid raft associated proteins, antigens, etc. [0141] b. RNA species: mRNA, miRNA, tRNA, rRNA, siRNA, and lncRNA and possible other RNA species [0142] c. DNAs: mitochondrial DNA (mtDNA), single stranded DNA (ssDNA), double stranded DNA (dsDNA) [0143] d. Lipids: cholesterol, sphingomyelin, hexosylcermides and others [0144] e. Lectins, glycans, proteoglycans, glycoproteins.

[0145] Extracellular Vesicles are membrane bound particles that carry cargo of soluble and insoluble substances mentioned above. The term Extracellular Vesicles refers a group of secreted or shedded vesicles of various species. These are divided in the following subtypes (Xu et al. JIC 2016): [0146] 1. Microvesicles or Shed Microvesicles; size range50-1500 nm [0147] 2. Exosomes; size range30-120 nm [0148] 3. Vesicles; size range <500 nm

[0149] Culture medium or expansion medium is the medium in which the hOMSC are cultured and expanded. 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's medium (LGDMEM), streptomycin, penicillin, gentamycin, amphotericin B, glutamine and serum, for example fetal calf serum (FCS).

[0150] According to some embodiments, the culture expansion medium comprises low LGDMEM supplemented with 100 g/ml streptomycin, 100 U/ml penicillin, (Biological Industries, Beit-Haemek, Israel), glutamine 2 mM (Invitrogen) and 10% fetal calf serum (FCS, Gibco).

[0151] Basal medium is the culture medium without serum.

[0152] Conditioned medium according to the present invention refers to the medium collected from hOMSC cultures comprising hOMSC-derived substances secreted or released into the medium in which they are grown or maintained.

[0153] The conditioned medium comprising the hOMSC secretome may optionally be concentrated using methods known in the art to increase the concentration of the secretome constituents and then preserved, for example in a frozen state. Alternatively, the conditioned medium can be lyophilized and the secretome preserved as a frozen powder and reconstituted in water for injection or saline or other known in the art solution for injection.

[0154] The concentrated conditioned medium containing the secretome or the lyophilized secretome, may be supplemented with any additive or preservative known in the art and then stored, according to some embodiments in a condition and temperature to maintain the substances in their native and effective form.

[0155] The secretomes of the present invention may be admixed with at least one excipient or carrier that is pharmaceutically acceptable and compatible with the secretome' ingredients 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 tricalciumphosphate putty or particles, and combinations thereof. Excipient and carriers may also include extracellular matrix components such as proteins (collagens, elastin, attachment proteins e.g. fibronectin, vitronectin, albumin, etc.); glycoproteins (osteopontin, bone sialoproteins, 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.

[0156] In addition, if desired, the composition can contain minor amounts of auxiliary substances such as emulsifying agents, pH buffering agents etc.

[0157] The term treatment as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0158] The term administering or administration of a composition to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a composition can be administered enterally or parenterally. Enterally refers to administration via the gastrointestinal tract including per os, sublingually or rectally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A composition can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

[0159] The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be 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.

Examples

[0160] The results described below were obtained in part from secretomes of cell populations derived from the lamina propria (not including the epithelial part) of the human gingiva which is an integral part of the oral mucosa lining the oral cavity. hOMSC isolated from the palate and alveolar mucosa exhibit the same properties.

hOMSC Isolation and Culture

[0161] hOMSC were obtained from oral mucosa biopsies particularly of gingival origin from donors aged 25-80 years as described above and in WO 2008/132722.

[0162] Briefly, gingival or alveolar mucosa biopsies 3-421 mm were minced and explants were cultured in 25 cm.sup.2 tissue culture flasks in low glucose Dulbecco's modified Eagle's medium (LGDMEM) supplemented with 100 g/ml streptomycin, 100 U/ml penicillin, (Biological Industries, Beit-Haemek, Israel), glutamine 2 mM (Invitrogen) and 10% fetal calf serum (FCS) (Gibco) as described by Marinka-Kalmani et al 2010 (ibid). This medium is referred as culture medium or expansion medium. In some cases the streptomycin and penicillin are replaced with gentamycin, and amphotericin B is also included in the expansion medium.

Secretome Generation

[0163] Cultures of hOMSC having a cumulative population doubling between 5-80 that are expanded in expansion medium are used for generating the hOMSC secretome. The expansion medium is removed and the cultures are washed exhaustively with PBS and then either 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.

