Activated stem cell derived products for stimulation of skin regeneration

Abstract

Disclosed herein are means, methods, and compositions of matter useful for stimulation of elastin / collagen production and suppression of matrix metalloprotease activity. The disclosure further provides compositionally useful preparations derived from regenerative cells that have been manipulated for optimum growth factor and anti-aging properties. In one embodiment cellular mixtures derived from regenerative cells contacted with activating signals. Said activating signals include stimulation of toll like receptors and other "danger" sensing molecules. In one embodiment regenerative factors are harvested from stem cells and compounded into composition preparations.

Description

RBIOC.007WO PATENTACTIVATED STEM CELL DERIVED PRODUCTS FOR STIMULATION OF SKINREGENERATIONINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified, for example, in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57, and Rules 4.18 and 20.6, including U.S. Provisional Application No. 63 / 695813 filed September 17, 2024, which is hereby incorporated by reference in its entirety.RELATED FIELD

[0002] The present disclosure relates to the field of antiaging medicine and stem cell biology. Some aspects of the present disclosure relate to the field of skin regeneration. Some aspects of the present disclosure relate to the use of regenerative cell-derived components and secreted factors for the purposes of skin regeneration and rejuvenation.BACKGROUND

[0003] The epidermis is the outer layer of the skin, which serves to protect the body against numerous external environment factors such as mechanical traumas, temperature, pathogenic microorganisms, xenobiotics, and UV radiation. Several mechanisms exist that act as barriers against factors the action of which accelerate the aging process and skin deterioration. One is the keratinization process initiated by epidermal keratinocytes that originate from the basal layer through the prickle and granular layer to the horny layer, inside which they start to transform to komeocytes.

[0004] Keratinization involves a series of biochemical and morphological changes, leading to among others, the cell proliferation ability loss, degeneration of the cell organelle parts and appearing of the new ones, changes in the cell membrane chemical compositions and also appearing of numerous proteins (e.g., involucrine, filagrine binding proteins) and lipids (sterols or phospholipids being the ceramides precursors).

[0005] The epidermis granular layer keratinocytes degenerate by apoptosis, while the comeocytes located on the horny layer, are subjected to peeling under action of proteolytic enzymes that degrade the comeodesmosomes, i.e., specialized inter-cellular links present in the epidermis horny layer. The ceramides, being the main inter-cellular cement component, are also subjected to changes: degradation to sphingosine and fat acids, which results in the intercellular cement liquefaction. Consequently, the corneocytes are mechanically removed from the epidermis surface.SUMMARY

[0006] Aspects of the disclosure include a method for making an anti-inflammatory composition comprising at least one regenerative factor. In some embodiments, the method includes extracting at least one cell from a tissue, exposing the at least one cell to conditions allowing for production of at least one regenerative factor, isolating the at least one regenerative factor, and admixing the at least one regenerative factor into an anti-inflammatory composition, wherein the at least one cell comprises a mesenchymal stem cell (MSC) and / or a cell derived from a mesenchymal stem cell. In some embodiments, the tissue is selected from the group consisting of adipose, nail cuticle, deciduous tooth, hair follicle, skin, bone marrow, placenta, umbilical cord blood, mobilized peripheral blood, peripheral blood, urine, breast milk, menstrual blood, fallopian tube, or a combination thereof. In some embodiments, the at least one regenerative factor includes an exosome. In some embodiments, the exosome includes at least one of a diameter of between 2 nm and 200 nm, a molecular weight of at least about 100 kDa, a hydrodynamic radius of at most about 100 nm, or a combination thereof. In some embodiments, the conditions allowing for production of an at least one regenerative factor comprises exposing the at least one cell to an agent that resembles cellular injury or cellular damage. In some embodiments, the agent that resembles cellular injury or cellular damage is a toll-like receptor agonist. In some embodiments, the toll-like receptor agonist is selected from a group consisting of Lipopolysaccharide (LPS), Pam3CSK4, lipoteichoic acid (LTA), zymosan, flagellin, polyinosinic:polycytidylic acid (Poly(I:C)), imiquimod, resiquimod (R848), loxoribine, gardiquimod, CL075, CL097, isatoribine (ANA-245), vesatolimod (GS- 9620), monophosphoryl lipid A (MPL), lipid IVa, eritoran (primarily an antagonist but with agonist-like effects in some contexts), CpG oligodeoxynucleotides (CpG-ODN), SM360320,CU-T12-9, GS-9688, R07020531, JNJ-4964, CBLB612, ISA-201, OPN-305 (primarily antagonist but with agonist potential in trials), TMX-101, DSP-0509, IMO-2055 (Amplivax), IC-31, E-6020, PF-4878691, RO6870868, RO6864018, high mobility group box 1 (HMGB1), peptidoglycan, bacterial DNA, fibrinogen, fibronectin, extracellular matrix proteins, 852A, VTX-2337, Bacillus Calmette-Guerin (BCG), Poly ICLC, synthetic imidazoquinoline, smallmolecule selective TLR8 agonist, mannose-capped lipoarabinomannan (ManLAM), phenol- soluble modulin, inulin acetate, retrocyclin, CL429, CpG-ODN: AG-OVA, P-glucans (general), Sparassis crispa P-glucan, Phellinus linteus -glucan, Platycodon grandiflorum P- glucan, Cordyceps militaris P-glucan, Angelica gigas Nakai P-glucan, Saccharomyces cerevisiae P-glucan, Laminarin (P-glucan), Curdlan (P-glucan), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shock protein 22 (HSP22), heat shock protein gp96, or a combination thereof In some embodiments, the exposing the at least one cell to conditions allowing for production of the at least one regenerative factor comprise exposing the at least one cell to at least one stimuli selected from the group consisting of hypoxia, hyperthermia, hypotonic challenge, oxidative stress, inflammatory stimuli, tolllike receptors, or any combination thereof. In some embodiments, the exposing the at least one cell to conditions allowing for production of the at least one regenerative factor comprise exposing the at least one cell to a histone deacetylase inhibitor, an amino acid, a leukemia inhibitory factor, a DNA methyltransferase inhibitor, ascorbic acid, insulin, lithium, transferrin, sodium bicarbonate, sodium selenite, a MAPK inhibitor, SB431542, CHIR99021, Y-27632, Y- thiazovivin, FGF-1, FGF-2, FGF-5, sodium borate, erythropoietin, IL-3, IL-6, IL-8, IL-10, IL-18, IL-20, IL-25, IGF-1, dexamethasome, holo-transferrin, interferon gamma, cytoplasm of an undifferentiated cell, or any combination thereof. In some embodiments, the at least one cell expresses CD73, and further expresses at least one of: TLR9, CD4, CD8, CD25, CD34, CD39, CD56, CD77, CD105, CTLA-4, GITR, lipocalin 3, lipocalin 12, lipocalin 13, IL-1, IL-3, IL-6, IL-7, IL-10, IL-17, IL-32, IL-35, TLR-2, TLR-3, TLR-4, TLR-5, TLR-7, TLR-8, angiopoietin receptor, BDNF receptor, cartilage intermediate layer protein 2, C-C chemokine receptor type 10, CCR5, CXCR4, RAGE, TNFa, TNFb, TNF-alpha receptor p55, TNF-alpha receptor p75, stem cell factor receptor, FRAS1 -related extracellular matrix protein 3, GM-CSF receptor alpha, HGF receptor, HLA-DR, HLA-G, IGF-1 receptor, IL12-p40, ILT- 3, indolamide 2,3 deoxygenase, Leukemia Inhibitory Factor Receptor, mannose binding lectin,MEGF10, mucin-2, mucolipin 1, nesprin-2, netrin 2-like, neuropilin- 1 , neuronal pentraxin receptor, occludin, oxytocin receptor, oxysterol -binding protein 1, PDGF-BB receptor, plasticity -related protein 2, plexin A2, plexin A4, poliovirus receptor, prolactin receptor, VPR- Binding Protein, TGF-b, thrombopoietin receptor, transferrin receptor, Tmc5, zinc transporter 9, seminal vesicle antigen-like 3, sarcoma antigen NY-SAR-41, vimentin, VEGF receptor, fibrosin 1, or any combination thereof. In some embodiments, the at least one cell does not express CD 14 or CD45.

[0007] Aspects of the disclosure relate to a topical anti-inflammatory composition. In some embodiments, the composition includes an activator of endogenous progeni tor / stem cells. In some embodiments, the composition further includes an angiogenic factor, a neurogenic factor, an antiapoptotic factor; and an immune modulatory factor. In some embodiments, the composition further includes at least one of platelet rich plasma, human chorionic gonadotropin, one or more antioxidants, one or more antiseptic agents, one or more anti-inflammatory agents, one o r more delivery vehicles, one or more known composition agents, or any combination thereof. In some embodiments, the one or more delivery vehicle is selected from the group consisting of phospholipids, palmityl myristate, DMSO, a polymer, liposomes, Trojan peptides, chariot peptides, small elastic vesicles, microspheres, chitosan suspensions, a matrix, nanoparticle, or a combination thereof. In some embodiments, the one or more known composition agent is selected from the group consisting of beta-glucan, propylene glycol, butylene glycol, polyethylene glycol, olive oil, dimethyl isosorbide, dimethylformamide, methyl salicylate, long chain oleic acids, lactic acid, senolytic agent, senomorphic agent, or a combination thereof. In some embodiments, the one or more known composition agent is selected from BCL2, BCL2-XL, survivin, or any combination thereof. In some embodiments, the angiogenic factor is selected from the group consisting of VEGF, HGF- 1, FGF-1, FGF-2, angiopoietin, interleukin-20, or any combination thereof. In some embodiments, the neurogenic factor is selected from the group consisting of NGF, BDNF, CNTF, neurotrophins, or any combination thereof. In some embodiments, the antiapoptotic factor is selected from the group consisting of molecules capable of increasing bcl-2 expression, molecules capable of decreasing BAD expression, molecules capable of increasing expression of bcl-2Xl, molecules capable of decreasing bcl-2Xs, molecules capable of decreasingexpression of members of the caspase family, molecules capable of increasing survivin expression, molecules capable of increasing livin expression; or any combination thereof.

[0008] Aspects of disclosure further relate to a composition for topical skin application. In some embodiments, the composition includes a conditioned medium comprising endogenous progenitor / stem cells, wherein said endogenous progenitor / stem cells are stimulated with at least one regenerative factor, and a dermatologically acceptable carrier. In some embodiments, the at least one regenerative factor is a histone deactylase inhibitor. In some embodiments, the endogenous progenitor / stem cells expresses CD73 and TLR9. In some embodiments, the endogenous progenitor / stem cells resemble cellular injury or cell damage by a toll-like receptor agonist.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts a non-limiting example quantification for the amount of collagen (ug / mL) produced over a period of 48, 72, and 96 hours for cells following culturing with: a) saline (control); b) CpGDNA (20 ng / ml); c) beta glucan (100 ng / ml) or d) combination of CpG and beta glucan.DETAILED DESCRIPTION

[0010] Disclosed herein is the use of tissue derived regenerative cells and derivatives thereof for production of compositions for topical skin application.

[0011] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.Terms

[0012] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications areincorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0013] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0014] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article, unless the context dictates otherwise. By way of example, “an element” means one element or more than one element.

[0015] The terms “first,” “second,” and “third” used in combination with substances are intended to designate distinguishable features to similar substances and do not imply any particular order unless otherwise specified.

[0016] As used herein, the term “including” as well as other forms such as “includes” and “included” is not limiting.

[0017] As used herein, “approximately” and “about” mean that a number or other measure referred to as “approximately” or “about” comprises the recited number plus or minus 1-10% of that recited number. For example, “about” 100 degrees can mean 95-100 degrees or as few as 99-101 degrees depending on the context. Whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range, i.e., meaning only 1, inly 2, only 3, etc. up to and including only 20.

[0018] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0019] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a nonhuman primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0020] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0021] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or anycombination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0022] As used herein, the term “compound” has its usual meaning and thus can refer to a chemical or biological material. Non-limiting examples of a compound include an atom, small molecule, large molecule, peptide, protein, drug, or any combination thereof.

[0023] As used herein, the term “composition” has its usual meaning and thus can refer to a chemical or biological compound or substance, or a mixture or combination of two or more such compounds or substances. In some embodiments, the composition comprises an atom, small molecule, large molecule, peptide, protein, salt, buffer, or any combination thereof. In some embodiments, the composition is formulated for a specific use, such as for administration to a subject.

[0024] As used herein, the terms “pharmaceutical composition” or “pharmaceutical formulation” refer to a chemical or biological compound or substance, or a mixture or combination of two or more such compounds or substances, intended for use in the cure, treatment, prevention or diagnosis of a disease or symptom.

[0025] As used herein, the term “pharmaceutical agent” has its usual meaning and thus can refer to a compound or composition with a known therapeutic function. In some embodiments, that function has use in the cure, treatment, prevention or diagnosis of a disease or symptom. In some embodiments, a pharmaceutical agent comprises a drug. In some embodiments, a pharmaceutical agent comprises a composition, the composition comprising a drug.

[0026] As used herein, “carrier” refers to a substance that serves as a vehicle for improving the efficacy of delivery or the effectiveness of a pharmaceutical composition, or both.

[0027] As used herein, “pharmaceutically acceptable” when used to define a carrier, whether diluent or excipient, refers to a substance that is compatible with other ingredients in a formulation and does not exert deleterious effects to the recipient thereof. As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also include one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol,isomalt, maltitol, or lactitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The formulation, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These formulations can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0028] The term “pharmaceutically acceptable salts” has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di- , and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0029] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history ofthe subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0030] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.

[0031] As used herein, the terms “treating” or “treatment” have their plain and ordinary meaning as understood in light of the specification, and refer to an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (e.g., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may include a single administration or may include a series of administrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment of a disease or disorder may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0032] Treat” and “treating” are not limited to the case where the subject (e g. patient) is cured and the disease is eradicated. Rather, the present disclosure also contemplates treatment that merely reduces symptoms, and / or delays disease progression.

[0033] As used herein, the term “therapeutic target” has its plain and ordinary meaning as understood in light of the specification and refers to a gene or gene product that,-l iupon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the disease phenotype. As used throughout, “modulation” is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).

[0034] The term “administering” includes contact with a skin, tissue, mucus, or fluid of a subject. It includes topical contact, administration as a suppository, intravaginal, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration can be by any route unless specifically stated, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “coadminister” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0035] As used herein, “excipient” refers to a pharmacologically inactive substance or compound that is formulated in combination with a pharmacologically active ingredient of a pharmaceutical composition, wherein an excipient may include a bulking agent, a filler, a diluent, or a product for drug solubility, drug absorption, or for modulating a pharmacokinetic property of an active ingredient of a pharmaceutical composition.

[0036] As used herein, “therapeutically effective amount” refers to the amount or quantity of a pharmaceutical composition that elicits a desired clinical or biological response in a cell, a tissue, an organ, an animal or a human, or an amount or quantity that is clinically useful for reducing, eliminating, or otherwise affecting a disease, condition, or medical complication in a subject.

[0037] As used herein, “administration” (or other forms such as “administering”) refers to the act of providing a substance or compound to a subject in need thereof.

[0038] The term “active ingredient” as used herein is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a specialor customized meaning) and refers to a component that confers a function. In some embodiments, the component provides direct or indirect effect in the cure, mitigation, treatment, diagnosis, or prevention of disease or condition in the body of humans or animals. “Anti-inflammatory” means a substance that reduces inflammation. Many analgesics remedy pain by reducing inflammation. Many steroids— specifically glucocorticoids— reduce inflammation by binding to cortisol receptors. Non-steroidal anti-inflammatory drugs (NSAIDs) alleviate pain by counteracting the cyclooxygenase (COX) enzyme. On its own COX enzyme synthesizes prostaglandins, creating inflammation. Many herbs have antiinflammatory qualities, including but not limited to hyssop and willow bark (the latter of which contains salicylic acid, the active ingredient in aspirin), as well as birch, licorice, wild yam and ginseng. Cytokines such as IL-4, IL- 10, TGF-beta, and IL-20 are known to reduce inflammation.

[0039] “Antioxidants” means any of a variety of substances that prevent or slow the breakdown of another substance by oxygen. Synthetic and natural antioxidants are used to slow the deterioration of gasoline and rubber, and such antioxidants as vitamin C (ascorbic acid), butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA) are typically added to foods to prevent them from becoming rancid or from discoloring. Nutrients such as beta-carotene (a vitamin A precursor), vitellogenin, vitamin C, vitamin E, and selenium have been found to act as antioxidants. They act by scavenging free radicals, molecules with one or more unpaired electrons, which rapidly react with other molecules, starting chain reactions in a process called oxidation. Free radicals are a normal product of metabolism; the body produces its own antioxidants (e.g., the enzyme superoxide dismutase) to keep them in balance. However, stress, aging, and environmental sources such as polluted air and cigarette smoke can add to the number of free radicals in the body, creating an imbalance. The highly reactive free radicals can damage healthy DNA and have been linked to changes that accompany aging (such as age-related macular degeneration, a leading cause of blindness in older people) and with disease processes that lead to cancer, heart disease, and stroke.

[0040] An “antiseptic” is a substance that kills or prevents the growth and reproduction of various microorganisms, including bacteria, fungi, protozoa, and viruses on the external surfaces of the body. The objective of antiseptics is to reduce the possibility of sepsis, infection or putrefaction by germs. Antibacterials have the same objective but only actagainst bacteria. Antibiotics perform a similar function, preventing the growth or reproduction of bacteria within the body.

[0041] Antiseptics include, but are not limited to, alcohol, iodine, hydrogen peroxide, and boric acid. There is great variation in the ability of antiseptics to destroy microorganisms and in their effect on living tissue. For example, mercuric chloride is a powerful antiseptic, but it irritates delicate tissue. In contrast, silver nitrate kills fewer germs but can be used on the delicate tissues of the eyes and throat. There is also a great difference in the time required for different antiseptics to work. Iodine, one of the fastest-working antiseptics, kills bacteria within 30 sec. Other antiseptics have slower, more residual action. Since so much variability exists, systems have been devised for measuring the action of an antiseptic against certain standards. The bacteriostatic action of an antiseptic compared to that of phenol (under the same conditions and against the same microorganism) is known as its phenol coefficient.

[0042] “Chitosan” is a beta-l,4-linked glucosamine polymer which, unlike chitin, contains few, if any, N-acetyl residues. It may be obtained from chitin, a polysaccharide found in the exoskeletons of crustaceans such as shrimp, lobster, and crabs. The shells may be ground into a pulverous powder. This powder is then deacetylated which allows the chitosan to absorb lipids.

[0043] “Collagen” means any of a variety of substances that contains the alpha chains of the collagen polypeptide with a sequence that generally follows the pattern Gly-X- Y, where Gly for glycine, X for proline, and Y for proline or hydroxyproline. Collagen proteins also contain significant amounts of glycine and proline. Hydroxyproline and hydroxylysine are not inserted directly by ribosomes. They are derivatized from proline and lysine in enzymatic processes of post- translational modification, for which vitamin C is required. This is related to why vitamin C deficiencies can cause scurvy, a disease that leads to loss of teeth and easy bruising caused by a reduction in strength of connective tissue due to, a lack of collagen, or defective collagen. Cells called fibroblasts form the various fibers in connective tissue in the body including collagen. The white collagen that makes up the matrix of most connective tissue in mammals consists of inter- woven fibers of the protein collagen. The collagen fibers consist of globular units of the collagen sub-unit, tropocollagen. Tropocollagen sub-units spontaneously arrange themselves under physiological conditions into staggered arraystructures stabilized by numerous hydrogen and covalent bonds. Tropocollagen sub-units are left-handed triple helices where each strand is, further, a right-handed helix itself. Thus, tropocollagen may be considered to be a coiled coil.

[0044] Although collagen is responsible for skin elasticity, and its degradation leads to wrinkles that accompany aging, it occurs in many other places throughout the body, and in different forms known as types: Type I collagen— This is the most abundant collagen of the human body present in scar tissue, the end product when tissue heals by repair; Type II collagen— Auricular cartilage Type III collagen— This is the collagen of granulation tissue, and is produced quickly by young fibroblasts before the tougher type I collagen is synthesized; Type IV collagen— Basal lamina; Type V collagen— most interstitial tissue, assoc, with type I; Type VI collagen— most interstitial tissue, assoc, with type I; Type VII collagen— epithelia; Type VIII collagen— some endothelial cells; Type IX collagen— cartilage, assoc, with type II; Type X collagen— hypertrophic and mineralizing cartilage; Type XI collagen— cartilage; Type XII collagen-interacts with types I and in.