[0164] The concentration of the secretome components can be increased by concentrating the supernatant using devises and methods known in the art. As shown herein, according to some embodiments, a concentration ratio ranging from 1.1-10,000 folds can be envisaged to be useful and effective for achieving a desired therapeutic effect.

[0165] Array analyses at the protein and molecular levels indicate that hOMSC secretome has a unique composition signature that has not been found before in stem cell secretomes derived from other sources, including skin-derived stem cells and bone marrow-derived stem cells.

[0166] The secretome composition may be changed by subjecting hOMSC to various culture conditions and stimulation. Examples of such culture stimulation and conditions include but are not limited to: [0167] Chemical: e.g. hypoxia, hyperoxia, chemical drugs, various classes of chemical stimulators or inhibitors or various pathways, hypo or hyper-ionic concentration as for example Ca.sup.++ and/or glucose, various chemical drugs as statins, bisphosphonate, etc; [0168] Physical: e.g. ultrasound, mechanical vibration, electrical stimulation, continuous or intermittent strain, light, radiation; [0169] Substrate: e.g. attachment proteins, 3 dimensional matrices, beads for suspension cell culture; [0170] Biologics: e.g. growth factors, cytokines, hormone stimulation, differentiation factors, DNA and RNA species, genetic manipulations.

Example 1. Comparison of Stem Cell Markers of hOMSC to Those or Other Sources

[0171] hOMSC were obtained as described previously (Marynka-Kalmani et al. 2010 and WO 2008/132722). Foreskin SC (hSkin SC) were isolated by enzymatic digestion from the foreskin 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 calf serum.

[0172] Stem cell markers of hOMSC and foreskin stem cells (hSkin SC), were assessed by RT-PCR and immunochemistry (Marinka-Kalmani et al 2010, ibid and unpublished data).

[0173] As shown in FIG. 1, hOMSC are endowed with a higher expression of pluripotency and neural crest associated markers than hSkin SC.

[0174] The markers OCT4, SOX2, and NANOG are characteristic pluripotency associated markers; c-MYC and KLF4 are both pluripotency associated and early neural crest markers; and SNAIL is a characteristic neural crest stem cell marker.

[0175] The molecular data was confirmed at the protein level by immunofluorescence staining with antibodies against pluripotency- and neural crest-associated stem cell markers. indicating that the markers NANOG, SOX2, C-MYC, KLF4 and SNAIL are more abundant in hOMSC than in the hSkin SC. It was also found that the staining in hOMSC is restricted to the nuclei suggesting functional activity of these transcription factors. It is therefore concluded that there is clear higher expression of these markers in hOMSC compared to hSkin Sc.

Example 2. Global Analysis of hOMSC Secretome

[0176] The unique signature of the hOMSC secretome is confirmed by determining its protein and nucleic acid content. Three different method are used to obtain a broad spectrum of hOMSC secretome components: protein arrays, mass spectrophotometry (MS) and microRNA (miRNA) characterization.

[0177] hOMSC are generated and expanded in expansion medium as described above. The protein profile in the condition medium was assessed by mass spectrophotometry and by commercially available protein array kits. The sequence of RNA specifies contained within the hOMSC secretome is performed using methods known in art, to determine the genetic cargo of hOMSC secretome.

Protein Profile

[0178] The protein content of hOMSC secretome was analyzed by MS and protein array.

MS analysis: Four secretomes, from 4 different hOMSC cultures, each derived from a separate donor, are prepared as described above. 0.1 ml samples are digested by trypsin, analyzed by LC-MS/MS on Q exactive plus (Thermo Fisher) and analyzed by Discoverer software version 1.4 against the human and bovine uniprot database (for fetal calf serum). The identified proteins are filtered for false discovery rates (FDRs)<0.01 in the peptide- and protein-level using the target-decoy strategy.

[0179] The proteins are filtered to eliminate the common contaminants and single peptide identifications. Semi quantitation was done by calculating the peak area of each peptide. The area of the protein is the average of the three most intense peptides from each protein.

[0180] A total of 369 proteins were identified within hOMSC secretome (FIG. 11), including extracellular matrix proteins, glycoproteins, protein receptors, proteolytic enzymes for extracellular matrix proteins and proteolytic enzyme inhibitors, proteins involved in metabolism, proteins involved in stress responses, such as heat shock proteins, nuclear proteins, proteins involved in tissue development and repair, integral cell membrane proteins as integrins and clusters of differentiation (CD) proteins including exosomal markers CD63, immune-modulatory proteins and other clusters of proteins.

[0181] Noteworthy are a cluster of 13 proteins that are involved in the homeostasis, protection and repair of the nervous system, detailed in Table 1:

TABLE-US-00001 TABLE 1 hOMSC secretome proteins involved in the homeostasis of the nervous system. Relative Accession No. Protein Description Gene Name Expression P01034 Cystatin-C OS = Homo sapiens GN = CST3 PE = CST3 2.220E8 1 SV = 1 [CYTC_HUMAN] P09382 Galectin-1 OS = Homo sapiens GN = LGALS1 PE = LGALS1 6.870E8 1 SV = 2 [LEG1_HUMAN] P07093 Glia-derived nexin OS = Homo sapiens SERPINE2 3.003E8 GN = SERPINE2 PE = 1 SV = 1 [GDN_HUMAN] P01344 Insulin-like growth factor II OS = Homo sapiens IGF2 1.730E8 GN = IGF2 PE = 1 SV = 1 [IGF2_HUMAN] E7EV71 Latent-transforming growth factor beta-binding LTBP1 1.084E8 protein 1 OS = Homo sapiens GN = LTBP1 PE = 1 SV = 2 - [E7EV71_HUMAN] G3V511 Latent-transforming growth factor beta-binding LTBP2 5.890E7 protein 2 OS = Homo sapiens GN = LTBP2 PE = 1 SV = 1 [G3V511_HUMAN] H0YC99 Latent-transforming growth factor beta-binding LTBP3 4.056E7 protein 3 (Fragment) OS = Homo sapiens GN = LTBP3 PE = 1 SV = 1 [H0YC99_HUMAN] A0A0C4DH07 Latent-transforming growth factor beta-binding LTBP4 2.054E7 protein 4 OS = Homo sapiens GN = LTBP4 PE = 1 SV = 1 [A0A0C4DH07_HUMAN] P55145 Mesencephalic astrocyte-derived neurotrophic factor MANF 2.533E6 OS = Homo sapiens GN = MANF PE = 1 SV = 3 [MANF_HUMAN] Q09666 Neuroblast differentiation-associated protein AHNAK 1.658E7 AHNAK OS = Homo sapiens GN = AHNAK PE = 1 SV = 2 [AHNK_HUMAN] P36955 Pigment epithelium-derived factor OS = Homo sapiens SERPINF1 6.100E8 GN = SERPINF1 PE = 1 SV = 4 [PEDF_HUMAN] P48061 Stromal cell-derived factor 1 OS = Homo sapiens CXCL12 1.390E8 GN = CXCL12 PE = 1 SV = 1 [SDF1_HUMAN] P00441 Superoxide dismutase [Cu-Zn] OS = Homo sapiens SOD1 2.246E7 GN = SOD1 PE = 1 SV = 2 [SODC_HUMAN]

[0182] Of special interest are the existence of SOD1 and mesencephalic astrocyte derived-neurotrophic factor (MANF) in hOMSC secretome. These proteins suppress the intracellular stress, which is a landmark of neurodegenerative diseases and a cause of cell death. Moreover, MANF has been shown to be efficient in the treatment of Parkinson Disease in animal models (Voutilainen et al. 2015).

[0183] The protein composition of hOMSC secretome as determined by MS is unique. When hOMSC secretome is compared to that of mesenchymal stem cells derived from human bone marrow (BMSC), adipose tissue (ASC) or dental pulp (DPSC) (Tachida et al. 2015), it is found that hOMSC secretome contains 75 proteins that are not detected in any of the secretomes.

[0184] The 75 unique proteins identified are listed in Table 2:

TABLE-US-00002 TABLE 2 Unique hOMSC secretome proteins. Protein Accession No. Protein Accession No. Protein Accession No. 1 SV E7ENT3 DSTN F6RFD5 MFAP4 P55083 3 SV HOYAE9 ECH1 M0R248 MMP1 P03956 ACTG2 P63267 EDIL3 O43854 MMP14 P50281 ADAM10 014672 EFEMP1 A0A0UIRQV3 MT2A P02795 ADAMTSL1 Q8N6G6 ELN E7ETP7 NBL1 A3KFI5 ADM EP9L83 FLG P20930 OMD Q99983 ANXA4 Q6P452 GNB2 C9JIS1 PFN1 P07737 APOD P05090 GREM2 G9H772 PI16 Q6UXB8 CALM2 P0DP24 H3F3B K7EMV3 PSG5 E7EQY3 CD109 Q6YHK3 HBA1 P69905 PSMB6 P28072 CD59 E9PNW4 HIST1H2AH Q96KK5 PTGDS P41222 CDH6 DHRF86 HIST1H2BK O60814 RARRES2 Q99969 CFD P00746 HIST1H4A P62805 SLIT3 A0A0A0MSC8 COL15A1 A0A087X0K0 HMGN2 P05204 SPOCK1 Q08629 COL1A2 A0A087WTA8 HNRNPAB D6R9P3 SPTBN4 M0QZQ3 COLEC12 Q5KU26 HSP90AA1 P07900 STOM P27105 CTHRC1 Q96CG8 HSPA1A P0DMV8 TMSB10 P63313 CTSC H0YCY8 HSPG2 P98160 TMSB4X P62328 CTSL P07711 IGFBP5 P24593 TNFAIP6 P98066 CXCL12 P48061 JUP P14923 TNXB A0A087WWA5 DCD P81605 KHSRP M0R0I5 TPI1 P60174 DDAH2 O95865 LDHA P00338 TUBA1C Q9BQE3 DKK1 O94907 LMNB2 Q03252 UBC F5H6Q2 DSG1 Q02413 LTBP4 A0A0C4DH07 VIT Q6UXI7 DSP P15924 MAN1A1 P33908 WNT5A P41221

[0185] The protein CXCL12, which is unique to hOMSC secretome, is known to be instrumental in stem cell recruitment to injured organs and promote proliferation and migration of neural progenitor cells (Wu et al. 2009). This protein is highly abundant in hOMSC secretome being ranked 58 out of 369 proteins, namely in the upper 20% of the detected proteins.

[0186] Peripheral tissues under stress caused by disease or injury secrete CXCL12 to recruit endothelial progenitors and mesenchymal stem (stromal) cells from the bone marrow. This process is substantially depressed in injured diabetic tissues (Rodrigues et al. 2015, Tepper et al. 2010). Administration of the hOMSC secretome that contains CXCL12 as a main trophic factor at the injured tissue, enhances wound healing in general and in diabetic individuals in particular.

[0187] As shown in FIG. 12, a total of 294 hOMSC secretome proteins are common to the 1534 proteins listed for the secretomes derived from either BMSC. ASC or DPSC. However, the unique signature of a secretome is determined not only by its components but also by the relative abundance of these components. For example, Insulin Growth factor 2 (IGF2), that belongs to the insulin family of growth factors and has pleiotropic functions in tissue homeostasis and repair is a major component of hOMSC secretome, but is barely detected in BMSC and undetected in ASC and DPSC.

[0188] Protein array: The protein component of the secretome of hOMSC was further analyzed and compared to that of hSkin SC by a protein array kit of 80 proteins (RayBio G-Series Cytokine Array, RayBiotech, Inc, USA). Protein analysis revealed the differences in the secretion of at least 21 proteins that were either over- or under-expressed in hOMSC secretome compared to that of hSkin SC (FIGS. 2 and 3). Notable are the proteins P1GF, MSCF, VEGF and HGF for the over abundant cytokines and the proteins leptin, ENA-78 and MCP-3 for the under abundant in hOMSC compared with hSkin SC.

[0189] The secretome of hOMSC and hSkin SC was further compared to that published for human mesenchymal stem cells derived from young human bone marrow that used the same antibody array kit that was used to determine the secretome of hOMSC and hSkin SC described above (Park et al. 2009). Considering the lower detection limit at the value of 50, comparison of the tables in FIGS. 2 and 4 shows that growth factors and neurotrophic agents as P1GF, EGF, SDF-1, BDNF, GDNF, IGF1, Angiogenin and many other are undetectable in the secretome of bone marrow derived mesenchymal stem cells but are expressed in hOMSC.

[0190] Some of the factors and agents, such as, for example, HGF, are highly expressed in hOMSC. The differences between hOMSC secretome and that of skin SC is shown in FIG. 3, which illustrates the proportions between the quantities of various chemokines within hOMSC secretome and skin SC one. The data presented in FIGS. 1-4 clearly demonstrate that the 3 stem cell types have a different secretory profile as of the proteins tested.