[0045] A “gel” is a semisolid material formed from a colloidal solution. By weight, gels are mostly liquid, yet they behave like solids. An example is gelatin.

[0046] The term “natural product” means any of a variety of organic chemical moieties whose molecular arrangement is derived from enzymatic transformations in a living organism excluding amino acids, proteins, polypeptides, nucleic acids and sequences, and saturated fatty acids. Examples include, but are not limited to lipids (i.e., that are not saturated fatty acids), carbohydrates / saccharides and polysaccharides, the steroids and their derivatives, the terpenes and their derivatives, vitamins, carotenoids, and natural medicines such as taxol, etc. The term “synthetic natural product” is a natural product not obtained from its natural source.

[0047] The term “gene” as used herein, refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or protein precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence, as long as the desired protein activity is retained.

[0048] “Nucleoside.” as used herein, refers to a compound consisting of a purine [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine (U), or cytidine (C)] basecovalently linked to a pentose, whereas “nucleotide” refers to a nucleoside phosphorylated at one of its pentose hydroxyl groups.

[0049] “Peptides”, herein defined as polymers formed from the linking, in a defined order, of .alpha.- amino acids; including but not limited to milk peptides, ribosomal peptides, nonribosomal peptides, peptones, cell derived peptides, stem cell derived peptides, immune modulatory peptides and peptide fragments.

[0050] “Variant” in regard to amino acid sequences is used to indicate an amino acid sequence that differs by one or more amino acids from another, usually related amino acid. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “non- conservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNAStar software.

[0051] “Purified” refers to molecules, including but not limited to nucleic, ribonucleic, lipid or amino acid sequences, which are removed from their natural environment, isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.

[0052] “Cell” means the smallest structural unit of living matter capable of functioning autonomously, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable membrane. Cells include all somatic cells obtained or derived from a living or deceased animal body at any stage of development as well as germ cells, including sperm and eggs (animal reproductive body consisting of an ovum or embryo together with nutritive and protective envelopes). Included are both general categories of cells: prokaryotes and eukaryotes. The cells contemplated for use in this disclosure include all types of cells from all organisms in all kingdoms: plans, animals, protists, fungi, archaebacteria and eubacteria. Stem cells are cells capable, by successive divisions, of producing specialized cells on many different levels. For example, hematopoietic stem cellsproduce both red blood cells and white blood cells. From conception until death, humans contain stem cells, but in adults their power to differentiate is reduced.

[0053] “Differentiation” related to cells means the process by which cells becomes structurally and functionally specialized, which is a progressive restriction of the developmental potential and increasing specialization of function which takes place during the development of the embryo and leads to the formation of specialized cells, tissues, and organs.

[0054] ‘Dedifferentiation” related to cells means the reverse process of differentiation, where cells become less structurally and functionally specialized, which increases the developmental potential of the cell.

[0055] “Extract” as used in the context of the current disclosure means a preparation of any type of cell as defined above obtained by chemical or mechanical action, as by pressure, distillation, evaporation etc. Extracts can include all or any single component or combination of components of the cells, including concentrated preparations of the active components. Such components of the extracts include but are not limited to RNA, DNA, micro RNA, lipids, free amino acids, all amino acid base structures including peptides and proteins, carbohydrates, minerals or combinations thereof.

[0056] Growth media” are compositions used to grow microorganisms or cells in culture. There are different sorts of media for growing different sorts of cells. The biggest difference in growth media are between those used for growing cells in culture (cell culture uses specific cell types derived from plants or animals) and those used for growing microorganisms (usually bacteria or yeast). These differences arise due to the fact that cells derived from whole organisms and grown in culture are often incapable of growth without the provision of certain requirements, such as hormones or growth factors which usually occur in vivo. In the case of animal cells these requirements are often provided by the addition of blood serum to the medium. These media are often red or pink due to the inclusion of pH indicators. Growth media for embryonic stem cells preferably contains minimal essential medium, i.e., Eagle's: amino acids, salts (Ferric nitrate nonahydrate, Potassium chloride, Magnesium sulfate, Sodium chloride, Sodium dihydrogen phosphate), vitamins. (Ascorbic acid, Folic acid, Nicotinamide. Riboflavin, B-12) orDulbecco's: additionally iron, glucose; non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, L-glutamine, fetal bovine serum and Leukemia Inhibitory Factor (LIF). In the case of microorganisms, there are no such limitations as theyare often single cell organisms. One other major difference is that animal cells in culture are often grown on a flat surface to which they attach, and the medium is provided in a liquid form, which covers the cells. Bacteria such as Escherichia coli (E. coli, the most commonly used microbe in laboratories) may be grown on solid media or in liquid media, liquid nutrient medium is commonly called nutrient broth. The growth media for microorganisms may include nutrient broth or Luria-Bertani medium (L-B medium). Bacteria grown in liquid cultures often form colloidal suspensions. When agar (a substance which sets into a gel) is added to a liquid medium it can be poured into Petri dishes where it will solidify (these are called agar plates) and provide a solid medium on which microbes may be cultured.

[0057] “Lipid” means any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides that are insoluble in water but soluble in nonpolar organic solvents, and are oily to the touch. Major classes of lipids include the fatty acids, the glycerolderived lipids (including the fats and oils and the phospholipids), the sphingosine-derived lipids (including the ceramides, cerebrosides, gangliosides, and sphingomyelins), the steroids and their derivatives, the terpenes and their derivatives, certain aromatic compounds, and long- chain alcohols and waxes. In living organisms lipids serve as the basis of cell membranes and as a form of fuel storage. Often lipids are found conjugated with proteins or carbohydrates, and the resulting substances are known as lipoproteins and lipopolysaccharides. The fat-soluble vitamins can be classified as lipids.

[0058] Liposomes are spherical vesicles formed by mixing lipids with water or water solutions. They have found applications in the oral administration of some drugs (e.g., insulin and some cancer drugs), since they retain their integrity until they are broken down by the lipases in the stomach and small intestine.

[0059] “Prevent” and “preventing” include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0060] “Transport vehicle, delivery vehicle, and delivery agents” include substances capable of aiding penetration of intact skin or skin cells or other somatic cells. The term “transport vehicle” is used synonymously with the term “permeabilizing agents”. Such transport vehicles include, but are not limited to: phospholipids, palmitylmyristyrates, DMSO, polymer or chitosan suspensions or matrix, liposomes, Trojan peptides, chariot peptides, smallelastic vesicles, microspheres (functionalized vectors made from naturally derived materials such as collagen, glycosaminoglycans, chondroitin sulfate, chitosan or polysaccharides), nanoparticles (carries lipophilic substances and enhance bioavailability of the encapsulated material into skin), preloaded spherical beads and sponges, uni- and / or multi-lamellar vesicles (stabilize contents of extracts in cream base and help transport into skin), retinol molecular film fluid (thin uniform monolayer film that facilitates the transfer of actives through the stratum corneum), poly acrylo nitrile (polymers comprising a controlled release system that synchronizes the release of an active ingredient along with a fragrance as a sensory marker which conveys the efficacy of the product), beta-glucan (oat fiber which aids in penetration of the skin, (Redmond, Int. Journ. Cosmetic science 2005), propylene glycol (as drug carrier, work best with a mineral oil based cream / lotion etc), butylene glycol, polyethylene glycol, olive oil, dimethyl isosorbide, dimethylformamide, methyl salicylate (these all enhance absorption through skin), long chain oleic acids (disrupts the bilayer within the stratum corneum, vital for permeation of compositions in propylene glycol-based formulations), substances capable of adjusting pH, hydration and local metabolism in skin. Agents modifying these factors include a vehicle containing an active hydrophobic agent, de-ionization of active ingredients, increased hydration of the skin (water content of carrier solution / cream / medium), lactic acid (alters the pH).Epidermal Regeneration

[0061] The epidermis regeneration process is mediated through internal stem cells. The stem cells’ larger conglomeration is the hair follicle bulge, called the bulge area. In this place, such cells intensively divide and then migrate. A fraction migrates to the hair germinal matrix (where they participate in the hair appearing and growth), while the others go to the apical hair part, in order to home the epidermis basal layer.

[0062] During the migration to the epidermis, the stem cells meet the area located just over the sebaceous gland, where there are concentrated stem cells responsible for production of sebum. However, in order to enable the keratinization and epidermis regeneration process creating the protective layer for the entire organism, many factors are controlled. Besides the many vitamins, calcium ions, or water level, attention should be paid to a keratinization control process by the keratinocytes themselves. They synthesize and emitmany factors that control their process of proliferation and differentiation. They include, among others, EGF, KGF, TGF-alpha, TGF-beta, and IL-1.

[0063] Although knowledge exist regarding means of skin self-renewal, means of inducing regeneration of skin in older subjects has been limited. Some aspects of the current disclosure relate to methods of providing skin regenerative agents using extracts derived from adipose derived stem cell and other cellular populations.

[0064] Utilization of adult stem cells has generated interest in almost all areas of medicine. Studies in heart failure, liver failure, stroke, and wound healing, have demonstrated possibility of utilizing mesenchymal stem cells to induce therapeutic benefit.

[0065] Unfortunately, the widespread application of adult stem cells in medicine is still substantially lacking. This is due to lack of products that are approved by regulatory agencies, as well as in situations where such products exist, relatively limited. One of the major revelations in the area of adult stem cell research is that the majority of therapeutic effects are elicited not by MSC directly transdifferentiating into damaged tissue, but by virtue of immune modulation and trophic support to endogenous reparative mechanisms of the host. Accordingly, the utilization of stem cell conditioned media / concentration of stem cell derived products has been an area of active investigation.Membrane Macrovesicles and Exosomes

[0066] Membrane macrovesicles (MMV) are fragments of phospholipid bilayer plasma membrane ranging from 30 nm to 1000 nm shed from almost all cell types. MMV, therefore are a subtype of membrane-vesicles, and play a role in intercellular communication and can deliver mRNA, siRNA, and proteins between cells. They have been implicated in the process of cancer tumor immune suppression, metastasis, tumor-stroma interactions and angiogenesis along with having a role in tissue regeneration. They originate directly from the plasma membrane of the cell and reflect the antigenic content of the cells from which they originate.

[0067] In contrast to MMV, exosomes are vesicles of 30-100 nm in diameter, which are actively secreted by a wide range of cell types under both normal and pathological conditions. Exosomes can be regarded as a sub-class of MMV. First discovered in maturing mammalian reticulocytes, they were shown to be a mechanism for selective removal of manyplasma membrane proteins and to discard transferrin-receptors from the cell surface of maturing reticulocytes. Although the exosomal protein composition varies with the cell of origin, most exosomes contain the soluble protein Hsc 70 and many others. 31 proteins are found to be in common between colorectal cancer, mast cells and urine-derived exosomes. Certain cells of the immune system, such as dendritic cells and B cells, secrete exosomes that many scientists believe play a functional role in mediating adaptive immune responses to pathogens and tumours.

[0068] Exosomes are typically formed through inward budding of endosomal membranes giving rise to intracellular multivesicular bodies (MVB) that later fuse with the plasma membrane, releasing the exosomes to the exterior. In other words, an exosome is created intracellularly when a segment of the cell membrane spontaneously invaginates and is endocytosed. The internalized segment may be broken into smaller vesicles that are subsequently expelled from the cell. The latter stage occurs when the late endosome, containing many small vesicles, fuses with the cell membrane, triggering the release of the vesicles from the cell. The vesicles (once released may be called exosomes) consist of a lipid raft embedded with ligands common to the original cell membrane. However, a more direct release of exosomes has been described, Jurkat T-cells, are said to shed exosomes directly by outward budding of the plasma membrane. Exosomes secreted by cells under normal and pathological conditions contain proteins and functional RNA molecules including mRNA and siRNA, which can be shuttled from one cell to another, affecting the recipient cell's protein production. This RNA is called “exosomal shuttle RNA”.

[0069] Although exosomes have been previously utilized in the area of regenerative medicine, such as in treatment of heart failure in animal models, the use of exosomes from adipose tissue has not been previously disclosed for cosmetic and skin regeneration purposes.Adipose Tissue

[0070] Aspects of the present disclosure include compositions derived from tissue as a source of regenerative cells. In some embodiments, the tissue is adipose tissue. Autologous adipose tissue possesses the advantage of a large number of stem cells being available without need for major in vitro amplification. Additionally, autologous adipose tissue allows for production of individualized cellular products and cellular derived products that arepersonalized and therefore reducing hypothetical risk of infections. Certain aspects of the present disclosure also relate to the use of allogeneic adipose derived stem cells. In this case, scalability and standardization of product is feasible. The safety of adipose derived stem cells is illustrated by numerous clinical trials that have been conducted, which have all demonstrated safety without adverse events. To date, clinical trials on adipose derived cells have utilized ex vivo-expanded cells, which share properties with bone marrow derived MSC. Additionally, several clinical trials have utilized non-expanded stromal vascular fraction (SVF) cells, which are essentially fat cells devoid of adipocytes.

[0071] Preparations of MSC expanded from adipose tissue are equivalent or superior to bone marrow in terms of differentiation ability, angiogenesis-stimulating potential, and immune modulatory effects. Given the extra processing steps associated with ex vivo expansion of adipose cells, a simpler and perhaps safer procedure would be the use of primary adipose tissue- derived cells for therapy. SVF comprises the mononuclear cells derived from adipose tissue, which are acquired through a simple isolation procedure whereby fat is lipo- aspirated and subjected to enzymatic digestion. In veterinary medicine, over 4,000 horses and 4,000 dogs with various cartilage and bone injuries have been treated with autologous SVF without cellular expansion. In double blind studies of canine osteoarthritis, statistically significant improvements in lameness, range of motion, and overall quality of life have been described.

[0072] Given the abundance of pre-clinical and veterinary experience with autologous adipose-derived cells, there is great potential for autologous stem cell therapies using adipose-derived cells for a multitude of indications. Indeed, this prospect underlies the interest of commercial entities in devising bench top closed systems for autologous adipose cell therapy, such as Cytori’s Celution™ system and Tissue Genesis’ TGI 1000™ platform, which are presently entering clinical trials. Although the majority of studies have focused on in vitro expanded adipose derived cells, SVF derived from whole lipoaspirate alleviates the need for extensive processing of the cells, thereby also minimizing the number of steps where contamination could be introduced. The safety of adipose-derived cells is supported by autologus fat grafting, a common practice in composition surgery. An important consideration in clinical scenarios where bulk SVF is utilized is the potential regenerative, angiogenic and immune regulatory contributions of the numerous cellular populations that are present.Methods of deriving cord tissue mesenchymal stem cells from human umbilical tissue are provided. The cells are capable of self-renewal and expansion in culture, and have the potential to differentiate into cells of other phenotypes. In some embodiments, the method comprises (a) obtaining human umbilical tissue; (b) removing substantially all of blood to yield a substantially blood-free umbilical tissue, (c) dissociating the tissue by mechanical or enzymatic treatment, or both, (d) resuspending the tissue in a culture medium, and (e) providing growth conditions which allow for the growth of a human umbilicus- derived cell capable of selfrenewal and expansion in culture and having the potential to differentiate into cells of other phenotypes.

[0073] Tissue can be obtained from any completed pregnancy, term or less than term, whether delivered vaginally, or through other routes, for example surgical Cesarean section. Obtaining tissue from tissue banks is also considered within the scope of the present disclosure. The tissue can be rendered substantially free of blood by any means known in the art. For example, the blood can be physically removed by washing, rinsing, diluting and / or the like, before and / or after bulk blood removal for example by suctioning or draining. Other means of obtaining a tissue substantially free of blood cells can include, for example, enzymatic or chemical treatment. Dissociation of the umbilical tissues can be accomplished by any of the various techniques known in the art, including by mechanical disruption, for example, tissue can be aseptically cut with scissors, or a scalpel, or such tissue can be otherwise minced, blended, ground, or homogenized in any manner that is compatible with recovering intact or viable cells from human tissue.Isolation of Cells from Tissue

[0074] In some embodiments, the isolation procedure comprises an enzymatic digestion process. Many enzymes can be useful for the isolation of individual cells from complex tissue matrices to facilitate growth in culture. Ranging from weakly digestive (e.g. deoxyribonucleases and the neutral protease, dispase) to strongly digestive (e.g. papain and trypsin), such enzymes are available commercially. A nonexhaustive list of enzymes compatible herewith includes: mucolytic enzyme activities, metalloproteases, neutral proteases, serine proteases (such as trypsin, chymotrypsin, or elastase), and deoxyribonucleases. In some embodiments, the enzyme comprises a metalloprotease activity,neutral protease activity, mucolytic activity, or any combination thereof. For example, collagenases are known to be useful for isolating various cells from tissues. Deoxyribonucleases can digest single-stranded DNA and can minimize cell-clumping during isolation.

[0075] Enzymes can be used alone or in combination. Serine protease are preferably used in a sequence following the use of other enzymes as they may degrade the other enzymes being used. The temperature and time of contact with serine proteases should be monitored. Serine proteases may be inhibited with alpha 2 microglobulin in serum and therefore the medium used for digestion is preferably serum- free. EDTA and DNase are commonly used and may improve yields or efficiencies. In some embodiments, the method comprises an enzymatic treatment with collagenase and dispase, or collagenase, dispase, and hyaluronidase. In some embodiments, a mixture of collagenase and the neutral protease dispase are used in the dissociating step. In some embodiments, digestion is performed in the presence of at least one collagenase from Clostridium histolyticum, and either of the protease activities, dispase and therm oly sin.

[0076] The skilled artisan will appreciate that many such enzyme treatments are known in the art for isolating cells from various tissue sources. For example, the LIBERASE™ BLENDZYME (Roche) series of enzyme combinations of collagenase and neutral protease are very useful and may be used in the instant methods. Other sources of enzymes are known, and the skilled artisan may also obtain such enzymes directly from their natural sources. The skilled artisan is also well-equipped to assess new, or additional enzymes or enzyme combinations for their utility in isolating the cells of the disclosure. In some embodiments, enzyme treatments are about 0.5, about 1, about 1.5, or about 2 hours long or longer. In some embodiments, the tissue is incubated at about 37 degrees Celsius during the enzyme treatment of the dissociation step. In some embodiments, the digest is diluted to improve yields of cells, as cells may be trapped within a viscous digest.

[0077] In some embodiments, the cells described herein can be isolated from a tissue as described herein using mechanical separation methods. In some embodiments, methods employing enzymatic activity may also be used. In some embodiments, enzymatic activity is not used and is not required for successful isolation. In some embodiments, following dissociation of the tissue, a centrifugation step may be performed to separate thecells from residual tissue and debris. In some embodiments, the resulting cell pellet is then resuspended in a suitable culture medium.Expanding a Cell Population

[0078] In some embodiments, cells are further expanded / populated following their isolation from tissue. One skilled in the art will appreciate that each cell type may have its own optimal growth conditions. Given that, growth conditions may comprise a wide range of options as to culture medium, supplements, atmospheric conditions, and relative humidity for the cells. In some embodiments, cells are cultured at a temperature that is about 20, about 25, about 30, about 35, about 40, about 45, or any value that is between about 20 and about 45 degrees Celsius. In some embodiments, cells are cultured at a temperature that is about 35, about 36, about 37, about 38, about 39, or any value that is between about 35 and about 39 degrees Celsius. In some embodiments, the culture medium is Dulbecco's Modified Eagle Medium (DMEM) or Minimum Essential Medium Alpha (a-MEM). In some embodiments, the culture medium is supplemented with about 10% to about 20% Fetal Bovine Serum (FBS), L-Glutamine, and an antibiotic agent such as Penicillin-Streptomycin. In some embodiments, the cells are cultured at a temperature of about 37°C. In some embodiments, the cells are cultured in a humidified atmosphere comprising about 5% CO2. In some embodiments, the cells are cultured under normoxic conditions comprising about 20-21% oxygen or under hypoxic conditions comprising about 2-5% oxygen. In some embodiments, the cells are cultured at a relative humidity of greater than 95%. In some embodiments, the cells require no exogenous growth factors, except as are available in the supplemental serum provided with the Growth Medium. In some embodiments, the cells are mesenchymal stem cells, are derived from mesenchymal stem cell, or a combination thereof. In some embodiments, the cells are derived from a tissue selected from, but not limited to, adipose, nail cuticle, deciduous tooth, hair follicle, skin, bone marrow, placenta, umbilical cord blood, mobilized peripheral blood, peripheral blood, urine, breast milk, menstrual blood, fallopian tube, or a combination thereof.

[0079] Also provided herein are methods of deriving umbilical cells capable of expansion in the absence of particular growth factors. The methods are similar to the method above, however they further comprise culturing the cells in a culture medium lacking particulargrowth factors. In this sense, the method is selective for those cells capable of division in the absence of the particular growth factors.

[0080] In some embodiments, cells are grown and expanded in chemically-defined growth media with no serum added. In some embodiments, the growth media further comprises an at least one growth factors. Non-limiting examples of growth factors include: FGF, EGF, IGF, PDGF, or any combination thereof. In some embodiments, two, three or all four of the factors are added to serum-free or chemically-defined media. In some embodiments, LIF is added to serum-free medium to support or improve growth of the cells.

[0081] Also provided are methods wherein the cells can expand in the presence of about 1%, about 5%, about 10%, about 15% about 20%, about 25%, or any value that is between about 1% and about 20% oxygen in their atmosphere.

[0082] Also provided are methods to obtain cells that require L-valine. In some embodiments, the method comprises culturing cells in the presence of L-valine. After a cell is obtained, its need for L-valine can be tested and confirmed by growing on D-valine containing medium that lacks the L-isomer.

[0083] Methods are provided wherein the cells can undergo at least 25, 30, 35, or 40 doublings prior to reaching a senescent state. Methods for deriving cells capable of doubling to reach 10xl014cells or more are provided. In some embodiments, the methods derive cells that can double sufficiently to produce at least about 10 xlO14, about 10 xlO15, about 10 xlO16, or about 10 xlO17or more cells when seeded at from about 10 xlO3to about 10 xlO6cells / cm2in culture. In some embodiments, the cell numbers are produced within about 80, about 70, or about 60 days or less. In one embodiment, cord tissue mesenchymal stem cells are isolated and expanded, and possess one or more markers selected from a group comprising of CD 10, CD13, CD44, CD73, CD90, CD141, PDGFr-alpha, HLA- A,B,C, or any combination thereof. In addition, the cells do not produce one or more of CD31, CD34, CD45, CD117, CD141, HLA-DR,DP, DQ, or any combination thereof.

[0084] Said mesenchymal stem cells may be administered intravenously, or in a embodiment, intrathecally in a patient suffering radiation associated neurodegenerative manifestations. Although doses may be determined by one of skill in the art, and are dependent on various patient characteristics, intravenous administration may be performed atconcentrations ranging from about 1 to about 10 million MSC per kilogram, with a dose of approximately about 2 to about 5 million cells per kilogram.Exosomes and Microparticles

[0085] In methods and compositions disclosed herein, exosomes and microparticles may be used interchangeably. Exosomes from MSCs may be generated from a mesenchymal stem cell conditioned medium (MSC-CM). In some embodiments, the exosomes are used to reprogram immunocytes for tolerance induction ex vivo and / or in vivo. Microparticles may be isolated, for example, by being separated from non-associated components based on any property of the particle. In some embodiments, the microparticles may be isolated based on molecular weight, size, shape, composition biological activity, or any combination thereof.

[0086] The conditioned medium may be filtered or concentrated or both during, prior to or subsequent to separation. For example, it may be filtered through a membrane, for example one with a size or molecular weight cut-off. It may be subject to tangential force filtration or ultrafiltration. With regard to a donor tissue, cell, graft or solid organ transplant in a recipient patient, it is believed that the method according to the disclosure may be effective in preventing acute rejection of such transplant in the recipient and / or for long-term maintenance therapy to prevent rejection of such transplant in the recipient (e.g., inhibiting rejection of insulin-producing islet cell transplant from a donor in the patient recipient suffering from diabetes). Thus, the method of the disclosure is useful for preventing Host-Versus-Graft- Disease (HVGD) and Graft- Versus-Host-Disease (GVHD). Typically, the method of the present disclosure is applied to the patient before and / or after transplantation. As used herein, the term “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.By a “therapeutically effective amount” is meant a sufficient amount of cells generated with the present disclosure for the treatment of the disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total usage of these cells will be decided by the attending physicians within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and survival rate of the cells employed; the duration of the treatment; drugs used in combination or coincidental with the administered cells; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of cells at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0087] Aspects of the present disclosure provides novel stem cell types, methods of manufacture, and therapeutic uses. Some embodiments relate to a means of deriving stem cells possessing regenerative, immune modulatory, anti- inflammatory, and angiogenic / neurogenic activity from umbilical cord tissue such as Wharton’s Jelly. In some embodiments, manipulation of stem cell “potency” is disclosed through hypoxic manipulation, growth on non -xenogeneic conditions, and / or through the use of epigenetic modulators.

[0088] In some embodiments, mesenchymal stem cells are encapsulated by membranes and / or capsules prior to implantation. It is contemplated that any of the many methods of cell encapsulation available may be employed. In some embodiments, cells are individually encapsulated. In some embodiments, many cells are encapsulated within the same membrane. In embodiments in which the cells are to be removed following implantation, a relatively large size structure encapsulating many cells, such as within a single membrane, may provide a convenient means for retrieval. A wide variety of materials may be used in various embodiments for microencapsulation of Reprogrammed immune cells. Such materials include, for example, polymer capsules, alginate- poly-L-lysine-alginate microcapsules, barium poly- L-lysine alginate capsules, barium alginate capsules, polyacrylonitrile / polyvinylchloride (PAN / PVC) hollow fibers, and polyethersulfone (PES) hollow fibers. Techniques for microencapsulation of cells that may be used for administration of Reprogrammed immune cells are known to those of skill in the art and are described, for example, in Chang, P., et al., 1999; Matthew, H. W., et al., 1991; Yanagi, K., et al., 1989; Cai Z. H., et al., 1988; Chang, T.M., 1992 and in U.S. Pat. No. 5,639,275 (which, for example, describes a biocompatible capsule for long-term maintenance of cells that stably express biologically active molecules. Additional methods of encapsulation are in European Patent Publication No. 301,777 and U.S. Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of the foregoing are incorporated herein by reference in parts pertinent to encapsulation of Reprogrammed immune cells. The cells of the disclosure are cultured under hypoxia, in one embodiment, cultured in order to induce and / or augment expression of chemokine receptors. One such receptor is CXCR-4.

[0089] In some embodiments, the population of cells, including population of umbilical cord mesenchymal cells, may be enriched for CXCR-4, such as (or such as about) 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the population expressing CXCR-4, CD31, CD34, or any combination thereof. In addition or alternatively, <1%, <2%, <3%, <4%, <5%, <6%, <7%, <8%, <9%, or <10% of the population of cells may express CD14 and / or CD45. In some embodiments, the umbilical cord cells of the disclosure may further possess markers selected from the group consisting of STRO-1, CD105, CD54, CD56, CD106, HLA-I markers, vimentin, ASMA, collagen-1, fibronectin, LFA-3, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b / CD29, CD49c / CD29, CD49d / CD29, CD61, CD18, CD29, thrombomodulin, telomerase, CD10, CD13, STRO-2, VCAM-1, CD146, and THY-1, or any combination thereof. In some embodiments, the placental cells of the disclosure are admixed with endothelial cells. Said endothelial cells may express one or more markers selected from the group consisting of: a) extracellular vimentin; b) CD133; c) c-kit; d) VEGF receptor; e) activated protein C receptor; and f) any combination thereof. In some embodiments, the population of endothelial cells comprises endothelial progenitor cells.

[0090] The population of cells may be allogeneic, autologous, or xenogenic to an individual, including an individual being administered the population of cells. In some embodiments, the population of cells are matched by mixed lymphocyte reaction matching.

[0091] In some embodiments, senolytic agents are utilized to enhance therapeutic activity of regenerative cells. Various regenerative cell types exist. In one embodiment said regenerative cells are stem cells, in a specific embodiment said stem cells are of the mesenchymal lineage. The population of mesenchymal stem cells may be derived from tissue selected from the group consisting of the placental body, placenta, umbilical cord tissue,peripheral blood, hair follicle, cord blood, Wharton's Jelly, menstrual blood, endometrium, skin, omentum, amniotic fluid, and a combination thereof. In some embodiments, the population of cells, the population of umbilical mesenchymal stem cells, or the population of endothelial cells comprises human umbilical cord derived adherent cells. In some embodiments, the human umbilical cord derived adherent cells may express a cytokines selected from, but not limited to, FGF-1, FGF-2, HGF, interleukin-1 receptor antagonist, and any combination thereof. In some embodiments, the population of cells, the population of umbilical cord cells express arginase, indol eamine 2,3 deoxygenase, interleukin- 10, and / or interleukin 35. In some embodiments, the population of cells, the population of umbilical cord cells, or the population of endothelial cells express hTERT and Oct-4 but does not express a STRO-1 marker.

[0092] For the purpose of the disclosure, supernatants are collected from cultured cells, such as those derived from adipose tissue. In some embodiments, one such type of cell may include, the mesenchymal stem cell, which is utilized as a production source of growth factors. Said growth factor production can be upregulated by stimulation in vitro with agents such as cytokines, as well as coculture with cells from adipose tissue that elicit production of cytokines. In one embodiment of the disclosure, coculture of adipose derived mesenchymal stem cells is performed with monocytes. Said monocytes are activated by culture in activatory conditions. In one embodiment monocytes are cultured with TNF-alpha for approximately 1- 72 hours, more preferably from 12-48 hours, and more preferably approximately 24 hours. Concentrations of TNF-alpha are approximately 1-1000 ng / million cells, more preferably approximately 10 ng per million cells. Subsequent to culture said monocytes are added to mesenchymal stem cells, in one embodiment at a 1 to 1 ratio, although other ratios are envisioned in the scope of the current disclosure. Said mesenchymal stem cells and monocyte cultures are allowed to incubate for a period of approximately 72 hours or until reaching confluence. Media useful for the practice of the disclosure include DMEM, RPMI, AIM-V, OPTI-MEM, and EMEM. Media may be supplemented with nutrients or other factors to maintain viability of cells. Subsequent to culture, said media is harvested and utilized as a component of production of the composition as described herein. In one embodiment said media may be concentrated by means of lyophilization and subsequent dialysis to remove salt. In one embodiment of the disclosure, provided is a composition derived from conditionedmedia of adipose MSC cultured with monocytes, said media useful for formulation into a composition as described herein, said media containing one or more factors selected from a group comprising of: a) Interleukin-1 beta; b) Interleukin-6; c) alpha-2-Macroglobulin; d) Midkine; e) Chemokine (C-X-C motif) ligand 1 Chemokine (C-X-C motif) ligands; f) Chemokine (C-X-C motif) ligand 2; g) Chemokine (C-X-C motif) ligand 5; h) Chemokine (C- X-C motif) ligand 6; i) Interleukin-8; j) Chemokine (C-X-C motif) ligand 16; k) Chemokine (C-C motif) ligand 2; l)Chemokine (C-C motif) ligand 8; m) WNT1 -inducible- signaling pathway protein 2; n) Fibroblast growth factor 9; o) Platelet-derived growth factor D; p) Vascular endothelial growth factor A; and q) Growth differentiation factor 15. The growth factors / regenerative factors isolated from the conditioned media may be combined with other agents known to be useful in the field of cosmetics, for example liquorice and / or other ingredients that repairs the stratum corneum. Anti-oxidants may be added to the mixture useful for cosmetic purposes. According to the teachings of this disclosure, one skilled in the art would be aware of various methods for applying the compositions secreted by MSC, MSC-monocyte and manipulated cellular supernatants of this disclosure to cosmetic products, shampoos, nail strengthening ointments healthcare products and medicaments, and can obtain various desired cosmetic products, healthcare products and medicaments by these methods, so that it is not further described here. Generation of cream or lotion compositions and formulations are described herein, for use in the therapeutic renewal and rejuvenation of the skin of a patient. Specific target components may act, alone or in synergistic combination, to increase the generation of stem, epidermal, or other cells in the skin; to activate or increase collagen synthesis in the skin; to activate or increase endogenous hyaluronic acid synthesis in the epidermis; to activate or increase the hydration of the skin, and to activate or increase the stem cell and fibroblast migration within the epidermis to sites of needed repair on the skin. The use of the skin care composition cream, lotion, or other dermal application compositions of the present disclosure can yield progress towards dramatically younger looking skin, rehydration and a decrease in signs of aging such as dryness, thin skin, deep wrinkles and dull appearances.

[0093] In some embodiments of the disclosure bone marrow MSC may be utilized in conjunction with adipose derived MSC, or as a substitute, in the preparation of a composition that is based on basal secretion of growth factors in conditioned media or stimulated production of growth factors in conditioned media. Specifically, generation of bonemarrow derived MSC may be performed as follows. Bone marrow is aspirated (10-30 ml) under local anesthesia (with or without sedation) from the posterior iliac crest, collected into sodium heparin containing tubes and transferred to a Good Manufacturing Practices (GMP) clean room. Bone marrow cells are washed with a washing solution such as Dulbecco's phosphate-buffered saline (DPBS), RPMI, or PBS supplemented with autologous patient plasma and layered on to 25 ml of Percoll (1.073 g / ml) at a concentration of approximately 1- 2 ' 107cells / ml. Subsequently the cells are centrifuged at 900 for approximately 30 min or a time period sufficient to achieve separation of mononuclear cells from debris and erythrocytes. Said cells are then washed with PBS and plated at a density of approximately 1 ' 106cells per ml in 175 cm2tissue culture flasks in DMEM with 10% FCS with flasks subsequently being loaded with a minimum of 30 million bone marrow mononuclear cells. The MSCs are allowed to adhere for 72 h followed by media changes every 3-4 days. Adherent cells are removed with 0.05% trypsin-EDTA and replated at a density of 1 ' 106per 175 cm2.

[0094] Aspects of the present disclosure relate to the findings as disclosed herein that adipose tissue SVF contains cells which when mixed together under stimulatory conditions secrete large amounts of factors capable of inducing skin regeneration. SVF was originally described as the proliferative component of adipose tissue by Hollenberg et al. in 1968. The cells comprising SVF morphologically resemble fibroblasts and were demonstrated to differentiate into pre-adipocytes and functional adipose tissue in vitro. Although it was suggested that non-adipose differentiation of SVF may occur under specific conditions, the notion of “adipose-derived stem cells” was not widely recognized until a seminal paper in 2001, where Zuk et al demonstrated the SVF contains large numbers of mesenchymal-like stem cells (MSC-like) cells that could be induced to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic lineages. Subsequent to the initial description, the same group reported that in vitro expanded SVF derived cells had surface marker expression similar to bone marrow derived MSC, displaying expression of CD29, CD44, CD71, CD90, CD105 / SH2, and SH3 and lacking CD31, CD34, and CD45 expression. MSC are defined as adherent, non-hematopoietic cells expressing the surface markers CD90, CD105, and CD73, while lacking expression of CD 14, CD34, and CD45, and having the ability to differentiate into adipocytes, chondrocytes, and osteocytes in vitro after treatment with the appropriate growthfactors. Since these early discoveries, research in SVF biological properties has yielded many insights that support the practice of the current disclosure.

[0095] In one embodiment of the disclosure, human lipoaspirates from donors undergoing selective suction-assisted lipectomy are collected. Said liposuctioned aspirate is washed extensively with D- Hanks solution to remove contaminating blood and local anesthetics. The cellular components are subsequently washed two times and plated in T-75 tissue culture flasks at a density of approximately 2 x 106 / ml. Cells are subsequently grown in media containing 57% DMEM / F-12, 40%MCDB-201, 2% fetal calf serum, 10 ng / ml epidermal growth factor, 10 ng / ml platelet-derived growth factor BB, 100 U / ml penicillin, and 100 g / ml streptomycin. Once adherent cells were more than 70% confluent, cells are detached with 0.125% trypsin and 0.01% EDTA, and replated at a 1 :3 dilution under the same culture conditions. Cells at 50% adherence are incubated with monocytes derived from adipose tissue, said monocytes are selected by magnetic activated sorting of processed lipoaspirate using positive selection for CD 14. Monocytes are activated by exposure to TNF-alpha at a concentration of 10 ng per ml for a culture period of 24 hours. Monocytes are admixed at a 1 : 1 ratio with cultured MSC for a period of 48 hours. Conditioned media is subsequently extracted and concentrated 25-fold by lyophilization and subsequent desalting. Media is then utilized for formulation into composition solutions.

[0096] The disclosure teaches means of inducing the production of collagen and proliferation of dermal fibroblasts. Skin has high elasticity, in part because of the collagen secreted by fibroblast cells in the dermis of the skin, which forms the scaffold of the skin. In some embodiments the cellular conditioned media of any one of the embodiments of the present disclosure reduces wrinkles by increasing both the number and volume of fibroblast in the skin. In s o m e em b odi m ent s , the conditioned media of any one of the embodiments of the present disclosure promotes hydroxyproline synthesis, facilitates the synthesis of collagen and collagenase, and secretes, hyaluronic acid and glycoproteins. The media produced in the disclosure in is generated to specifically stabilize collagenous fibers.

[0097] Augmentation of regenerative proteins for the use in the practice of the current disclosure may be accomplished by transfecting adipose mesenchymal stem cells with genes associated with self- renewal. Said genes are involved in pathways associated with embryonic development (Wnt / 0- catenin, Notch / Delta-like, BMP / SMADs), the hox genes andtheir partners (Cdx, Hoxa9, Hoxal 0, Hoxb4, Meis, Pbx ), and polycomb / trithorax group genes (Bmil, Mil). In addition, a number of transcription factors involved in blood cell differentiation have also been shown to be necessary for self-renewal (Gata-2, Gfil, JunB, Pu.l, Myb, Cbp, Myc, and Zfx). How these diverse pathways are integrated in vivo is not understood; it has been postulated that epigenetic modifications such as chromatin and histone methylation and acetylation play a key role, and that the switch between MSC self-renewal and differentiation is regulated by competition between transcription factor complexes, akin to the interplay among Gata-1, c / ebpa, and Pu. l that mediates the myeloid / erythroid lineage decision . Furthermore, enhancement of self-renewal properties of mesenchymal stem cells from which culture supernatant is collected can be performed by addition of epigenetic modulators. Additionally, epigenetic modulators can be added to the composition formulation itself to enhance efficacy. The present disclosure is not limited to the use of any particular epigenetic inhibitors. Indeed, the use of variety of epigenetic inhibitors is contemplated, including, but not limited to synthetic epigenetic inhibitors and epigenetic inhibitors isolated or derived from natural sources. Examples of epigenetic inhibitors include, but are not limited to histone deacetylase inhibitors, DNA methyltransferase inhibitors and some vitamins. In some embodiments, the epigenetic inhibitors comprises sodium phenylbutyrate or a natural extract containing butyrate or butyric acid made from natural foods such as butter from animal fats or milk (e.g. cows milk or cheese), plant oils (e.g. Heracleum giganteum (cow parsnip) and Pastinaca sativa (parsnip)), or Kombucha tea (includes Butyric Acid as a result of fermentation containing butyrate). Extract preparation may include fermentation by obligate anaerobic bacteria (e.g. Clostridium butyricum, Clostridium kluyveri.

[0098] Clostridium pasteurianum, Fusobacterium nucleatum, Butyrivibrio fibrisolvens, Eubacterium limosum). Animal fat or plant oil product extracts may be prepared by chemical or physical processes inducing the liberation of butyric acid from the glyceride by hydrolysis. The extract could also be prepared by the fermentation of sugar or starch in the natural foods by the addition of Bacillus subtilis, with calcium carbonate added to neutralize the acids formed. Epigenetic modulators may be added directly into the tissue culture condition of the MSC, or may be added as part of the formulation that will be applied to the skin. In one embodiment, camphor is added at therapeutic concentrations and frequencies, said therapeutic implying sufficient to stimulate collagen production and inhibit elastase, utilization of camphoras part of composition use is described in the following reference, which can be used to guide one of skill in the art in combining camphor to enhance regenerative properties of the disclosure disclosed herein.