MicroRNA analysis: Conditioned medium was collected from three different hOMSC cultures, each derived from a different donor, as described above for the preparation of the secretome, but without performing the concentration step. The conditioned medium was centrifuged for 4 minutes at 12000 G at 4 C., total miRNA was extracted according to the procedure below: [0191] 1. 7 ml of Trizol reagent is added to 10 ml of sample and vortexed. Then the samples are left to stand for 5 minutes at room temperature. [0192] 2. 1.4 ml of chloroform is added and the sample shaken vigorously for 15 seconds. [0193] 3. Samples are centrifuged at 15,300G at 4 C., for 15 minutes. [0194] 4. The upper aqueous phase is transferred to a new tube, carefully avoiding the interphase. [0195] 5. miRNA is isolated using mirVana PARIS miRNA Isolation Kit (Ambion) according the manufacturer's protocol. The mirVana kit utilizes two sequential GFFs.
miRNA is eluted in 50 l RNase-free water.

[0196] The concentration and purity of miRNA was assessed using NanoDrop light spectrophotometer (NanodropTechnologies, Wilmington, DE, USA). The wavelength dependent extinction coefficient represents the microcomponent of all RNA in solution as shown the Table 3:

TABLE-US-00003 TABLE 3 Wavelength-dependent extinction coefficient values Extinction Sample Volume Coefficient # ng/l (l) at 260/280 nm 1 38.5 17 2.11 2 30.9 17 2.15 3 40.7 17 2.1
Characterization of microRNAs Expression

[0197] miRNA expression profiling was performed using the nCounter miRNA Expression Assay (described in https://www.nanostring.com), that provides a method for detecting 800 miRNAs without the use of reverse transcription or amplification by using molecular barcodes called nCounter Reporter Probes. All data analysis and normalization were performed using the nSolver Software Analysis (complimentary download from NanoString Technologies) in which specific miRNA counts are normalized to a selection of stably expressed miRNAs based on CVS statistics calculated across all experimental samples or with the use of the Spike-in controls.

[0198] The normalized data shown in Table 4 indicates 39 miRNAs that are expressed in the 3 hOMSC secretomes, each derived from a different donor. Each of this miRNAs has a relative expression value higher than 20, which is an accepted lower threshold for miRNA detection when the methodology described above is used.

TABLE-US-00004 TABLE 4 miRNAs having a relative value >20, identified in 3 unique hOMSC secretomes. Expressed miRNA name also in Reference hsa-miR-4454 + hsa-miR-7975 MIMAT0018976 hsa-miR-23a-3p MIMAT0000078 hsa-let-7b-5p MIMAT0000063 hsa-miR-612 MIMAT0003280 hsa-miR-125b-5p BMSC MIMAT0000423 hsa-miR-3144-3p MIMAT0015015 hsa-miR-199a-3p + hsa-miR-199b-3p MIMAT0000232 hsa-miR-191-5p ASC & BMSC MIMAT0000440 hsa-miR-100-5p ASC & BMSC MIMAT0000098 hsa-miR-127-3p ASC & BMSC MIMAT0000446 hsa-miR-1260a MIMAT0005911 hsa-miR-378h MIMAT0018984 hsa-miR-379-5p MIMAT0000733 hsa-miR-376a-3p MIMAT0000729 hsa-let-7i-5p BMSC MIMAT0000415 hsa-miR-526a + hsa-miR-518c-5p + MIMAT0002845 hsa-miR-518d-5p hsa-miR-212-3p MIMAT0000269 hsa-miR-520c-3p MIMAT0002846 hsa-miR-28-5p MIMAT0000085 hsa-miR-758-3p + hsa-miR-411-3p MIMAT0003879 hsa-miR-29a-3p MIMAT0000086 hsa-miR-1206 MIMAT0005870 hsa-miR-1286 MIMAT0005877 hsa-miR-514a-3p MIMAT0002883 hsa-miR-548ah-5p MIMAT0018972 hsa-miR-184 MIMAT0000454 hsa-miR-543 MIMAT0004954 hsa-miR-626 MIMAT0003295 hsa-miR-339-3p MIMAT0004702 hsa-miR-1234-3p MIMAT0005589 hsa-miR-155-5p MIMAT0000646 hsa-miR-888-5p MIMAT0004916 hsa-miR-542-3p MIMAT0003389 hsa-miR-514b-5p MIMAT0015087 hsa-miR-548m MIMAT0005917 hsa-miR-30e-5p MIMAT0000692 hsa-miR-1290 MIMAT0005880 hsa-miR-1255a MIMAT0005906