[0099] Additionally, cultured MSC may be endowed with certain properties by genetic modification. Since in one embodiment of the disclosure the active product that is administered to the skin for regeneration is supernatant and not cells, certain genetic modifications to the cells may be performed without risk of modifications being transferred to recipient. In one embodiment MSC are transfected with anti-apoptotic proteins to enhance in vitro longevity. This may be accomplished by transfection with at least one anti-apoptotic protein as a therapy to inhibit or prevent apoptosis. In one embodiment, MSC are contacted with apoptotis cells. In another embodiment, MSC that have been contacted with an apoptotic cell express high levels of anti- apoptotic molecules. In some instances, the MSC that have been contacted with an apoptotic cell secrete high levels of at least one anti-apoptotic protein, including but not limited to, STC-1, BCL-2, XIAP, Survivin, and Bcl-2XL. Methods of transfecting antiapoptotic genes into MSC have been previously described which can be applied to the current disclosure, said antiapoptotic genes that can be utilized for practice of the disclosure, in a nonlimiting way, include GATA-4, FGF-2, bcl-2, and HO-1. Based upon the disclosure provided herein, MSC can be obtained from any source. The MSC may be autologous with respect to the recipient (obtained from the same host) or allogeneic with respect to the recipient. In addition, the MSC may be xenogeneic to the recipient (obtained from an animal of a different species). In one embodiment of the disclosure MSC are pretreated with agents to induce expression of antiapoptotic genes, one example is pretreatment with exendin-4 as previously described. In a further non-limiting embodiment, MSC used in the present disclosure can be isolated, from the bone marrow of any species of mammal, including but not limited to, human, mouse, rat, ape, gibbon, bovine. In a non-limiting embodiment, the MSC are isolated from a human, a mouse, or a rat. In another non-limiting embodiment, the MSC are isolated from a human adipose derived MSC.

[0100] Based upon the present disclosure, MSC can be isolated and expanded in culture in vitro to obtain sufficient numbers of cells for use in the methods described herein provided that the MSC are cultured in a manner that promotes contact with a tumor endothelial cell. For example, MSC can be isolated from human bone marrow and cultured in completemedium (DMEM low glucose containing 4 mM L-glutamine, 10% FBS, and 1% penicillin / streptomycin) in hanging drops or on non-adherent dishes. The disclosure, however, should in no way be construed to be limited to any one method of isolating and / or to any culturing medium. Rather, any method of isolating and any culturing medium should be construed to be included in the present disclosure provided that the MSC are cultured in a manner that provides MSC to express increased amounts of at least one anti-apoptotic protein. Culture conditions for growth of clinical grade MSC have been described in the literature and are incorporated by reference.

[0101] In other embodiments, the epigenetic inhibitors comprise a natural extract of red grapes containing the phytoalexin resveratrol and / or pterostilbene, including an extract from juice or fermented juice (wine) of red grapes. Extracts could be prepared by mechanical disruption of grapes, separation of the skin from the flesh and seeds, and either extracting phytoalexin by chemical or mechanical methods, or be prepared from fresh or fermented grape juice by chemical or physical methods including boiling, fractionation, affinity chromatography, freeze- drying or gel separation.

[0102] In other embodiments, the epigenetic inhibitors comprise a natural extract containing Cyanocobalamin (vitamin B-12) made from organisms containing enzymes required for the synthesis ofB12 such as bacteria and archaea, or natural products which harbor such B12 producing bacteria including meat (especially liver and shellfish), eggs, and milk products. Extracts can be prepared by chemical or physical methods such as homogenization followed by fractionation, affinity chromatography, freeze-drying or gel separation.

[0103] In other embodiments, the epigenetic inhibitors comprise a natural extract containing one or several variants of vitamin B, made from either potatoes, bananas, lentils, chilli peppers, tempeh, liver, turkey, tuna, nutritional yeast (or brewer's yeast), beer or marmite. Extracts can be prepared by chemical or physical methods such as homogenization followed by e.g. fractionation, affinity chromatography, freeze-drying or gel separation. In other embodiments, the epigenetic inhibitors comprise a natural extract containing retinoids or retinoid precursors, made from either animal sources (e.g. milk and eggs) which contain retinyl esters, or from plants (e.g. carrots, spinach) which contain pro-vitamin A carotenoids. The extract may be modified by hydrolysis (animal sources) of retinyl esters to result in retinol, while plant extracts containing pro-vitamin A carotenoids can be cleaved to produce retinal(retin aldehyde), which can be further be reversibly reduced to produce retinol or it can be irreversibly oxidized to produce retinoic acid. The best described active retinoid metabolites are 11-cis-retinal and the all-trans and 9-cis-isomers of retinoic acid, which may be added to this extract.

[0104] Examples of other DNA methyltransferase inhibitors include, but are not limited to, 5- Azacytidine. 5-Aza-20-deoxycytidine, Arabinosyl-5-azacytidine, 5-6-Dihydro- 5-azacytidine, 5- Fluoro-20-deoxycytidine, EGX30P, Epigallocatechin-3 -gallate, Green tea polyphenol, Hydralazine, MG98, Procainamide, Procaine, and Zebularine. Examples of other histone deacetylase inhibitors include, but are not limited to Apicidin, Butyrates. Phenylbutyrate, m- Carboxy cinnamic acidbishydroxamide (CBHA). Cyclic hydroxamic-acid- containing peptide 1 (CHAP 1), TSA-Trapoxin Hybrid, Depudecin Epoxide, Depsipeptide FR901228, Benzamidine, LAQ824, Oxamflatin, MGCD0103, PXD101. Pyroxamide, Suberic Bishydroxamic Acid (SBHA), Suberoylanilide Hydroxamic Acid (SAHA), Trichostatin A (TSA), Trapoxin A, and Valproic acid. Other agents that enhance self-renewal may be utilized such as inhibitors of GSK- 3, one such inhibitor being lithium. Formulations and use of lithium for stimulation of stem cells are described in the following papers which are incorporated by reference. Without being bound to theory, addition of lithium and salts thereof may be incorporated into the composition mixture with the purpose of preventing apoptosis of progenitor cells.

[0105] Additionally, combinations of epigenetic acting agents together with lithium are envisioned within the practice of the disclosure to stimulate effects of conditioned media, or to enhance ability of cells to generate conditioned media. Previous combinations of the epigenetic modulator valproic acid with lithium have been published, which can guide one of skill in the art in practice of the disclosure. Use of lithium to induce dedifferentiation or rejuvenation of cells has previously been performed in experiments in which lithium can enhance inducible pluripotent stem cell generation, the generation of these cells being essentially a dedifferentiation of adult stem cells into a pluripotent state.

[0106] Agents that can be utilized in the practice of the disclosure include known composition agents that can be admixed with the cellular conditioned media generated by the disclosure, said composition agents include botanicals (which may be extracted from one or more of a root, stem bark, leaf, seed or fruit of a plant or plurality of plants). Some botanicalsmay be extracted from a plant biomass (e.g., root, stem, bark, leaf, etc.) using one more solvents. Botanicals may comprise a complex mixture of compounds and lack a distinct active ingredient. Another category of composition agents that can be admixed with the product of the disclosure include vitamin compounds and derivatives and combinations thereof, such as a vitamin B3 compound, a vitamin B5 compound, a vitamin B6 compound, a vitamin B9 compound, a vitamin A compound, a vitamin C compound, a vitamin E compound, and derivatives and combinations thereof (e.g., retinol, retinol esters, niacinamide, folic acid, panthenol, ascorbic acid, tocopherol, and tocopherol acetate). Vitamins have been used to prevent or reverse skin damage, and in particular, skin damage associated with inflammation due to UV radiation. Furthermore reference is made to U.S. Pat. Nos. 5,574,063, 5,545,398, 5,409,693, and 5,376,361 which describe the use of fatty acid esters of ascorbic acid (e.g., vitamin C palmitate) or tocotrienol (vitamin E) for treatment and prevention of skin damage. Anti-oxidants / radical scavengers may be utilized to augment composition effects of the disclosure, said agents include ascorbic acid (vitamin C) and its salts, ascorbyl esters of fatty acids, ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbyl sorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherol acetate, other esters of tocopherol, butylated hydroxy benzoic acids and their salts, 6-hydroxy-2, 5,7,8- tetramethylchroman-2-carboxylic acid (commercially available under the trade name Trolox.TM.), gallic acid and its alkyl esters, especially propyl gallate, uric acid and its salts and alkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g., N,N-di ethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g., glutathione), dihydroxy fumaric acid and its salts, lysine, pidolate, arginine pidolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, Superoxide dismutase, silymarin, tea extracts, grape skin / seed extracts, melanin, and rosemary extracts may be used. Other anti-oxidants / radical scavengers are selected from tocopherol sorbate and other esters of tocopherol. For example, the use of tocopherol sorbate in topical compositions and applicable to the present disclosure is described in U.S. Pat. No. 4,847,071.

[0107] In one embodiment of the disclosure exosomes are selected from cultures of adipose derived stem cells, or stromal vascular fraction. Said exosomes are purified by anion exchange chromatography. In this way, unexpectedly, it is demonstrated in this application that exosomes are resolved in a homogeneous peak after anion exchange chromatography. Thisresult is completely unexpected given that exosomes are complex supram olecul ar objects composed, among other things, of a membrane, surrounding an internal volume comprising soluble proteins. In addition, exosomes contain membrane proteins. Therefore, a object of this disclosure relates to a method of preparing, particularly of purifying, vesicle membranes, from a adipose cell or adipose stem cell conditioned media, comprising at least one anion exchange chromatography step. To apply the disclosure, a strong or weak, preferably strong, anion exchange may be performed. In addition, in a specific embodiment, the chromatography is performed under pressure. Thus, more specifically, it may consist of high performance liquid chromatography (HPLC). Different types of supports may be used to perform the anion exchange chromatography. More preferably, these may include cellulose, poly(styrene- divinylbenzene), agarose, dextran, acrylamide, silica, ethylene glycol-methacrylate copolymer, or mixtures thereof, e g., agarose-dextran mixtures. To illustrate this, it is possible to mention the different chromatography equipment composed of supports as mentioned above, particularly the following gels: SOURCE. POROS.RTM.. SEPHAROSE.RTM., SEPHADEX.RTM., TRISACRYL.RTM., TSK-GEL SW OR PW.RTM., SUPERDEX RTM. TO YOPEARL.HW and SEPHACRYL RTM., for example, which are suitable for the application of this disclosure.

[0108] Therefore, in a specific embodiment, this disclosure relates to a method of preparing membrane vesicles from a conditioned media, comprising at least one step during which the conditioned media is treated by anion exchange chromatography on a support selected from cellulose, poly(styrene-divinylbenzene), silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer, alone or in mixtures, optionally functionalized.

[0109] In addition, to improve the chromatographic resolution, within the scope of the disclosure, it is preferable to use supports in bead form. Ideally, these beads have a homogeneous and calibrated diameter, with a sufficiently high porosity to enable the penetration of the objects under chromatography (i.e. the exosomes). In this way, given the diameter of exosomes (generally between 50 and 100 nm), to apply the disclosure, it is preferable to use high porosity gels, particularly between 10 nm and 5 um, more preferably between approximately 20 nm and approximately 2 um, even more preferably between about 100 nm and about 1 um.

[0110] For the anion exchange chromatography, the support used must be functionalized using a group capable of interacting with an anionic molecule. Generally, this group is composed of an amine which may be ternary or quaternary, which defines a weak or strong anion exchanger, respectively.[OlH] Within the scope of this disclosure, it is particularly advantageous to use a strong anion exchanger. In this way, according to the disclosure, a chromatography support as described above, functionalized with quaternary amines, is used. Therefore, according to a more specific embodiment of the disclosure, the anion exchange chromatography is performed on a support functionalized with a quaternary amine. Even more preferably, this support should be selected from poly(styrene-divinylbenzene), acrylamide, agarose, dextran and silica, alone or in mixtures, and functionalized with a quaternary amine.

[0112] Examples of supports functionalized with a quaternary amine include the gels SOURCEQ. MONO Q, Q SEPHAROSE.RTM., POROS.RTM. HQ and POROS.RTM. QE, FRACTOGEL.RTM.TMAE type gels and TOYOPEARL SUPER.RTM.Q gels.

[0113] A particularly support to perform the anion exchange chromatography comprises poly(styrene-divinylbenzene). An example of this type of gel which may be used within the scope of this disclosure is SOURCE Q gel, particularly SOURCE 15 Q (Pharmacia). This support offers the advantage of very large internal pores, thus offering low resistance to the circulation of liquid through the gel, while enabling rapid diffusion of the exosomes to the functional groups, which are particularly important parameters for exosomes given their size.

[0114] The biological compounds retained on the column may be eluted in different ways, particularly using the passage of a saline solution gradient of increasing concentration, e.g. from 0 to 2 M. A sodium chloride solution may particularly be used, in concentrations varying from 0 to 2 M, for example. The different fractions purified in this way are detected by measuring their optical density (OD) at the column outlet using a continuous spectrophotometric reading. As an indication, under the conditions used in the examples, the fractions comprising the membrane vesicles were eluted at an ionic strength comprised between approximately 350 and 700 mM, depending on the type of vesicles.

[0115] Different types of columns may be used to perform this chromatographic step, according to requirements and the volumes to be treated. For example, depending on the preparations, it is possible to use a column from approximately 100 mu.l up to 10 ml or greater.In this way, the supports available have a capacity which may reach 25 mg of proteins / ml, for example. For this reason, a 100 .mu.l column has a capacity of approximately 2.5 mg of proteins which, given the samples in question, allows the treatment of culture supernatants of approximately 2 1 (which, after concentration by a factor of 10 to 20, for example, represent volumes of 100 to 200 ml per preparation). It is understood that higher volumes may also be treated, by increasing the volume of the column, for example.

[0116] In addition, to perform this disclosure, it is also possible to combine the anion exchange chromatography step with a gel permeation chromatography step. In this way, according to a specific embodiment of the disclosure, a gel permeation chromatography step is added to the anion exchange step, either before or after the anion exchange chromatography step. Preferably, in this embodiment, the permeation chromatography step takes place after the anion exchange step. In addition, in a specific variant, the anion exchange chromatography step is replaced by the gel permeation chromatography step. The present application demonstrates that membrane vesicles may also be purified using gel permeation liquid chromatography, particularly when this step is combined with an anion exchange chromatography or other treatment steps of the conditioned media, as described in detail below.

[0117] To perform the gel permeation chromatography step, a support selected from silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer or mixtures thereof, e.g., agarose- dextran mixtures, are preferably used. As an illustration, for gel permeation chromatography, a support such as SUPERDEX.RTM 200HR (Pharmacia), TSK G6000 (TosoHaas) or SEPHACRYL.RTM. S (Pharmacia) is preferably used.

[0118] The process according to the disclosure may be applied to different conditioned medias. In particular, these may consist of a biological fluid from a subject (bone marrow, peripheral blood, etc.), a culture supernatant, a cell lysate, a pre-purified solution or any other composition comprising membrane vesicles.

[0119] In this respect, in a specific embodiment of the disclosure, the conditioned media is a culture supernatant of membrane vesicle-producing cells.

[0120] In addition, according to a embodiment of the disclosure, the conditioned media is treated, prior to the chromatography step, to be enriched with membrane vesicles (enrichment stage). In this way, in a specific embodiment, this disclosure relates to a method of preparing membrane vesicles from a conditioned media, characterized in that it comprisesat least: b) an enrichment step, to prepare a sample enriched with membrane vesicles, and c) a step during which the sample is treated by anion exchange chromatography and / or gel permeation chromatography.

[0121] According to a embodiment, the conditioned media is a culture supernatant treated so as to be enriched with membrane vesicles. In particular, the conditioned media may be composed of a pre-purified solution obtained from a culture supernatant of a population of membrane vesicle-producing cells or from a biological fluid, by treatments such as centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography, particularly with clarification and / or ultrafiltration and / or affinity chromatography.

[0122] Therefore, a method of preparing membrane vesicles according to this disclosure more particularly comprises the following steps: a) culturing a population of membrane vesicle (e g. exosome) producing cells under conditions enabling the release of vesicles, b) a step of enrichment of the sample in membrane vesicles, and c) an anion exchange chromatography and / or gel permeation chromatography treatment of the sample.

[0123] As indicated above, the sample (e.g. supernatant) enrichment step may comprise one or more centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography steps on the supernatant. In a first specific embodiment, the enrichment step comprises (i) the elimination of cells and / or cell debris (clarification), possibly followed by (ii) a concentration and / or affinity chromatography step. In an other specific embodiment, the enrichment step comprises an affinity chromatography step, optionally preceded by a step of elimination of cells and / or cell debris (clarification). A enrichment step according to this disclosure comprises (i) the elimination of cells and / or cell debris (clarification), (ii) a concentration and (iii) an affinity chromatography.

[0124] The cells and / or cell debris may be eliminated by centrifugation of the sample, for example, at a low speed, preferably below 1000 g, between 100 and 700 g, for example, centrifugation conditions during this step are approximately 300 g or 600 g for a period between 1 and 15 minutes, for example.

[0125] The cells and / or cell debris may also be eliminated by filtration of the sample, possibly combined with the centrifugation described above. The filtration may particularly be performed with successive filtrations using filters with a decreasing porosity. For this purpose, filters with a porosity above 0.2 mu m, e g. between 0.2 and 10 mu m, arepreferentially used. It is particularly possible to use a succession of filters with a porosity of 10 .mu.m, 1 .mu.m, 0.5 um followed by 0.22 um.

[0126] A concentration step may also be performed, in order to reduce the volumes of sample to be treated during the chromatography stages. In this way, the concentration may be obtained by centrifugation of the sample at high speeds, e.g. between 10,000 and 100,000 g, to cause the sedimentation of the membrane vesicles. This may consist of a series of differential centrifugations, with the last centrifugation performed at approximately 70,000 g. The membrane vesicles in the pellet obtained may be taken up with a smaller volume and in a suitable buffer for the subsequent steps of the process.

[0127] The concentration step may also be performed by ultrafiltration. In fact, this ultrafiltration allows both to concentrate the supernatant and perform an initial purification of the vesicles. According to an embodiment, the conditioned media (e.g., the supernatant) is subjected to an ultrafiltration, preferably a tangential ultrafiltration. Tangential ultrafiltration consists of concentrating and fractionating a solution between two compartments (filtrate and retentate), separated by membranes of determined cut-off thresholds. The separation is carried out by applying a flow in the retentate compartment and a transmembrane pressure between this compartment and the filtrate compartment. Different systems may be used to perform the ultrafiltration, such as spiral membranes (Millipore®, Amicon®), flat membranes or hollow fibers (Amicon®, Millipore®, Sartorius, Pall, GF, Sepracor). Within the scope of the disclosure, the use of membranes with a cut-off threshold below 1000 kDa, preferably between 300 kDa and 1000 kDa, or even more preferably between 300 kDa and 500 kDa, is advantageous.

[0128] The affinity chromatography step can be performed in various ways, using different chromatographic support and material. It is advantageously a non-specific affinity chromatography, aimed at retaining (i.e., binding) certain contaminants present within the solution, without retaining the objects of interest (i.e., the exosomes). It is therefore a negative selection. Preferably, an affinity chromatography on a dye is used, allowing the elimination (i.e., the retention) of contaminants such as proteins and enzymes, for instance albumin, kinases, dehydrogenases, clotting factors, interferons, lipoproteins, or also co-factors, etc. More preferably, the support used for this chromatography step is a support as used for the ion exchange chromatography, functionalized with a dye. As specific example, the dye may beselected from Blue SEPHAROSE RTM. (Pharmacia), YELLOW 86, GREEN 5 and BROWN 10 (Sigma). The support is more preferably agarose. It should be understood that any other support and / or dye or reactive group allowing the retention (binding) of contaminants from the treated conditioned media can be used in the instant disclosure.

[0129] In a specific embodiment of the disclosure, the conditioned media is obtained by subjecting a membrane vesicle-producing cell culture supernatant to at least one filtration stage.

[0130] In another specific embodiment of the disclosure, the conditioned media is obtained by subjecting a membrane vesicle-producing cell culture supernatant to at least one centrifugation step.