[0199] The profile of these 39 miRNAs was compared to those published (Baglio et al, 2015) of bone marrow derived mesenchymal stem cell (BMSC) and adipose derived mesenchymal stem cells (ASC). The results demonstrate that the hOMSC secretome miRNA profile is unique. Only 5 and 3 miRNAs are shared between hOMSC secretome miRNAs and the miRNAs detected in BMSC and ASC secretome, respectively. Three out of the 5 miRNAs shared by hOMSC and BMSC secretomes are also shared with ASC secretome. Thus, 34 miRNAs are not contained within the miRNA composition of adult BMSC and ASC secretomes which are considered to be endowed with high therapeutic capacity.

Example 3. Therapeutic Capacity of hOMSC in Wound Healing

[0200] Wound healing in diabetics is delayed due to impaired local and systemic signaling and inappropriate tissue response to wound healing cues. This multifactorial impaired wound healing processes brings about delayed cell migration, reduced new vasculature and connective tissue formation. Stem cells because of their multifactorial secretome have been proposed as cutting-edge tools for the treatment of diabetic wound.

[0201] Transgenic mice that lack the leptin receptor (db/db mice), have increased food intake and as a result, become obese and develop type II diabetes, were used as having disease etiology similar to type II diabetes in humans. Diabetic db/db mice exhibit the slowest rate of skin wound closure amongst other known models of diabetes in mice (Michaels J et al. 2007).

[0202] Full thickness dermal wounds 6 mm in diameter were performed on the back of diabetic (blood glucose >300 mg/dcl) db/db mice. A silicon donut-shaped ring having an internal diameter of 8 mm was sutured at the periphery of the wound to prevent wound contraction and served as a standard reference object to calculate the rate of wound closure at the macroscopic level. The animals were divided into 3 groups of 5-10 animal in each: i) a negative control group injected with PBS that served as the vehicle for cell delivery; ii) a hOMSC-treated group; and iii) a hSkin SC treated group. The cells were injected intradermally at 4 equidistant sites, 510.sup.5 cells/site. The animals were photographed every 2-4 days to determine the rate of wound closure. To do this the wound area in each animal at each time point was determined by image analysis on the photographs and the wound area was normalized by determining the area delineated by the inner circumference of the donut-shaped ring as it appeared on the same photograph (FIG. 5).

[0203] The results demonstrated in FIG. 6A (quantitative results) and 6B (representative qualitative phototgraphs) indicate that the rate of wound healing until wound closure was statistically significant (p<0.05) higher in the hOMSC-treated animals compared to the untreated (PBS vehicle) or the hSkin SC-treated ones. Wound closure in all animals treated with hOMSC occurred 16 days after wounding whereas complete wound closure in all animals of the untreated group took place 26 days after wounding. Furthermore, no significant statistical differences were observed between the untreated group and the hSkin SC treated one.

[0204] In a separate study, the natural rate of wound healing was tested also in healthy untreated wild type (WT) animals. For this purpose, the same experimental setting as described above was used. Animals were age-matched with their db/db counterparts.

[0205] As depicted in FIG. 7, the rate of wound healing in the hOMSC-treated mice was similar to that WT-untreated mice. The average time to complete wound closure in the db/db diabetic mice treated with hOMSC was 14.31.4 days, that of the WT-untreated animals was 15.141.06 days and that of db/db diabetic untreated mice was 22.752.16. These data indicate that hOMSC have the capacity to overcome the deleterious effect of the diabetic status on wound healing and reverse the rate of diabetic wound healing to normal.

[0206] The lack of stimulatory effect of hSkin SC on the of diabetic wound healing was surprising and therefore the capacity of another SC population, namely adipose tissue-derived stem cells (hADSC) was tested in the same experimental setting. The results shown in FIGS. 8 and 9 demonstrate that the rate of wound healing in the hADSC-treated animals was statistical significantly lower than that in the hOMSC-treated animals but higher than that of the untreated- or hSkin SC-treated animals (p<0.05). Nevertheless, complete wound healing in all the animals treated with hADSC occurred 24 days after wounding as for the animals treated with hSkin SC.

[0207] It was therefore concluded that Nave hOMSC are superior to other stem cells in enhancing diabetic wound healing.

Example 4. The Therapeutic Potential of hOMSC Secretome

[0208] To test whether the therapeutic effect of hOMSC can be at least partially attributed to their unique secretome, 110.sup.6 hOMSC were maintained in serum free medium for 24 hours.