[0131] In an embodiment of the disclosure, the conditioned media is obtained by subjecting a membrane vesicle-producing cell culture supernatant to at least one ultrafiltration step. In another embodiment of the disclosure, the conditioned media is obtained by subjecting a membrane vesicle-producing cell culture supernatant to at least one affinity chromatography step. A more specific membrane vesicle preparation process within the scope of this disclosure comprises the following steps: a) the culture of a population of membrane vesicle (e g. exosome) producing cells under conditions enabling the release of vesicles, b) the treatment of the culture supernatant with at least one ultrafiltration or affinity chromatography step, to produce a conditioned media enriched with membrane vesicles (e.g. with exosomes), and c) an anion exchange chromatography and / or gel permeation chromatography treatment of the conditioned media. In an embodiment, step b) above comprises a filtration of the culture supernatant, followed by an ultrafiltration, preferably tangential. In another embodiment, step b) above comprises a clarification of the culture supernatant, followed by an affinity chromatography on dye, preferably on Blue Sepharose™ 6 Fast Flow. In addition, after step c), the material harvested may, if applicable, be subjected to one or more additional treatment and / or filtration stages d), particularly for sterilization purposes. For this filtration treatment stage, filters with a diameter less than or equal to 0.3 pm are preferentially used, or even more preferentially, less than or equal to 0.25 pm. Such filters have a diameter of 0.22 pm, for example. After step d), the material obtained is, for example, distributed into suitable devices such as bottles, tubes, bags, syringes, etc., in a suitable storage medium. The purified vesicles obtained in this way may be stored cold, frozen or used extemporaneously. In someembodiments, a specific preparation process within the scope of the disclosure comprises at least the following steps: c) an anion exchange chromatography and / or gel permeation chromatography treatment of the conditioned media, and d) a filtration step, particularly sterilizing filtration, of the material harvested after stage c). In a first variant, the process according to the disclosure comprises: c) an anion exchange chromatography treatment of the conditioned media, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). In another variant, the process according to the disclosure comprises: c) a gel permeation chromatography treatment of the conditioned media, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). According to a third variant, the process according to the disclosure comprises: c) an anionic exchange treatment of the conditioned media followed or preceded by gel permeation chromatography, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c).

[0132] Another means of concentrating exosomes is through their selective affinity to lectins such as GNA, NPA, cyanovirin and ConA. In one particular embodiment of the disclosure tissue culture media from a culture of adipose derived MSC, alone, or with other cells, is passed through a porous hollow fiber membrane wherein lectin molecules which bind to high mannose glycoproteins are immobilized within the porous exterior portion of the membrane, collecting pass-through tissue culture media and either discarding it, or reusing it. In an embodiment, the method of the present disclosure is carried out by using an affinity cartridge, with devices of this general type being disclosed in U.S. Pat. Nos. 4,714,556, 4,787,974 and 6,528,057, the disclosures of which are incorporated herein by reference. In this device, tissue culture media from an adipose derived MSC alone or MSC together with other cells is passed through the lumen of a hollow fiber ultrafiltration membrane that is in intimate contact, on the non-blood wetted side of the membrane, with immobilized lectins, which form a means to accept and immobilize viruses and toxic and / or infectious fragments thereof. Thus, the device retains intact exosomes while allowing other components to pass through the lumen. In one embodiment, concentration of exosomes is performed by a device which includes multiple channels of hollow fiber ultrafiltration membrane that forms a filtration chamber. An inlet port and an effluent port are in communication with the filtration chamber. The ultrafiltration membrane is preferably an anisotropic membrane with the tight or retention side facing the bloodstream. The membrane is conveniently formed of any number of polymersknown to the art, for example, polysulfone, polyethersulfone, polyamides, polyimides, cellulose acetate, and polyacrylamide. In some embodiments, the membrane has pores 200-500 nm in diameter, which will allow passage of exosomes and microvesicles. In some embodiments, the exosome has a diameter between 2 nm and 200 nm. In some embodiments, the exosome has a molecular weight of at least about 100 kDa. In some embodiments, the exosome is a hydrodynamic radius of at most about 100 nm. The device in one embodiment comprises a cartridge comprising a tissue culture media-processing chamber formed of interior glass wall. Around chamber is an optional exterior chamber. A temperature controlling fluid can be circulated into chamber through port and out of port. The device includes an inlet port for the tissue culture media and an outlet port for the effluent. The device also provides one or more ports, for accessing the extra channel space in the cartridge. The technology to immobilize enzymes, chelators, and antibodies in dialysis-like cartridges has been developed (Ambrus et al. Science 201(4358): 837 839, 1978; Ambrus et al. Ann Intern Med 106(4): 531 537, 1987; Kalghatgi et al. Res Commun Chem Pathol Pharmacol 27(3): 551 561, 1980) and is incorporated herein by reference. These cartridges can be directly perfused with tissue culture media from stem cell cultures through direct access to the tissue culture system, and, when desired returned to the tissue culture without further manipulations.

[0133] Prototypic cartridges have been used to metabolize excess phenylalanine (Kalghatgi et al., 1980, supra; Ambrus, 1978, supra) or to remove excess aluminum from patients' blood (Anthone et al. J Amer Soc Nephrol 6: 1271 1277, 1995). An illustration of preparing proteins for immobilization to the hollow fibers for the method of the present disclosure is presented in U.S. Pat. Nos. 4,714,556 and 4,787,974, 5,528,057. For binding of lectins to the ultrafiltration membrane, the polymers of the ultrafiltration membrane are first activated, i.e., made susceptible for combining chemically with proteins, by using processes known in the art. Any number of different polymers can be used. To obtain a reactive polyacrylic acid polymer, for example, carbodiimides can be used (Valuev et al., 1998, Biomaterials, 19:41 3). Once the polymer has been activated, the lectins can be attached directly or via a linker to form in either case an affinity matrix. Suitable linkers include, but are not limited to, avidin, strepavidin, biotin, protein A, and protein G. The lectins may also be directly bound to the polymer of the ultrafiltration membrane using coupling agents such as bifunctional reagents, or may be indirectly bound. In some embodiments, GNA covalentlycoupled to agarose can be used to form an affinity matrix. Once exosomes are bound they may be eluted off using a variety of chemicals known in the art for elution of nanovesicles containing high glycoprotein content from lectins.

[0134] In one embodiment of the disclosure, regenerative cells are immortalized or semi-immortalized and utilized as a source of conditioned media. Immortalization may be accomplished by one or more methods known to one of skill in the art. In one embodiment regenerative cells are treated with factors / genes selected from a list comprising of ABCB1, ABCG2, ABI1, ABL1, ABL2, ACKR3, ACSL3, ACSL6, ACVR1B, ACVR2A, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH1A1, ALDH2, ALK, AMER1, ANGPT1, ANGPT2, ANKRD23, APC, AR, ARAF, AREG, ARFRP1, ARHGAP26, ARHGEF12, ARID1A, ARID1B, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BBC3, BCL10, BCL11A, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL3, BCL6, BCL7A, BCL9, BCOR, BCORL1, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRINP3, BRIP1, BTG1, BTG2, BTK, BUB1B, Cl lorfiO, C15orf65, C2orf44, CA6, CACNA1D, CALR, CAMTAI, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD19, CD22, CD274, CD38, CD4, CD70, CD74, CD79A, CD79B, CD83, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDK7, CDK8, CDK9, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDX2, CEBPA, CHCHD7, CHD2, CHD4, CHEK1, CHEK2, CHIC2, CHN1, CHORDCI, CIC, CIITA, CLP1, CLTC, CLTCL1, CNBP, CNOT3, CNTRL, COL 1 Al, COPB1, COX6C, CRBN, CREB1, CREB3L1, CREB3L2, CREBBP, CRKL, CRLF2, CRTC1, CRTC3, CSF1R, CSF3R, CTCF, CTLA4, CTNNA1, CTNNB1, CUL3, CXCR4, CYLD, CYP17A1, CYP2D6, DAXX, DDB2, DDIT3, DDR1, DDR2, DDX10, DDX3X, DDX5, DDX6, DEK, DICER1, DIS3, DLL4, DNM2, DNMT1, DNMT3A, DOT1L, DP YD, DUSP4, DUSP6, EBF1, ECT2L, EDNRB, EED, EGFR, EIF4A2, ELF4, ELK4, ELL, ELN, EML4, EP300, EPHA3, EPHA5, EPHA7, EPHA8, EPHB1, EPHB2, EPHB4, EPS15, ERBB2, ERBB3, ERBB4, ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, EREG, ERG, ERN1, ERRFI1, ESRI, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR, FAF1, FAIM3, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS, FAT1, FBXO11, FBXW7, FCRL4, FEV, FGF10, FGF14, FGF19, FGF2, FGF23, FGF3,FGF4, FGF6, FGFR1, FGFR10P, FGFR2, FGFR3, FGFR4, FH, FHIT, FIP1L1, FKBP1 A, FLCN, FLU, FLT1, FLT3, FLT4, FNBP1, F0XA1, FOXL2, FOXO1, FOXO3, FOXO4, FOXP1, FRS2, FSTL3, FUBP1, FUS, GABRA6, GAS7, GATA1, GATA2, GATA3, GATA4, GATA6, GID4, GLI1, GMPS, GNA11, GNA12, GNA13, GNAQ, GNAS, GNRH1, GOLGA5, GOPC, GPC3, GPHN, GPR124, GRIN2A, GRM3, GSK3B, GUCY2C, H3F3A, H3F3B, HCK, HDAC1, HERPUD1, HEY1, HGF, HIP1, HIST1H1E, HIST1H3B, HIST1H4I, HLF, HMGA1, HMGA2, HMGN2P46, HNF1A, HNMT, HNRNPA2B1, HNRNPK, H00K3, H0XA11, H0XA13, H0XA9, HOXC11, HOXC13, H0XD11, H0XD13, HRAS, HSD3B1, HSP90AA1, HSP90AB1, IAPP, ID3, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL2, IL21R, IL3RA, IL6, IL6ST, IL7R, INHBA, INPP4B, IRF2, IRF4, IRS2, ITGAV, ITGB1, ITK, ITPKB, JAK1, JAK2, JAK3, JAZF1, JUN, KAT6A, KAT6B, KCNJ5, KDM1A, KDM5A, KDM5C, KDM6A, KDR, KDSR, KEAP1, KEL, KIAA1549, KIF5B, KIR3DL1, KIT, KLF4, KLHL6, KLK2, KMT2A, KMT2C, KMT2D, KRAS, KTN1, LASPI, LCK, LCP1, LGALS3, LGR5, LHFP, LIFR, LM01, LM02, LOXL2, LPP, LRIG3, LRP1B, LUC7L2, LYL1, LYN, LZTR1, MAF, MAFB, MAGED1, MAGI2, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAPK1, MAPK11, MAX, MCL1, MDM2, MDM4, MDS2, MECOM, MED12, MEF2B, MEN1, MET, MITF, MKI67, MKL1, MLF1, MLH1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MLLT6, MMP9, MN1, MNX1, MPL, MRE11A, MS4A1, MSH2, MSH6, MSI2, MSN, MST1R, MTCP1, MTF2, MTOR, MUC1, MUC16, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, MYH11, MYH9, NACA, NAE1, NBN, NCKIPSD, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFE2L2, NFIB, NFKB2, NFKBIA, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NOTCH3, NPM1, NR4A3, NRAS, NSD1, NT5C2, NTRK1, NTRK2, NTRK3, NUMA1, NUP214, NUP93, NUP98, NUTM1, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PAK3, PALB2, PARK2, PARP1, PATZ1, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDCD1, PDCD1LG2, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PDK1, PECAM1, PERI, PHF6, PHOX2B, PICALM, PIK3C2B, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIM1, PLAG1, PLCG2, PML, PMS1, PMS2, POLDI, POLE, POTI, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PREX2, PRF1, PRKAR1A, PRKCI, PRKDC, PRLR, PRPF40B, PRRT2, PRRX1, PRSS8, PSIP1, PSMD4, PTBP1, PTCHI, PTEN, PTK2, PTPN11, PTPRC, PTPRD, QKI, RABEP1, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAFI, RALGDS,RANBP17, RANBP2, RAP1 GDS1 , RARA, R131 , RBM10, RBM15, RCOR1 , RECQL4, REL, RELN, RET, RHOA, RHOH, RICTOR, RIPK1, RMI2, RNF213, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RPS6KB1, RPTOR, RUNX1, RUNX1T1, S1PR2, SAMHD1, SBDS, SDC4, SDHA, SDHAF2, SDHB, SDHC, SDHD, SEPT5, SEPT6, SEPT9, SET, SETBP 1, SETD2, SF1, SF3A1, SF3B1, SF3B2, SFPQ, SGK1, SH2B3, SH3GL1, SLAMF7, SLC34A2, SLC45A3, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1, SMARCE1, SMC1A, SMC3, SMO, SNCAIP, SNX29, S0CS1, SOXIO, SOX11, SOX2, SOX9, SPECC1, SPEN, SPOP, SPTA1, SRC, SRGAP3, SRSF2, SRSF3, SS18, SS18L1, SSX1, STAG2, STAT3, STAT4, STAT5B, STEAP1, STIL, STK11, SUFU, SUZ12, SYK, TAF1, TAF15, TALI, TAL2, TBL1XR1, TBX3, TCEA1, TCF12, TCF3, TCF7L2, TCL1A, TEK, TERC, TERT, TET1, TET2, TFE3, TFEB, TFG, TFPT, TFRC, TGFB1, TGFBR2, THRAP3, TIMP1, TJP1, TLX1, TLX3, TM7SF2, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF9, TNFSF11, TOPI, TOP2A, TP53, TP63, TPBG, TPM3, TPM4, TPR, TRAF2, TRAF3, TRAF3IP3, TRAF7, TRIM26, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTK, TTL, TYMS, U2AF1, U2AF2, UBA1, UBR5, USP6, VEGFA, VEGFB, VHL, VPS51, VTI1A, WAS, WEE1, WHSCI, WHSC1L1, WIFI, WISP3, WNT11, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT6, WNT7B, WRN, WT1, WWTR1, XBP1, XPA, XPC, XPO1, YWHAE, YWHAZ, ZAK, ZBTB16, ZBTB2, ZMYM2, ZMYM3, ZNF217, ZNF331, ZNF384, ZNF521, ZNF703 and ZRSR2. Mesenchymal stem cells for generation of the composition as described herein may be extracted from tissues containing cells selected from a group of cells comprising of: endothelial cells, epithelial cells, dermal cells, endodermal cells, mesodermal cells, fibroblasts, osteocytes, chondrocytes, natural killer cells, dendritic cells, hepatic cells, pancreatic cells, stromal cells, salivary gland mucous cells, salivary gland serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, ceruminous gland cells, eccrine sweat gland dark cells, eccrine sweat gland clear cells, apocrine sweat gland cells, gland of Moll cells, sebaceous gland cells, bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate gland cells, bulbourethral gland cells, Bartholin's gland cells, gland of Littre cells, uterus endometrium cells, isolated goblet cells, stomach lining mucous cells, gastric gland zymogenic cells, gastric gland oxyntic cells, pancreatic acinar cells, paneth cells, type II pneumocytes, clara cells, somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, intermediate pituitary cells, magnocellular neurosecretory cells,gut cells, respiratory tract cells, thyroid epithelial cells, parafollicular cells, parathyroid gland cells, parathyroid chief cell, oxyphil cell, adrenal gland cells, chromaffin cells, Leydig cells, theca interna cells, corpus luteum cells, granulosa lutein cells, theca lutein cells, juxtaglomerular cell, macula densa cells, peripolar cells, mesangial cell, blood vessel and lymphatic vascular endothelial fenestrated cells, blood vessel and lymphatic vascular endothelial continuous cells, blood vessel and lymphatic vascular endothelial splenic cells, synovial cells, serosal cell (lining peritoneal, pleural, and pericardial cavities), squamous cells, columnar cells, dark cells, vestibular membrane cell (lining endolymphatic space of ear), stria vascularis basal cells, stria vascularis marginal cell (lining endolymphatic space of ear), cells of Claudius, cells of Boettcher, choroid plexus cells, pia-arachnoid squamous cells, pigmented ciliary epithelium cells, nonpigmented ciliary epithelium cells, corneal endothelial cells, peg cells, respiratory tract ciliated cells, oviduct ciliated cell, uterine endometrial ciliated cells, rete testis ciliated cells, ductulus efferens ciliated cells, ciliated ependymal cells, epidermal keratinocytes, epidermal basal cells, keratinocyte of fingernails and toenails, nail bed basal cells, medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, cuticular hair root sheath cells, hair root sheath cells of Huxley's layer, hair root sheath cells of Henle's layer, external hair root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, basal cell of epithelia, urinary epithelium cells, auditory inner hair cells of organ of Corti, auditory outer hair cells of organ of Corti, basal cells of olfactory epithelium, coldsensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue- sensitive cone cells, photoreceptor green-sensitive cone cells, photoreceptor red-sensitive cone cells, proprioceptive primary sensory neurons, touch- sensitive primary sensory neurons, type I carotid body cells, type II carotid body cell (blood pH sensor), type I hair cell of vestibular apparatus of ear (acceleration and gravity), type II hair cells of vestibular apparatus of ear, type I taste bud cells cholinergic neural cells, adrenergic neural cells, peptidergic neural cells, inner pillar cells of organ of Corti, outer pillar cells of organ of Corti, inner phalangeal cells of organ of Corti, outer phalangeal cells of organ of Corti, border cells of organ of Corti, Hensen cells of organ of Corti, vestibular apparatus supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, enteric glial cells, astrocytes, neurons, oligodendrocytes, spindle neurons,anterior lens epithelial cells, crystallin-containing lens fiber cells, hepatocytes, adipocytes, white fat cells, brown fat cells, liver lipocytes, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, kidney distal tubule cells, kidney collecting duct cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gall bladder epithelial cells, ductulus efferens nonciliated cells, epididymal principal cells, epididymal basal cells, ameloblast epithelial cells, planum semilunatum epithelial cells, organ of Corti interdental epithelial cells, loose connective tissue fibroblasts, corneal keratocytes, tendon fibroblasts, bone marrow reticular tissue fibroblasts, nonepithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblast / cementocytes, odontoblasts, odontocytes, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, hyalocytes, stellate cells (ear), hepatic stellate cells (Ito cells), pancreatic stelle cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, nuclear bag cells of muscle spindle, nuclear chain cells of muscle spindle, satellite cells, ordinary heart muscle cells, nodal heart muscle cells, Purkinje fiber cells, smooth muscle cells, myoepithelial cells of iris, myoepithelial cell of exocrine glands, melanocytes, retinal pigmented epithelial cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonium cells, spermatozoa, ovarian follicle cells, Sertoli cells, thymus epithelial cell, and / or interstitial kidney cells.

[0135] In one embodiment MSC donor lots are generated from umbilical cord tissue. Means of generating umbilical cord tissue MSC have been previously published and are known to those skilled in the art. The term “umbilical tissue derived cells (UTC)” refers, for example, to cells as described in U.S. Pat. No. 7,510,873, U.S. Pat. No. 7,413,734, U.S. Pat. No. 7,524,489, and U.S. Pat. No. 7,560,276. The UTC can be of any mammalian origin e.g. human, rat, primate, porcine and the like. In one embodiment of the disclosure, the UTC are derived from human umbilicus, umbilicus-derived cells, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, have reduced expression of genes for one or more of short stature homeobox 2; heat shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); elastin (supravalvular aortic stenosis, Williams- Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2 (growth arrest-specific homeobox); sine oculis homeobox homolog 1 (Drosophila); crystallin, alpha B; disheveled associated activator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogen binding protein); src homology three (SH3) and cysteine rich domain; cholesterol 25- hydroxylase; runt-related transcription factor 3; interleukin 11 receptor, alpha; procollagen C- endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2; neuroblastoma, suppression of tumorigenicity 1; insulin-like growth factor binding protein 2, 36kDa; Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1; potassium intermediate / small conductance calcium-activated channel, subfamily N, member 4; integrin, beta 7; transcriptional co-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response 3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-keto reductase family 1, member C3 (3 -alpha hydroxy steroid dehydrogenase, type II); biglycan; transcriptional coactivator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C / guanylate cyclase C (atrionatriuretic peptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2 / adenovirus E1B 19 kDa interacting protein 3 -like; AE binding protein 1; and cytochrome c oxidase subunit Vila polypeptide 1 (muscle). In addition, these isolated human umbilicus-derived cells express a gene for each of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and tumor necrosis factor, alphainduced protein 3, wherein the expression is increased relative to that of a human cell which is a fibroblast, a mesenchymal stem cell, an iliac crest bone marrow cell, or placenta-derived cell. In some embodiments, the cells are capable of self- renewal and expansion in culture, and have the potential to differentiate into cells of other phenotypes.