[0209] Then, the medium was collected and concentrated with a concentration filter having a cutoff of 1,000 Dalton (Amicon). The concentrated medium contains the components of the secretome of nave hOMSC as analyzed by MS. This hOMSC-derived concentrated conditioned medium was injected in the diabetic wound healing model described in Example 2 and FIG. 5. Each diabetic db/db mice at each site marked by an arrow in FIG. 5 was injected with 50 l of conditioned medium representing the secretion of 210.sup.5 hOMSC over a period of 24 hours. Thus, a total 200 l of concentrated conditioned medium was injected into the periphery of the wound of each animal. The administration of the conditioned medium was performed only at the beginning of the experimental period. The animals were followed macroscopically as described above and sacrificed at closure. The wound area including the silicon ring were retrieved and processed for histologic analysis.

[0210] The rate of wound closure is shown in FIG. 10. The results indicate that a one-time administration of hOMSC-secretome confined within the concentrated hOMSC conditioned medium was as effective as hOMSC to enhance diabetic wound healing. Histomorphometric analysis of the number of blood vessels and the amount of collagen in the center of the healed wound revealed: i) no statistical differences between the amount of collagenous connective tissue in the hOMSC-treated animals and that in the hOMSC secretome-treated animals; and ii) increase in the number of blood vessels in the hOMSC secretome-treated animals compared to the untreated or hSkin SC-treated animals and reduction in the number of blood vessels compared to hOMSC-treated animals.

[0211] Without wishing to be bound to any theory or mode of action, it is suggested that continued or multiple treatment with secretome will be required compared to treatment with stem cells which continue to secrete substances. Nevertheless, secretome composition is safer and easier to manipulate, characterize, maintain and administer, then cells.

[0212] Collectively, the results indicate that the hOMSC-secretome retains the efficiency of hOMSC to enhance the healing of diabetic foot ulcer and therefore they might be used as a self-standing therapeutic tool or as an adjunctive for cell therapy particularly whenever de novo vascularization is required.

[0213] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

REFERENCES

[0214] Antonyak M A, Cerionee R A (2015) Emerging picture of the distinct traits and functions of microvesicles and exosomes. PNAS 112(12), 3589-3590. [0215] Baglio S R, Rooijers K, Koppers-Lalic D. Verweij F J, M P, Zini N, Naaijkens B, Perut F, Niessen H W M, Baldini N and Pegtel D M (2015). Human bone marrow and adiposemesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. 6, 127-147. [0216] Desrochers L M, Antonyak M A, Cerione R A (2016) Extracellular vesicles: Satellites of information transfer in cancer and stem cell biology. Development Cell 37, 301-309. [0217] DiPietro L A (2003). Differential injury responses in oral mucosal and cutaneous wounds. J. Dent. Res. 82, 621-626. [0218] Ganz J, Arie I, Ben-Zur T, Dadon-Nachum M, Pour S, Araidy S, Pitaru S, Offen D (2014). Astrocyte-like cells derived from human oral mucosa stem cells provide neuroprotection in vitro and in vivo. Stem Cells Transl. Med. 3, 375-86. [0219] Ganz J, Arie I, Buch S, Zur T B, Barhum Y, Pour S, Araidy S, Pitaru S, Offen D (2014). Dopaminergic-like neurons derived from oral mucosa stem cells by developmental cues improve symptoms in the hemi-parkinsonian rat model. PLoS One 9(6), e100445. [0220] Gue W., Gao Y., Li N., Shao F., Wang C., Wang P., Yang Z., Li R., and He J. (2017). Exosomes: New players in cancer (Review). Oncology Reports 38: 665-675. [0221] Hu L, Wang J, Zhou X, Xiong Z, Zhao J, Yu R, Huang F, Zhang H, Chen L (2016). Exosomes derived from human adipose mesenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Scientific Reports 6, 32993. [0222] Kanada M, Bachmann M H, Hardy J W, Frimannson D O, Bronsart L, Wang A, Sylvester M D, Schmidt T L, Kaspar R L, Butte M J, Matin A C, Contag C H (2015). Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc. Natl. Acad. Sci. USA 112, E1433-E1442. [0223] Konala V B, Mamidi M K, Bhonde R, Das A K, Pochampally R, Pal R (2016). The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration. Cytotherapy 18(1), 13-24. [0224] Lai, R. C., Arslan, F., Lee, M. M., Sze, N. S. K., Choo, A., Chen, T. S., Salto-Tellez, M., Timmers, L., Lee, C. N., El Oakley, R. M., et al. (2010). Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res. 4, 214-222. [0225] Lopez-Verrilli M A, Caviedes A, Cabrera A, Sandoval S, Wyneken U, Khoury M (2016) Mesenchymal stem cell-derived exosomes from different sources selectively promote neuritic outgrowth. Neuroscience 320, 129-139. [0226] Marynka-Kalmani K, Treves S, Yafee M, Rachima H, Gafni Y, Cohen M A, Pitaru S (2010). The lamina propria of adult human oral mucosa harbors a novel stem cell population. Stem Cells 28(5), 984-95. [0227] Michaels J 5th, Churgin S S, Blechman K M, Greives M R, Aarabi S, Galiano R D, Gurtner G C (2007). db/db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. Wound Repair Regen. 15(5), 665-70 [0228] Park C W, Kim K S, Bae S, Son H K, Myung P K, Hong H J, Kim H (2009). Cytokine secretion profiling of human mesenchymal stem cells by antibody array. Int J Stem Cells 2(1), 59-68. [0229] Rodrigues M, Wong V W, Rennert C R, Davis C R, Longaker M T, Gurtner G C (2015). Progenitor cell dysfunction underlie some diabetic complications. The American Journal of Pathology 2015, 85(8). [0230] Skog J, Wurdinger T, van Rijn S, Meijer D H, Gainche L, Sena-Esteves M, Curry W T Jr, Carter B S, Krichevsky A M, Breakefield X O (2008). Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell. Biol. 10(12), 1470-6. [0231] Tachida Y, Sakurai H, Okutsu J, Suda K, Sugita R, Yaginuma Y, Ogura Y, Shimada K, Isono F, Kubota K and Kobayashi H. (2015). Proteomic Comparison of the Secreted Factors of Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue and Dental Pulp. Journal of Proteomics and Bioinformatics; 8(12), 266-273. [0232] Tepper O M, Car J, Robert J. Allen Jr, Chang C C, Li C D, Tanaka R, Gupta S M, Levine J P, Saa deh P B, Warren S M. (2010). Decreased circulating cell number and failed mechanisms of stromal cell-derived factor-1 mediated bone marrow mobilization impair diabetic tissue repair. Diabetes 59, 1974-1983. [0233] Treves-Manusevitz S, Hoz L, Rachima H, Montoya G, Tzur E, Vardimon A, Narayanan A S, Amar S, Arzate H, Pitaru S (2013). Stem cells of the lamina propria of human oral mucosa and gingiva develop into mineralized tissues in vivo. J. Clin. Periodontol. 40(1), 73-81. [0234] Villarroya-Beltri C, Baixauli F, Gutierrez-Vazquez C, Sanchez-Madrid F, Mittelbrunn M (2014) Sorting it out: regulation of exosome loading. Semin Cancer Biol 28, 3-13. [0235] Voutilainen M H, Arumae U, Airavaara M, Saarma M. (2015). Therapeutic potential of the endoplasmatic reticulum located and secreted CDNF/MANF family of neurotrophic factors in Parkinson's Disease FEBS Letter 589, 3739-3748. [0236] Wu Y., Peng H., Cui M., Whitney N. P., Huang Y., and Zheng C. (2009), CXCL2 increases human neural progenitor cell proliferation through Akt-1/FOXO3a signaling pathway. Journal of Neurochemistry 109, 1157-1167. [0237] Xu R, Greening D W, Zhu H J, Takahashi N, Simpson R J (2016). Extracellular vesicle isolation and characterization: toward clinical application. J, Clin, Invest.; 126(4), 1152-62. [0238] Zander C, Heidbreder M, Kasperek Y, Noll T, Seitz O, Saldamli B, Sudhoff H, Sader R, Kaltschmidt C, Kaltschmidt B (2009). Adult Palatum as a Novel Source of Neural Crest-Related Stem Cells. Stem Cells; 27, 1899-1910. [0239] Zhang Q, Nguyen P, Xu Q, Park W, Lee S, Furuhashi A, Le A D (2017). Neural Progenitor-Like Cells Induced from Human Gingiva-Derived Mesenchymal Stem Cells Regulate Myelination of Schwann Cells in Rat Sciatic Nerve Regeneration. Stem Cells Transl. Med. 6(2), 458-470.