[0136] Methods of deriving cord tissue mesenchymal stem cells from human umbilical tissue are provided. In some embodiments, the cells are capable of self-renewal andexpansion in culture, and have the potential to differentiate into cells of other phenotypes. In some embodiments, the method includes (a) obtaining human umbilical tissue; (b) removing substantially all of blood to yield a substantially blood-free umbilical tissue, (c) dissociating the tissue by mechanical or enzymatic treatment, or both, (d) resuspending the tissue in a culture medium, and (e) providing growth conditions which allow for the growth of a human umbilicus-derived cell capable of self-renewal and expansion in culture and having the potential to differentiate into cells of other phenotypes. Tissue can be obtained from any completed pregnancy, term or less than term, whether delivered vaginally, or through other routes, for example surgical Cesarean section. Obtaining tissue from tissue banks is also considered within the scope of the present disclosure.

[0137] In some embodiments, the tissue is rendered substantially free of blood by any means known in the art. For example, the blood can be physically removed by washing, rinsing, and diluting and the like, before or after bulk blood removal for example by suctioning or draining. Other means of obtaining a tissue substantially free of blood cells might include enzymatic or chemical treatment.

[0138] In some embodiments, dissociation of the umbilical tissues can be accomplished by any of the various techniques known in the art, including by mechanical disruption, for example, tissue can be aseptically cut with scissors, or a scalpel, or such tissue can be otherwise minced, blended, ground, or homogenized in any manner that is compatible with recovering intact or viable cells from human tissue.

[0139] In some embodiments, the isolation procedure also utilizes an enzymatic digestion process. Many enzymes are known in the art to be useful for the isolation of individual cells from complex tissue matrices to facilitate growth in culture. As discussed above, a broad range of digestive enzymes for use in cell isolation from tissue is available to the skilled artisan. Ranging from weakly digestive (e.g. deoxyribonucleases and the neutral protease, dispase) to strongly digestive (e.g. papain and trypsin), such enzymes are available commercially. A nonexhaustive list of enzymes compatible herewith includes mucolytic enzyme activities, metalloproteases, neutral proteases, serine proteases (such as trypsin, chymotrypsin, or elastase), and deoxyribonucleases. In some embodiments, enzyme activities may be selected from metalloproteases, neutral proteases and mucolytic activities. For example, collagenases are known to be useful for isolating various cells from tissues. Deoxyribonucleases can digestsingle-stranded DNA and can minimize cell-clumping during isolation. Enzymes can be used alone or in combination. Serine protease are preferably used in a sequence following the use of other enzymes as they may degrade the other enzymes being used. The temperature and time of contact with serine proteases must be monitored. Serine proteases may be inhibited with alpha 2 microglobulin in serum and therefore the medium used for digestion is preferably serum-free. EDTA and DNase are commonly used and may improve yields or efficiencies. In some embodiments, methods may include enzymatic treatment with, for example, collagenase and dispase, or collagenase, dispase, and hyaluronidase, and such methods are provided wherein in certain embodiments, a mixture of collagenase and the neutral protease dispase are used in the dissociating step. In some embodiments, methods employing digestion in the presence of at least one collagenase from Clostridium histolyticum, and either of the protease activities, dispase and thermolysin. In some embodiments, methods may employ digestion with both collagenase and dispase enzyme activities. In some embodiments, methods may include digestion with a hyaluronidase activity in addition to collagenase and dispase activities. The skilled artisan will appreciate that many such enzyme treatments are known in the art for isolating cells from various tissue sources. For example, the LIBERASE™ BLENDZYME (Roche) series of enzyme combinations of collagenase and neutral protease are very useful and may be used in the instant methods. Other sources of enzymes are known, and the skilled artisan may also obtain such enzymes directly from their natural sources. The skilled artisan is also well-equipped to assess new, or additional enzymes or enzyme combinations for their utility in isolating the cells of the disclosure, enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer. In some embodiments, the tissue is incubated at 37 °C. during the enzyme treatment of the dissociation step. Diluting the digest may also improve yields of cells as cells may be trapped within a viscous digest.

[0140] In one embodiment of the disclosure provides for a skin cream. In some embodiments, the skin cream includes at least one regenerative factor. In some embodiments, the at least one regenerative factor includes cellular regenerative factors obtained from conditioned media. Skin creams of the disclosure may comprise several ingredients, some of which are beneficial to the skin, and others that promote absorption of the regeneratively active ingredients into the skin. For example, U.S. Pat. No. 4,362,747 describes a cream pack formulation which comprises a mixture of the following components: (1) propylene glycol andpoly oxy ethylene; (2) monopalmitate and glyoxyldiureide; (3) alcohol, beeswax, sorbitan monopalmitate, and polyoxyethylene; (4) alcohol, dimethicone copolyol, glyceryl monosterarate / polyoxyethylene; and (5) stearate and zinc or titanium oxide. U.S. Pat. No. 5,391,373 describes a skin cream comprising sodium lactate, a micellar complex of plant extracts, vitamin B, and glycosphingolipids, a protein complex of serum proteins, animal proteins, and glycogen, a carbohydrate based complex of dextran, glycine and glucosamine, a long-chain fatty acid ester of retinol, a long-chain fatty acid ester of ascorbic acid and a short chain fatty acid ester of tocopherol. Other non-limiting examples of composition agent additives include sugar amines, phytosterols, hexamidine, hydroxy acids, ceramides, amino acids, and polyols. In addition, the generated compositions may be admixed with carriers for proper entry of said therapeutic / regenerative molecules into the skin. In the practice of the disclosure, dermatologically acceptable carriers include carriers that are safe for use in contact with human skin tissue and do not exacerbate existing skin conditions. Suitable carriers that may be contemplated may include water and / or water miscible solvents. In some embodiments, the skin cream may comprise from about 1% to about 95% by weight of water and / or water miscible solvent. The composition may comprise from about 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% water and / or water miscible solvents. Examples of water miscible solvents include monohydric alcohols, dihydric alcohols, polyhydric alcohols, glycerol, glycols, polyalkylene glycols such as polyethylene glycol, and mixtures thereof. When the skin cream is in the form of an emulsion, water and / or water miscible solvents are carriers typically associated with the aqueous phase. Furthermore, carriers may also include oils. The skin cream may comprise from about 1% to about 95% by weight of one or more oils. The skin cream may comprise from about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 3% of one or more oils. Oils may be used to solubilize, disperse, or carry materials that are not suitable for water or water soluble solvents. Suitable oils include silicones, hydrocarbons, esters, amides, ethers, and mixtures thereof. The oils may be volatile or nonvolatile. In one embodiment, silicone oils are utilized as carriers, silicone oils include poly siloxanes. Poly siloxanes that are currently in useinclude the polydimethylsiloxanes family, which are also known as dimethi cones, examples of which include the DM-Fluid series from Shin-Etsu, the Vicasil.RTM. series sold by Momentive Performance Materials Inc., and the Dow Corning. RTM. 200 series sold by Dow Corning Corporation. Specific examples of suitable polydimethylsiloxanes include Dow Corning.RTM. 200 fluids (also sold as Xiameter.RTM. PMX-200 Silicone Fluids) having viscosities of 0.65, 1.5, 50, 100, 350, 10,000, 12,500 100,000, and 300,000 centistokes. Other agents that are useful for carrier properties include hydrocarbon oils. Suitable hydrocarbon oils include straight, branched, or cyclic alkanes and alkenes. The chain length may be selected based on desired functional characteristics such as volatility.

[0141] In one embodiment of the disclosure provides for a composition for topical skin application. In some embodiments, the composition for topical skin application includes at least one regenerative factor. In some embodiments, the at least one regenerative factor includes cellular regenerative factors obtained from conditioned media. In some embodiments, the conditioned medium includes endogenous progenitor / stem cells. In some embodiments, the endogenous progenitor / stem cells are stimulated with at least one regenerative factor. In some embodiments, the at least one regenerative factor is a histone deactylase inhibitor. In some embodiments, the endogenous progenitor / stem cells expresses CD73 and TLR9. In some embodiments, the endogenous progenitor / stem cells resemble cellular injury or cell damage by a toll-like receptor agonist. Compositions for topical skin application of the disclosure may comprise several ingredients, some of which are beneficial to the skin, and others that promote absorption of the regeneratively active ingredients into the skin. For example, the composition for topical skin application may include a mixture of the following components: (1) propylene glycol and polyoxyethylene; (2) monopalmitate and glyoxyldiureide; (3) alcohol, beeswax, sorbitan monopalmitate, and polyoxyethylene; (4) alcohol, dimethicone copolyol, glyceryl monosterarate / polyoxyethylene; and (5) stearate and zinc or titanium oxide. U.S. Pat. No. 5,391,373 describes a composition for topical skin application comprising sodium lactate, a micellar complex of plant extracts, vitamin B, and glycosphingolipids, a protein complex of serum proteins, animal proteins, and glycogen, a carbohydrate based complex of dextran, glycine and glucosamine, a long-chain fatty acid ester of retinol, a long-chain fatty acid ester of ascorbic acid and a short chain fatty acid ester of tocopherol. Other non-limiting examples of composition agent additives include sugar amines, phytosterols, hexamidine, hydroxy acids,ceramides, amino acids, and polyols. Tn addition, the generated compositions may be admixed with carriers for proper entry of said therapeutic / regenerative molecules into the skin. In the practice of the disclosure, dermatologically acceptable carriers include carriers that are safe for use in contact with human skin tissue and do not exacerbate existing skin conditions. Suitable carriers that may be contemplated may include water and / or water miscible solvents. In some embodiments, the composition for topical skin application includes from about 1% to about 95% by weight of water and / or water miscible solvent. The composition may comprise from about 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% water and / or water miscible solvents. Examples of water miscible solvents include monohydric alcohols, dihydric alcohols, polyhydric alcohols, glycerol, glycols, polyalkylene glycols such as polyethylene glycol, and mixtures thereof. When the composition for topical skin application is in the form of an emulsion, water and / or water miscible solvents are carriers typically associated with the aqueous phase. Furthermore, carriers may also include oils. The composition for topical skin application may comprise from about 1% to about 95% by weight of one or more oils. The composition for topical skin application may comprise from about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 3% of one or more oils. Oils may be used to solubilize, disperse, or carry materials that are not suitable for water or water soluble solvents. Suitable oils include silicones, hydrocarbons, esters, amides, ethers, and mixtures thereof. The oils may be volatile or nonvolatile. In one embodiment, silicone oils are utilized as carriers, silicone oils include polysiloxanes. Polysiloxanes that are currently in use include the polydimethylsiloxanes family, which are also known as dimethicones, examples of which include the DM-Fluid series from Shin-Etsu, the Vicasil.RTM. series sold by Momentive Performance Materials Inc., and the Dow Corning.RTM. 200 series sold by Dow Coming Corporation. Specific examples of suitable polydimethylsiloxanes include Dow Corning.RTM. 200 fluids (also sold as Xiameter.RTM. PMX-200 Silicone Fluids) having viscosities of 0.65, 1.5, 50, 100, 350, 10,000, 12,500 100,000, and 300,000 centistokes. Other agents that are useful for carrier properties include hydrocarbon oils. Suitable hydrocarbon oils include straight, branched, or cyclic alkanes andalkenes. The chain length may be selected based on desired functional characteristics such as volatility.

[0142] Suitable volatile hydrocarbons may have between 5-20 carbon atoms or, alternately, between 8- 16 carbon atoms. Other suitable oils include esters. The suitable esters typically contained at least 10 carbon atoms. These esters include esters with hydrocarbyl chains derived from fatty acids or alcohols (e.g., mono-esters, polyhydric alcohol esters, and di- and tri-carboxylic acid esters). The hydrocarbyl radicals of the esters hereof may include or have covalently bonded thereto other compatible functionalities, such as amides and alkoxy moieties (e g., ethoxy or ether linkages, etc.). In another embodiment of the disclosure amides are utilized as oils. Useful amides include compounds having an amide functional group while being liquid at 20. degree. C. and insoluble in water. Suitable amides include N-acetyl-N- butylaminopropionate, isopropyl N- lauroylsarcosinate, and N,N, -diethyltoluamide. Other suitable amides are disclosed in U.S. Pat. No. 6,872,401. Furthermore, ethers can be utilized for delivery of the disclosure as carriers.

[0143] Suitable ethers include saturated and unsaturated fatty ethers of a polyhydric alcohol, and alkoxylated derivatives thereof. Exemplary ethers include C. sub.4-20 alkyl ethers of polypropylene glycols, and di-C. sub.8-30 alkyl ethers. Suitable examples of these materials include PPG- 14 butyl ether, PPG- 15 stearyl ether, dioctyl ether, dodecyl octyl ether, and mixtures thereof.

[0144] Within the purpose of administering the disclosure, the composition as described herein may include an agent capable of emulsifying the composition. The composition may include from about 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, or 1% to about 20%, 10%, 5%, 3%, 2%, or 1% emulsifier. Emulsifiers may be nonionic, anionic or cationic. Nonlimiting examples of emulsifiers are disclosed in U.S. Pat. No. 3,755,560, U.S. Pat. No. 4,421,769, and McCutcheon's, Emulsifiers and Detergents, 2010 Annual Ed., published by M. C. Publishing Co. Other suitable emulsifiers are further described in the Personal Care Product Council's International Composition Ingredient Dictionary and Handbook, Thirteenth Edition, 2006, under the functional category of “Surfactants—Emulsifying Agents.” Linear or branched type silicone emulsifiers may also be used. Particularly useful polyether modified silicones include KF-6011, KF-6012, KF-6013, KF-6015, KF-6015, KF- 6017, KF-6043, KF-6028, and KF-6038 from Shin Etsu. Also particularly useful are the polyglycerolated linear or branched siloxane emulsifiers including KF-6100, KF-6104, and KF- 6105 from Shin Etsu. Emulsifiersalso include emulsifying silicone elastomers. Suitable silicone elastomers may be in the powder form, or dispersed or solubilized in solvents such as volatile or nonvolatile silicones, or silicone compatible vehicles such as paraffinic hydrocarbons or esters. Suitable emulsifying silicone elastomers may include at least one polyalkyl ether or polyglycerolated unit.

[0145] Viscosity of the composition is particularly important dependent on use or area of the body to be administered. Structuring agents may be used to increase viscosity, thicken, solidify, or provide solid or crystalline structure to the skin care composition. Structuring agents are typically grouped based on solubility, dispersibility, or phase compatibility. Examples of aqueous or water structuring agents include polymeric agents, natural or synthetic gums, polysaccharides, and the like. In one embodiment, the composition may comprises from about 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5% to about 25%, 20%, 10%, 7%, 5%, 4%, or 2%, by weight of the composition, of one or more structuring agents. Furthermore, polysaccharides and gums may be suitable aqueous phase thickening agents. Suitable classes of polymeric structuring agents include but are not limited to carboxylic acid polymers, polyacrylamide polymers, sulfonated polymers, high molecular weight poly alkylglycols or polyglycerins, copolymers thereof, hydrophobically modified derivatives thereof, and mixtures thereof. Silicone gums are another oil phase structuring agent. Another type of oily phase structuring agent includes silicone waxes. Silicone waxes may be referred to as alkyl silicone waxes which and are semi-solids or solids at room temperature. Other oil phase structuring agents may be one or more natural or synthetic waxes such as animal, vegetable, or mineral waxes.

[0146] The administration of the cellular regenerative factors / conditioned media may be utilized in conjunction with known approaches in the art to augment efficacy of composition effect. For example, administration by liposomes may be performed as thought in U.S. Pat. No. 6,146,650 which describes the use of liposomes to deliver collagen, avocado oil, aloe and vital nutrients such as Vitamins A, C, D and E to the skin. Also, U.S. Pat. No. 6,281,236 describes compositions containing allantoin and an emulsifier such as natural beeswax for the treatment of skin. Utilization of the disclosure in combination with procedures based on inhibiting existing damaged tissue is disclosed in the current disclosure, for example, U.S. Pat. No. 6,319,942, teaches the use of alkanolamines such as dimethylaminoethanol (DMAE) for the treatment of scars; U.S. Pat. No. 6,296,861, teaches the use of conjugatedlinoleic acid and fatty acid esters of vitamin C for treatment of skin damage; U.S. Pat. No. 6,191,121, teaches the use of poly enoylphosphatidyl choline to treat skin damage; U.S. Pat. Nos. 5,965,618 and 5,709,868, teaches the treatment of scar tissue using lipoic acid, and additionally, alpha-hydroxy acids, fatty acid esters of vitamin C, and tocopherol (vitamin E); and U.S. Pat. Nos. 5,554,647 and 5,643,586 teaches the use of catecholamine or acetylcholine precursors for treatment of skin damage. In another embodiment of the disclosure, biologically useful stem cells are disclosed, of the mesenchymal or related lineages, which are therapeutically reprogrammed cells having minimal oxidative damage and telomere lengths that compare favorably with the telomere lengths of undamaged, pre-natal or embryonic stem cells (that is, the therapeutically reprogrammed cells of the present disclosure possess near prime physiological state genomes). Moreover the therapeutically reprogrammed cells of the present disclosure are immunologically privileged and therefore suitable for therapeutic applications. Additional methods of the present disclosure provide for the generation of hybrid stem cells. Furthermore, the present disclosure includes related methods for maturing stem cells made in accordance with the teachings of the present disclosure into specific host tissues. For use in the current disclosure, the practitioner is thought that ontogeny of mammalian development provides a central role for stem cells. Early in embryogenesis, cells from the proximal epiblast destined to become germ cells (primordial germ cells) migrate along the genital ridge. These cells express high levels of alkaline phosphatase as well as expressing the transcription factor Oct4. Upon migration and colonization of the genital ridge, the primordial germ cells undergo differentiation into male or female germ cell precursors (primordial sex cells). For the purpose of this disclosure disclosure, only male primordial sex cells (PSC) will be discussed, but the qualities and properties of male and female primordial sex cells are equivalent and no limitations are implied. During male primordial sex cell development, the primordial stem cells become closely associated with precursor sertoli cells leading to the beginning of the formation of the seminiferous cords. When the primordial germ cells are enclosed in the seminiferous cords, they differentiate into gonocytes that are mitotically quiescent. These gonocytes divide for a few days followed by arrest at G0 / G1 phase of the cell cycle. In mice and rats these gonocytes resume division within a few days after birth to generate spermatogonial stem cells and eventually undergo differentiation and meiosis related to spermatogenesis. It is known that embryonic stem cells are cells derived from the inner cellmass of the pre-im plantation blastocyst-stage embryo and have the greatest differentiation potential, being capable of giving rise to cells found in all three germ layers of the embryo proper. From a practical standpoint, embryonic stem cells are an artifact of cell culture since, in their natural epiblast environment, they only exist transiently during embryogenesis.

[0147] Manipulation of embryonic stem cells in vitro has lead to the generation and differentiation of a wide range of cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerves, skeletal muscle, chondrocytes, adipocytes, liver and pancreatic islets. Growing embryonic stem cells in co-culture with mature cells can influence and initiate the differentiation of the embryonic stem cells to a particular lineage. Maturation is a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation and / or dedifferentiation. In one example of the maturation process, a cell, or group of cells, interacts with its cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches and these niches, or microenvironments, house stem cells that direct and regulate organogenesis. At the time of birth, maturation has progressed such that cells and appropriate cellular niches are present for the organism to function and survive post-natally. Developmental processes are highly conserved amongst the different species allowing maturation or differentiation systems from one mammalian species to be extended to other mammalian species in the laboratory. During the lifetime of an organism, the cellular composition of the organs and organs systems are exposed to a wide range of intrinsic and extrinsic factors that induce cellular or genomic damage. Ultraviolet light not only has an effect on normal skin cells but also on the skin stem cell population. Chemotherapeutic drugs used to treat cancer have a devastating effect on hematopoietic stem cells. Reactive oxygen species, which are the byproducts of cellular metabolism, are intrinsic factors that compromises the genomic integrity of the cell. In all organs or organ systems, cells are continuously being replaced from stem cell populations. However, as an organism ages, cellular damage accumulates in these stem cell populations. If the damage is inheritable, such as genomic mutations, then all progeny will be effected and thus compromised. A single stem cell clone can contribute to generations of lineages such as lymphoid and myeloid cells for more than a year and therefore have the potential to spread mutations if the stem cell is damaged. The body responds to a compromised stem cell by inducing apoptosis thereby removing it from the pool and preventing potentially dysfunctionalor tumorigenic properties. Apoptosis removes compromised cells from the population, but it also decreases the number of stem cells that are available for the future. Therefore, as an organism ages, the number of stem cells decrease. In addition to the loss of the stem cell pool, there is evidence that aging decreases the efficiency of the homing mechanism of stem cells. Telomeres are the physical ends of chromosomes that contain highly conserved, tandemly repeated DNA sequences. Telomeres are involved in the replication and stability of linear DNA molecules and serve as counting mechanism in cells; with each round of cell division the length of the telomeres shortens and at a pre-determined threshold, a signal is activated to initiate cellular senescence. Stem cells and somatic cells produce telomerase, which inhibits shortening of telomeres, but their telomeres still progressively shorten during aging and cellular stress. In one teaching, or embodiment, of the disclosure, therapeutically reprogrammed cells, in some embodiments mesenchymal stem cells, are provided. Therapeutic reprogramming refers to a maturation process wherein a stem cell is exposed to stimulatory factors according the teachings of the present disclosure to yield enhanced therapeutic activity. In some embodiments, enhancement of therapeutic activity may be increase proliferation, in other embodiments, it may be enhanced chemotaxis. Other therapeutic characteristics include ability to under resistance to apoptosis, ability to overcome senescence, ability to differentiate into a variety of different cell types effectively, and ability to secrete therapeutic growth factors which enhance viability / activity, of endogenous stem cells. In order to induce therapeutic reprogramming of cells, in some cases, as disclosed herein, of wharton’s jelly originating cells, the disclosure teaches the utilization of stimulatory factors, including without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic programming of cells. These stimulatory factors induce, among other results, genomic methylation changes in the donor DNA. Embodiments of the present disclosure include methods for preparing cellular extracts from whole cells, cytoplasts, and karyplasts, although other types of cellular extracts are contemplated as being within the scope of the present disclosure. In a non-limiting example, the cellular extracts of the present disclosure are prepared from stem cells, specifically embryonic stem cells. Donor cells are incubated with the chemicals, biochemicals or cellular extracts for defined periods of time, in a non-limiting example for approximately one hour to approximately two hours, and those reprogrammed cells that express embryonic stem cell markers, such as Oct4, after a culture period are then ready for transplantation,cry opreservation or further maturation. In another embodiment of the present disclosure, hybrid stem cells are provided which can be used for cellular regenerative / reparative therapy. The hybrid stem cells of the present disclosure are pluripotent and customized for the intended recipient so that they are immunologically compatible with the recipient. Hybrid stem cells are a fusion product between a donor cell, or nucleus thereof, and a host cell. Typically the fusion occurs between a donor nucleus and an enucleated host cell. The donor cell can be any diploid cell, including but not limited to, cells from pre-embryos, embryos, fetuses and post-natal organisms. More specifically, the donor cell can be a primordial sex cell, including but not limited to, oogonium or differentiated or undifferentiated spermatogonium, or an embryonic stem cell. Other non-limiting examples of donor cells are therapeutically reprogrammed cells, embryonic stem cells, fetal stem cells and multipotent adult progenitor cells. Preferably the donor cell has the phenotype of the intended recipient. The host cell can be isolated from tissues including, but not limited to, pre- embryos, embryos, fetuses and post-natal organisms and more specifically can include, but is not limited to, embryonic stem cells, fetal stem cells, multipotent adult progenitor cells and adipose- derived stem cells. In a non-limiting example, cultured cell lines can be used as donor cells. The donor and host cells can be from the same individual or different individuals. In one embodiment of the present disclosure, lymphocytes are used as donor cells and a two-step method is used to purify the donor cells. After the tissues was disassociated, an adhesion step was performed to remove any possible contaminating adherent cells followed by a density gradient purification step. The majority of lymphocytes are quiescent (in GO phase) and therefore can have a methylation status than conveys greater plasticity for reprogramming. Multipotent or pluripotent stem cells or cell lines useful as donor cells in embodiments of the present disclosure are functionally defined as stem cells by their ability to undergo differentiation into a variety of cell types including, but not limited to, adipogenic, neurogenic, osteogenic, chondrogenic and cardiogenic cell.

[0148] In some embodiments, host cell enucleation for the generation of hybrid stem cells according to the teachings of the present disclosure can be conducted using a variety of means. In a non- limiting example, ADSCs were plated onto fibronectin coated tissue culture slides and treated with cells with either cytochalasin D or cytochalasin B. After treatment, the cells can be trypsinized, re-plated and are viable for about 72 hours post enucleation. Host cells and donor nuclei can be fused using one of a number of fusion methods known to thoseof skill in the art, including but not limited to electrofusion, microinjection, chemical fusion or virus-based fusion, and all methods of cellular fusion are envisioned as being within the scope of the present disclosure. The hybrid stem cells made according to the teachings of the present disclosure possess surface antigens and receptors from the enucleated host cell but has a nucleus from a developmentally younger cell. Consequently, the hybrid stem cells of the present disclosure will be receptive to cytokines, chemokines and other cell signaling agents, yet possess a nucleus free from age-related DNA damage. The therapeutically reprogrammed cells and hybrid stem cells made in accordance with the teachings of the present disclosure are useful in a wide range of therapeutic applications for cellular regenerative / reparative therapy. For example, and not intended as a limitation, the therapeutically reprogrammed cells and hybrid stem cells of the present disclosure can be used to replenish stem cells in animals whose natural stem cells have been depleted due to age or ablation therapy such as cancer radiotherapy and chemotherapy. In another non-limiting example, the therapeutically reprogrammed cells and hybrid stem cells of the present disclosure are useful in organ regeneration and tissue repair. In one embodiment of the present disclosure, therapeutically reprogrammed cells and hybrid stem cells can be used to reinvigorate damaged muscle tissue including dystrophic muscles and muscles damaged by ischemic events such as myocardial infarcts. In another embodiment of the present disclosure, the therapeutically reprogrammed cells and hybrid stem cells disclosed herein can be used to ameliorate scarring in animals, including humans, following a traumatic injury or surgery. In this embodiment, the therapeutically reprogrammed cells and hybrid stem cells of the present disclosure are administered systemically, such as intravenously, and migrate to the site of the freshly traumatized tissue recruited by circulating cytokines secreted by the damaged cells. In another embodiment of the present disclosure, the therapeutically reprogrammed cells and hybrid stem cells can be administered locally to a treatment site in need or repair or regeneration.

[0149] In one embodiment, umbilical cord samples were obtained following the delivery of normal term babies with Institutional Review Board approval. A portion of the umbilical cord was then cut into approximately 3 cm long segments. The segments were then placed immediately into 25 ml of phosphate buffered saline without calcium and magnesium (PBS) and 1. times, antibiotics (100 U / ml penicillin, 100 ug / ml streptomycin, 0.025 ug / ml amphotericin B). The tubes were then brought to the lab for dissection within 6 hours. Each 3cm umbilical cord segment was dissected longitudinally utilizing aseptic technique. The tissue was carefully undermined and the umbilical vein and both umbilical arteries were removed. The remaining segment was sutured inside out and incubated in 25 ml of PBS, 1. times, antibiotic, and 1 mg / ml of collagenase at room temperature. After 16-18 hours the remaining suture and connective tissue was removed and discarded. The cell suspension was separated equally into two tubes, the cells were washed 3 times by diluting with PBS to yield a final volume of 50 ml per tube, and then centrifuged. Red blood cells were then lysed using a hypotonic solution. Cells were plated onto 6-well plates at a concentration of 5-20. times.10E6 cells per well. UC-MSC were cultured in low-glucose DMEM (Gibco) with 10% FBS (Hyclone), 2 mM L-Glutamine (Gibco), 100 U / ml penicillin, 100 ug / ml streptomycin, 0.025 ug / ml amphotericin B (Gibco). Cells were washed 48 hours after the initial plating with PBS and given fresh media. Cell culture media were subsequently changed twice a week through half media changes. After 7 days or approximately 70-80% confluence, cells were passed using HyQTase (Hyclone) into a 10 cm plate. Cells were then regularly passed 1 :2 every 7 days or upon reaching 80% confluence. Alternatively, 0.25% HQ trypsin / EDTA (Hyclone) was used to passage cells in a similar manner.

[0150] In some embodiments of the disclosure, administration of cells of the disclosure is performed for suppression of an inflammatory and / or autoimmune disease. In these situations, it may be necessary to utilize an immune suppressive / or therapeutic adjuvant. Immune suppressants are known in the art and can be selected from a group comprising of: cyclosporine, rapamycin, campath- 1H, ATG, Prograf, anti IL-2r, MMF, FTY, LEA, cyclosporin A, diftitox, denileukin, levamisole, azathioprine, brequinar, gusperimus, 6- mercaptopurine, mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, and trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6- mercaptopurine, thiamiprine, and thiaguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, and tegafur) fluocinolone, triaminolone, anecortave acetate, fluoromethoIone, medrysone, prednislone, etc. In another embodiment, the use of stem cell conditioned media may be used to potentiate an existing anti-inflammatory agent. Anti-inflammatory agents may comprise one or more agents including NSAIDs, interleukin-1 antagonists, dihydroorotate synthase inhibitors, p38 MAPkinase inhibitors, TNF-a inhibitors, TNF-a sequestration agents, and methotrexate. More specifically, anti-inflammatory agents may comprise one or more of, e.g., anti-TNF-a, lysophylline, alpha 1-antitrypsin (AAT), interleukin- 10 (IL-10), pentoxyfilline, COX-2 inhibitors, 21 -acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluoromethoIone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (eg., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetyl salicylate,diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1- naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), epsilon. -acetamidocaproic acid, s- adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, a- bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, zileuton, candelilla wax, alpha bisabolol, aloe vera, Manjistha, Guggal, kola extract, chamomile, sea whip extract, glycyrrhetic acid, glycyrrhizic acid, oil soluble licorice extract, monoammonium glycyrrhizinate, monopotassium glycyrrhizinate, dipotassium glycyrrhizinate, 1-beta-glycyrrhetic acid, stearyl glycyrrhetinate, and 3-stearyloxy- glycyrrhetinic acid.

[0151] Envisioned within the current disclosure is admixing of regenerative factors disclosed in the disclosure with existing creams and ointments that address underlying skin problems. Incorporated by reference are U.S. Pat. Nos. 5,958,397, 5,922,331, 5,817,621, 5,658,580, 5,362,488, 5,322,685, 5,254,331, 4,760,096, 4,297,374, 4,268,526, 4,087,555, and 4,007,266, which all describe the formulation of skin creams which address specific aspects related to skin care. For example, U.S. Pat. No. 5,817,621 describes a skin cream comprising a lipid ointment, vitamin A, a salicylic acid, D-camphor, a biogenic GABAergic substance, a dopaminergic substance, M- cholionolyics, pancreatin, ascorbic acid, pantothenic acid calcium salt, and vitamin D.sub.2 as a means to cause a high trophoprotective effect followed by a restoration of skin physiological functions. Other useful patents for practice of the disclosure include teaches of U.S. Pat. No. 7,608,642 describes pharmaceutical compositions and methods for managing wound and skin care, in particular methods and compositions that employ compounds that can promote skin cell renewal, wound healing, proliferation of fibroblasts and / or keratinocytes, and the production of collagen.

[0152] In some embodiments, the conditions allowing for production of an at least one regenerative factor includes exposing the at least one cell to an agent that resembles cellular injury and / or cellular damage. In some embodiments, the agent that resembles cellular injury and / or cellular damage is a toll-like receptor agonist. In some embodiments, the said toll-likereceptor agonist is selected from a group comprising of: Lipopolysaccharide (LPS), Pam3CSK4, lipoteichoic acid (LTA), zymosan, flagellin, polyinosinic:poly cytidylic acid (Poly(LC)), imiquimod, resiquimod (R848), loxoribine, gardiquimod, CL075, CL097, isatoribine (ANA-245), vesatolimod (GS-9620), monophosphoryl lipid A (MPL), lipid IVa, eritoran (primarily an antagonist but with agonist-like effects in some contexts), CpG oligodeoxynucleotides (CpG-ODN), SM360320, CU-T12-9, GS-9688, R07020531, JNJ- 4964, CBLB612, ISA-201, OPN-305 (primarily antagonist but with agonist potential in trials), TMX-101, DSP-0509, IMO-2055 (Amplivax), IC-31, E-6020, PF-4878691, RO6870868, RO6864018, high mobility group box 1 (HMGB1), peptidoglycan, bacterial DNA, fibrinogen, fibronectin, extracellular matrix proteins, 852A, VTX-2337, Bacillus Calmette-Guerin (BCG), Poly ICLC, synthetic imidazoquinoline, small-molecule selective TLR8 agonist, mannosecapped lipoarabinomannan (ManLAM), phenol-soluble modulin, inulin acetate, retrocyclin, CL429, CpG-ODN: AG-OVA, P-glucans (general), Sparassis crispa P-glucan, Phellinus linteus P-glucan, Platycodon grandiflorum P-glucan, Cordyceps militaris P-glucan, Angelica gigas Nakai P-glucan, Saccharomyces cerevisiae P-glucan, Laminarin (P-glucan), Curdlan (P- glucan), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shock protein 22 (HSP22), heat shock protein gp96.

[0153] Some embodiments relate to a composition related to a composition comprising a conditioned medium comprising endogenous progenitor / stem cells. In some embodiments, the conditioned medium is a nutrient-rich broth or medium that stem cells can be or were grown in. In some embodiments, the conditioned medium may further include at least one signaling protein that instruct the stem cells. In some embodiments, the at least one signaling protein may instruct the subject’s skin cells to produce more collagen, repair damage, or reduce inflammation. In some embodiments, the composition may further include at least one exosome. In some embodiments, the exosomes may be found within the conditioned medium. In some embodiments, the stem cells are stimulated to produce exosomes by CpG and valproic acid. In some embodiments, the stem cells are umbilical cord blood mesenchymal stem cells. In some embodiments, the CpG is synthetic oligodeoxynucleotide with the sequence 5'-TCCATGACGTTCCTGATGCT-3', phosphorothioate-modified for stability. In some embodiments, the composition further comprises a toll-like receptor agonist or stem cells treated with a toll-like receptor agonist. In some embodiments, the composition furthercomprises Pam3CSK4 or stem cells treated with Pam3CSK4. In some embodiments, the composition further comprises Poly(I:C) or stem cells treated with Poly(I:C). In some embodiments, the composition further comprises LPS or stem cells treated with LPS. In some embodiments, the composition further comprises Flagellin or stem cells treated with Flagellin. In some embodiments, the composition further comprises FSL-1 or stem cells treated with FSL-1. In some embodiments, the composition further comprises Imiquimod or stem cells treated with Imiquimod. In some embodiments, the composition further comprises ssRNA40 or stem cells treated with ssRNA40. In some embodiments, the composition further comprises Zymosan or stem cells treated with Zymosan. In some embodiments, the composition further comprises Gardiquimod or stem cells treated with Gardiquimod. In some embodiments, the composition comprises a toll-like receptor agonist and valproic acid or stem cells treated with a toll-like receptor agonist and valproic acid. In some embodiments, the composition further comprises a lithium source, such as, but not limited to LiCl, or stem cells treated with LiCl. In some embodiments, the composition is treated with an exogenous environmental element or the stem cells were previously treated to an exogenous environmental element. In some embodiments, the composition further comprises stem cells treated with hyperthermia. In some embodiments, the composition further comprises chorionic gonadotropin or stem cells treated with chorionic gonadotropin. In some embodiments, the composition further comprises a dermatological carrier. In some embodiments, the composition is formulated as a topical. In some embodiments, the composition is formulated as an injectable.

[0154] Some embodiments relate to a method for enhancing exosome production. In some embodiments, the method includes treating a population of mesenchymal stem cells with an effective amount of a CpG and valproic acid. In some embodiments, the CpG is ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT-3'. In some embodiments, the method further includes culturing a population of mesenchymal stem cells in a culture medium. In some embodiments, the method further comprises isolating exosomes from the culture medium. In some embodiments, the exosomes comprise a molecular cargo that stimulates the proliferation of the exosomes from the mesenchymal stem cells. In some embodiments, the composition includes a conditioned medium comprising endogenous progenitor / stem cells. In some embodiments, the endogenous progenitor / stem cells are stimulated with at least one regenerative factor. In some embodiments, the composition furthercomprises a dermatologically acceptable carrier. In some embodiments, the endogenous progenitor / stem cells are mesenchymal stem cells, n some embodiments, the at least one regenerative factor is selected from the group consisting of at least one toll-like receptor agonist, valproic acid (VP A), human chorionic gonadotropin, ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT-3', LiCl or a combination thereof. In some embodiments, the toll like receptor agonist is selected from a group consisting of Lipopolysaccharide (LPS), Pam3CSK4, lipoteichoic acid (LTA), zymosan, flagellin, polyinosinic:polycytidylic acid (Poly(LC)), imiquimod, resiquimod (R848), loxoribine, gardiquimod, CL075, CL097, isatoribine (ANA-245), vesatolimod (GS-9620), monophosphoryl lipid A (MPL), lipid IVa, eritoran (primarily an antagonist but with agonistlike effects in some contexts), CpG oligodeoxynucleotides (CpG-ODN), SM360320, CU-T12- 9, GS-9688, R07020531, JNJ-4964, CBLB612, ISA-201, OPN-305 (primarily antagonist but with agonist potential in trials), TMX-101, DSP-0509, IMO-2055 (Amplivax), IC-31, E-6020, PF-4878691, RO6870868, RO6864018, high mobility group box 1 (HMGB1), peptidoglycan, bacterial DNA, fibrinogen, fibronectin, extracellular matrix proteins, 852A, VTX-2337, Bacillus Calmette-Guerin (BCG), Poly ICLC, synthetic imidazoquinoline, small-molecule selective TLR8 agonist, mannose-capped lipoarabinomannan (ManLAM), phenol-soluble modulin, inulin acetate, retrocyclin, CL429, CpG-ODN: AG-OVA, ^-glucans (general), Sparassis crispa P-glucan, Phellinus linteus P-glucan, Platycodon grandiflorum P-glucan, Cordyceps militaris P-glucan, Angelica gigas Nakai P-glucan, Saccharomyces cerevisiae P- glucan, Laminarin (P-glucan), Curdlan (P-glucan), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shock protein 22 (HSP22), heat shock protein gp96, or a combination thereof. In some embodiments, the endogenous progenitor / stem cells expresses CD73 and TLR9. In some embodiments, the endogenous progenitor / stem cells resemble cellular injury or cell damage by a toll-like receptor agonist. In some embodiments, the composition is a topical anti-inflammatory composition. In some embodiments, the composition includes an activator of endogenous progenitor / stem cells. In some embodiments, the composition further includes an angiogenic factor, a neurogenic factor, an antiapoptotic factors, an immune modulatory factors, or a combination thereof. In some embodiments, the composition further includes at least one of: platelet rich plasma, human chorionic gonadotropin, one or more antioxidants, one or more antiseptic agents, one or more anti-inflammatory agents, one or more delivery vehicles, one or more known composition agents, or any combination thereof. In some embodiments, the one or more delivery vehicle is selected from the group consisting of phospholipids, palmityl myristate, DMSO, a polymer, liposomes, Trojan peptides, chariot peptides, small elastic vesicles, microspheres, chitosan suspensions, a matrix, nanoparticle, or a combination thereof. In some embodiments, the one or more known composition agent is selected from the group consisting of beta-glucan, propylene glycol, butylene glycol, polyethylene glycol, olive oil, dimethyl isosorbide, dimethylformamide, methyl salicylate, long chain oleic acids, lactic acid, senolytic agent, senomorphic agent, or a combination thereof. In some embodiments, the one or more known composition agent is selected from BCL2, BCL2-XL, survivin, or any combination thereof. In some embodiments, the angiogenic factor is selected from the group consisting of VEGF, HGF-1, FGF-1, FGF-2, angiopoietin, interleukin-20, or any combination thereof. In some embodiments, the neurogenic factor is selected from the group consisting of NGF, BDNF, CNTF, neurotrophins, or any combination thereof. In some embodiments, the antiapoptotic factor is selected from the group consisting of molecules capable of increasing bcl-2 expression, molecules capable of decreasing BAD expression, molecules capable of increasing expression of bcl-2Xl, molecules capable of decreasing bcl-2Xs, molecules capable of decreasing expression of members of the caspase family, molecules capable of increasing surviving expression, molecules capable of increasing living expression; or any combination thereof.

[0155] Some embodiments relate to a method for stimulating angiogenesis. In some embodiments, the method includes administering to a subject or target tissue with a composition as described herein. In some embodiments, stimulating angiogenesis promotes vascular repair. In some embodiments, stimulating angiogenesis is stimulated by umbilical cord mesenchymal stem cell release. In some embodiments, mesenchymal stem cells are digested using, for example, collagenase and dispase. In some embodiments, mesenchymal stem cells are stimulated to generate exosomes. In some embodiments, the mesenchymal stem cells are stimulated with, but not limited to, valproic acid, CpG (e g., ODN 2006 CpG), a tolllike receptor agonist, or a combination thereof. In some embodiments, the composition for stimulating angiogenesis includes valproic acid and ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT-3'.

[0156] Some embodiments relate to a method for enhancing biological activity of a fibroblast. In some embodiments, the method includes contacting the fibroblast with an exosome isolated from a mesenchymal stem cell, wherein the mesenchymal stem cell was treated with a composition as described herein. In some embodiments, the biological activity is fibroblast proliferation. In some embodiments, the biological activity is collagen production. In some embodiments, the contacting step results in a synergistic enhancement of the biological activity. In some embodiments, the composition comprises valproic acid and ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT-3'.

[0157] Some embodiments relate to a method for promoting wound healing or tissue repair. In some embodiments, the method includes administering to a target site a composition as described herein. In some embodiments, the method includes providing exosomes isolated from mesenchymal stem cells treated with valproic acid and ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT-3'.

[0158] Some embodiments relate to a method for enhancing a biological activity of a fibroblast. In some embodiments, the method includes contacting the fibroblast with a composition as described herein. In some embodiments, the method includes contacting the fibroblast with an exosome isolated from mesenchymal stem cells, wherein the mesenchymal stem cells were treated with a combination of hyperthermia and ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT-3'. In some embodiments, the biological activity is fibroblast proliferation or collagen production.

[0159] Some embodiments relate to a method for modulating monocyte cytokine production. In some embodiments, the method includes administering a composition as described herein. In some embodiments, the method includes activating a population of adipose derived mesenchymal stem cells ex vivo with a toll-like receptor agonist, and contacting a population of monocytes with the activated adipose derived mesenchymal stem cells or a secretion product thereof. In some embodiments, the modulation of monocyte cytokine production enhances an anti-inflammatory or regenerative response.

[0160] Some embodiments relate to a method for enhancing cytokine production in cells. In some embodiments, the method includes administering a composition as described herein. In some embodiments, the cells are mesenchymal stem cells. In some embodiments, the method includes contacting mesenchymal stem cells with a composition as describedherein. In some embodiments, the composition comprises valproic acid, and gonadotropin, and the mesenchymal stem cells are further exposed to hyperthermia.

[0161] Some embodiments relate to a method for synergistically enhancing cytokine production cells. In some embodiments, the cells are mesenchymal stem cells. In some embodiments, the method includes providing a composition as described herein. In some embodiments, the method includes contacting the mesenchymal stem cells with a toll-life receptor agonist and LiCl.

[0162] Some embodiments relate to a method for suppressing cytokine production in cells. In some embodiments, the cells include mesenchymal stem cells. In some embodiments, the method includes contacting the mesenchymal stem cells with LiCl in the absence of a toll-life receptor agonist.

[0163] Some embodiments relate to a method for synergistically amplifying cytokine production in mesenchymal stem cells. In some embodiments, the method includes contacting the mesenchymal stem cells with a composition as described herein. In some embodiments, the composition comprises a toll-life receptor agonist and the mesenchymal stem cells are further exposed to hyperthermia.

[0164] Adipose stem cells possess numerous therapeutic proteins, however, because they are derived from adult tissue, the anti-aging properties are limited. Accordingly, in one embodiment of the disclosure the potency of factors secreted from adipose stem cells and combination of adipose stem cells with other cells is increased by pretreatment with therapeutic agents in vitro, prior to isolation and purification of the supernatant. In one specific embodiment valproic acid is added to stimulate production of growth factors by adipose stem cells or cultures of adipose stem cells containing other cells. Means of utilizing valproic acid for stimulation of regenerative properties are known in the literature and one skilled in the art is referred to the following publications for reference [133, 142-151], Exemplary means of utilizing valproic acid for tissue culture of adipose derived MSC within the practice of the disclosure includes culture for a period of approximately 48 hours at concentrations of approximately 2.5 mmol / L. The growth factors produced from the cells, combined with the anti-aging factors and subsequently demonstrated to increase collagen synthesis in vitro from fibroblasts, as well as to possess antiaging properties.

[0165] Aspects of the present disclosure are further detailed in the following enumerated alternatives:

[0166] 1. A composition useful for enhancing skin appearance obtained by the steps of: a) extracting one or more cellular populations a tissue possessing regenerative properties; b) exposing said cells to conditions allowing for production of regenerative factors; c) extracting said regenerative factors; d) concentrating said regenerative factors; and e) admixing said regenerative factors in a solution useful for topical application.

[0167] 2 The composition of alternative 1, wherein said cellular population is comprised of cells selected from a group comprising any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of: a) monocytes; b) T regulatory cells; c) mesenchymal stem cells; d) endothelial progenitor cells; e) hematopoietic stem cells; f) VSEL; g) c-kit expressing mesenchymal stem cells; h) CD34 expressing mesenchymal stem cells; i) IL-3 receptor expressing mesenchymal stem cells; j) HGF receptor expressing mesenchymal stem cells and; k) M2 monocytes

[0168] 3. The composition of alternative 2, wherein said cellular populations are expanded ex vivo.

[0169] 4 The composition of alternative 2, wherein said cellular populations are derived from tissues selected from a group comprising any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of: a) adipose; b) nail cuticle; c) deciduous tooth; d) hair follicle; e) skin; f) bone marrow; g) placenta; h) umbilical cord blood; i) mobilized peripheral blood; j) peripheral blood; k) urine; 1) breast milk; m) menstrual blood; and n) fallopian tube.

[0170] 5 The composition of alternative 4, wherein said stromal vascular fraction is obtained by the steps of: a) infiltrating an area of the body containing adipose tissue with a solution containing xylocaine and epinephrine; b) extracting adipose tissue in utilizing a cannula attached to a syringe; c) washing said extracted adipose tissue in a saline based solution; d) admixing an enzyme capable of digesting adipose tissue with said washed adipose tissue; e) allowing sufficient time for said enzyme capable of digesting adipose tissue to digest adipose tissue; and f) washing said digested adipose tissue so as to pellet the cellular portion of said adipose tissue while discarding remains of said adipose tissue.

[0171] 6 The composition of alternative 2, wherein said monocytes are isolated by means of plastic adherence.

[0172] 7 The composition of alternative 2, wherein said monocytes are isolated by means of Magnetic Activated Cell Sorting (MACS).

[0173] 8. The composition of alternative 7, wherein said selection of monocytes by MACS is achieved by use of antibody targeting CD14.

[0174] 9 The composition of alternative 2, wherein said T regulatory cells are isolated from said adipose tissue by means of MACS.

[0175] 10. The composition of alternative 9, wherein said selection of T regulatory cells by MACS is achieved by use of antibody targeting CD25.

[0176] 11 The composition of alternative 2, wherein said mesenchymal stem cells are isolated from adipose tissue by means of plastic adherence.

[0177] 12. The composition of alternative 2, wherein said mesenchymal stem cells are isolated from adipose tissue by means of growth in mesenchymal stem cell promoting media.

[0178] 13. The composition of alternative 2, wherein said mesenchymal stem cells are isolated from adipose tissue by means of MACS.

[0179] 14. The composition of alternative 2, wherein said mesenchymal stem cells are isolated from adipose tissue by means of MACS using antibody targeting STRO-1.

[0180] 15. The composition of alternative 1, wherein said regenerative factors are concentrated by lyophilization.

[0181] 16. The composition of alternative 1, wherein said regenerative factors are exosomes.

[0182] 17. The composition of alternative 16, wherein said exosomes are concentrated by an affinity means.

[0183] 18. The composition of alternative 17, wherein said affinity means is column chromatography.

[0184] 19. The composition of alternative 17, wherein said affinity means involves exposing conditioned media through a column containing agents with selective affinity to exosomes.

[0185] 20. The composition of alternative 19, wherein said agents with selective affinity to exosomes are selected from a group comprising of: a) a protein; b) an antibody; c) a DNano particle; d) a lectin; and e) an aptamer.

[0186] 21. The composition of alternative 19, wherein said lectin is selected from a group comprising of: a) Galanthus nivalis agglutinin (GNA); b) Narcissus pseudonarcissus agglutinin (NPA); c) cyanovirin; and d) Conconavalin A.

[0187] 22. The composition of alternative 16, wherein said exosomes possess the following characteristics: (a) have a size of between 50 nm and 100 nm as determined by electron microscopy; (b) comprises a complex of molecular weight>100 kDa, for example comprising proteins of <100 kDa; (c) comprises a complex of molecular weight>300 kDa, for example comprising proteins of <300 kDa; (d) comprises a complex of molecular weight>1000 kDa; (e) has a size of between 2 nm and 200 nm, such as a size of between 50 nm and 150 nm or a size of between 50 nm and 100 nm, for example as determined by filtration against a 0.2 mu.M filter and concentration against a membrane with a molecular weight cut-off of 10 kDa; or (f) a hydrodynamic radius of below 100 nm, such as between about 30 nm and about 70 nm, between about 40 nm and about 60 nm, such as between about 45 nm and about 55 nm, such as about 50 nm, for example as determined by laser diffraction or dynamic light scattering.

[0188] 23. The composition of alternative 1, wherein said cells derived from adipose tissue are exposed to conditions selected from a group comprising of: a) hypoxia; b) hyperthermia; c) hypotonic challenge; d) oxidative stress; and e) inflammatory stimuli.

[0189] 24. The composition of alternative 23, wherein said exposure to said conditions is performed to augment production of regenerative factors.

[0190] 25. The composition of alternative 1, wherein said composition is admixed with platelet rich plasma.

[0191] 26. The composition of alternative 1, wherein human chorionic gonadotropin is admixed with said composition.

[0192] 27. The composition of alternative 1, wherein one or more antioxidants are added to said composition.

[0193] 28. The composition of alternative 1, wherein one or more antiseptic agents are added to said composition.

[0194] 29. The composition of alternative 1, wherein one or more antiinflammatory agents are added to said composition.

[0195] 30. The composition of alternative 1, wherein one or more delivery vehicles are admixed with said composition.

[0196] 31 The composition of alternative 1, wherein said delivery vehicle is selected from a group comprising of: a) phospholipids; b) palmitylmyristyrates; c) DMSO; d) a polymer or chitosan suspensions or matrix; d) liposomes; e) Trojan peptides; f) chariot peptides; g) small elastic vesicles; and h) microspheres.

[0197] 32. The composition of alternative 31, wherein said microspheres are made from naturally derived materials selected from a group comprising of: a) collagen; b) glycosaminoglycans; c) chondroitin sulfate; and d) chitosan or polysaccharides.

[0198] 33. The composition of alternative 1, wherein said composition is administered together with a nanoparticle delivery vehicle capable of transferring the epidermis.

[0199] 34. The composition of alternative 1, wherein said composition is admixed with agents selected from a group comprising of: a) beta-glucan; b) propylene glycol; c) butylene glycol; d) polyethylene glycol; e) olive oil; f) dimethyl isosorbide; g) dimethylformamide; h) methyl salicylate; i) long chain oleic acids; and j) lactic acid.

[0200] 35. A method of producing a cosmetic product comprising: a) obtaining an adherent population of regenerative cells; b) stimulating said adherent population of regenerative cells with one or more toll like receptors; c) allowing said adherent population of regenerative cells to produce growth factors into a tissue culture media; d) collecting said tissue culture media in the form of a cell free solution; e) optionally concentrating cell secreted factors from said culture media; and f) utilizing said growth factors in the form of a cosmetic or cosmetic ingredient.

[0201] 36. The method of alternative 35, wherein said cosmetic product is admixed with one or more senolytic agents.

[0202] 37. The method of alternative 35, wherein said cosmetic product is admixed with one or more senomorphic agents.

[0203] 38. The method of alternative 36, wherein said senolytic agent is an inhibitor of BCL2.

[0204] 39. The method of alternative 36, wherein said senolytic agent is an inhibitor of BCL2-XL.

[0205] 40. The method of alternative 36, wherein said senolytic agent is an inhibitor of survivin.

[0206] 41. The method of alternative 35, wherein said adherent regenerative cells are mesenchymal stem cells.

[0207] 42. The method of alternative 41, wherein said mesenchymal stem cells are capable of generating immune regulatory T cells from naive T cells.

[0208] 43. The method of alternative 42, wherein said immune regulatory T cells express CD4 or CD8 or a combination.

[0209] 44. The method of alternative 43, wherein said immune regulatory T cells are T regulatory cells.

[0210] 45. The method of alternative 44, wherein said T regulatory cells possess ability to suppress a conventional T cell in an antigen specific manner.

[0211] 46. The method of alternative 44, wherein said T regulatory cells possess ability to suppress a conventional T cell in an antigen non-specific manner.

[0212] 47. The method of alternative 44, wherein said T regulatory cells possess ability to suppress a conventional T cell in a contact dependent manner.

[0213] 48. The method of alternative 44, wherein said T regulatory cells possess ability to suppress a conventional T cell in a contact independent manner.

[0214] 49. The method of alternative 45-48, wherein said T regulatory cells areCD4 positive and CD25 positive.

[0215] 50. The method of alternative 45-48, wherein said conventional T cells areCD4 positive and CD25 negative.

[0216] 51. The method of alternative 45-48, wherein said T regulatory cells areCD4 positive and CTLA-4 positive.

[0217] 52. The method of alternative 45-48, wherein said conventional T cells areCD4 positive and CTLA- 4 negative.

[0218] 53. The method of alternative 45-48, wherein said T regulatory cells areCD4 positive and GITR positive.

[0219] 54. The method of alternative 45-48, wherein said conventional T cells areCD4 positive and GITR negative.

[0220] 55. The method of alternative 45-48, wherein said T regulatory cells areCD4 positive and CD39 and / or CD73 positive.

[0221] 56. The method of alternative 45-48, wherein said conventional T cells areCD4 positive and CD39 and / or CD73 negative.

[0222] 57. The method of alternative 45-48, wherein said T regulatory cells areCD4 positive and IL-7 receptor negative.

[0223] 58. The method of alternative 45-48, wherein said conventional T cells areCD4 positive and IL-7 receptor positive.

[0224] 59. The method of alternative 44, wherein said T regulatory cells are capable of inhibiting maturation of dendritic cells in response to ligation of a “danger signal”.

[0225] 60. The method of alternative 59, wherein said “danger signal” is activation of a toll like receptor (TLR).

[0226] 61. The method of alternative 60, wherein said TLR is TRL-4.

[0227] 62. The method of alternative 60, wherein said activation of dendritic cell maturation endows said dendritic cell ability to induce proliferation of naive T cells.

[0228] 63. The method of alternative 60, wherein said activation of dendritic cell maturation endows said dendritic cell ability to induce cytokine secretion of naive T cells.

[0229] 64. The method of alternative 60, wherein said activation of dendritic cell maturation endows said dendritic cell ability to induce cytotoxic activity to naive T cells.

[0230] 65. The method of alternative 60, wherein said activation of dendritic cell maturation endows said dendritic cell ability to induce differentiation of naive CD4 T cells into a helper phenotype.

[0231] 66. The method of alternative 65, wherein said helper phenotype is T helper1, characterized by expression of interferon gamma, interleukin 2, interleukin 7, interleukin 12, interleukin 15, interleukin 18, and interleukin 23.

[0232] 67. The method of alternative 66, wherein said Thl cells is characterized by expression of STAT4.

[0233] 68. The method of alternative 65, wherein said helper phenotype is T helper2, characterized by expression of IL-4, IL-5, IL-13, and IL-10.

[0234] 69. The method of alternative 68, wherein said T helper 2 cell is characterized by expression of STAT6.

[0235] 70. The method of alternative 65, wherein said T helper cell is a Th9 cell.

[0236] 71 . The method of alternative 70, wherein said Th9 cell is characterized by expression of the FOXO1 transcription factor.

[0237] 72. The method of alternative 70, wherein said Th9 cell is characterized by expression of interleukin- 9

[0238] 73. The method of alternative 65, wherein said T helper cell is a Thl7 cell.

[0239] 74. The method of alternative 73, wherein said Thl7 cell produces cytokines selected from a group comprising of: a) IL-17; b) IL-17A); c) IL-17F, and d) IL-6.

[0240] 75. The method of alternative 73, wherein said Thl7 cell expresses BATF.

[0241] 76. The method of alternative 73, wherein said Th 17 cell expressesRORgamma.

[0242] 77. A cosmetic composition capable of inhibiting antigen presenting cell function comprised of culture supernatant from a mesenchymal stem cell.

[0243] 78. The composition of alternative 77, wherein said antigen presenting cell is a B cell.

[0244] 79. The composition of alternative 78, wherein said B cell is a CD5 positiveB cell.

[0245] 80. The composition of alternative 77, wherein said antigen presenting cell is an endothelial cell.

[0246] 81. The composition of alternative 77, wherein said endothelial cell is activated with interferon gamma.

[0247] 82. The composition of alternative 77, wherein said antigen presenting cell is an epithelial cell.

[0248] 83. The composition of alternative 77, wherein said antigen presenting cell is activated with interferon gamma.

[0249] 84. The composition of alternative 77, wherein said antigen presenting cell is a monocyte.

[0250] 85. The composition of alternative 77, wherein said antigen presenting cell is a macrophage.

[0251] 86. The composition of alternative 85, wherein said antigen macrophage is an Ml macrophage.

[0252] 87. The composition of alternative 85, wherein said antigen macrophage is an M2 macrophage.

[0253] 88. The composition of alternative 86, wherein said Ml macrophage expresses markers selected from a group comprising of: CD80, CD86, CD64, CD 16 and CD32.

[0254] 89. The composition of alternative 86, wherein said M2 macrophage expresses markers selected from a group comprising of: CD68. CD163, and CD206.

[0255] 90. The composition of alternative 77, wherein said antigen presenting cell is a dendritic cell.

[0256] 91. The composition of alternative 90, wherein said dendritic cell is a myeloid dendritic cell.

[0257] 92. The composition of alternative 90, wherein said dendritic cell is a lymphoid dendritic cell.

[0258] 93. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of MHC I expression.

[0259] 94. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of MHC II expression.

[0260] 95. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of peptide generation from proteins inside said antigen presenting cell.

[0261] 96. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of peptide loading into MHC I.

[0262] 97. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of peptide loading into MHC II.

[0263] 98. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of T cell stimulatory cytokine production by said antigen presenting cell.

[0264] 99. The composition of alternative 98, wherein said T cell stimulatory cytokine is interleukin-2.

[0265] 100. The composition of alternative 98, wherein said T cell stimulatory cytokine is interleukin-7.

[0266] 101 The composition of alternative 98, wherein said T cell stimulatory cytokine is interleukin-12.

[0267] 102. The composition of alternative 98, wherein said T cell stimulatory cytokine is interleukin-15.

[0268] 103. The composition of alternative 98, wherein said T cell stimulatory cytokine is interleukin- 17.

[0269] 104. The composition of alternative 98, wherein said T cell stimulatory cytokine is interleukin-18.

[0270] 105. The composition of alternative 77, wherein said inhibition antigen presenting cell function is associated with suppression of costimulatory molecule expression.

[0271] 106. The composition of alternative 105, wherein said costimulatory molecule expression is CD40.

[0272] 107. The composition of alternative 105, wherein said costimulatory molecule expression is ICOS ligand.

[0273] 108. The composition of alternative 105, wherein said costimulatory molecule expression is CD80.

[0274] 109. The composition of alternative 105, wherein said costimulatory molecule expression is CD86.

[0275] 110. The composition of alternative 105, wherein said costimulatory molecule expression is CD 137.

[0276] 111. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of ability of said antigen presenting cell to form an immunological synapse with the T cell.

[0277] 112. The composition of alternative 77, wherein said inhibition of antigen presenting cell function is associated with suppression of ability of said antigen presenting cell to produce exosomes.

[0278] 113. The composition of alternative 112, wherein said exosomes possess ability to activate a T cell.

[0279] 114. The composition of alternative 112, wherein said exosomes possess MHC I.

[0280] 1 15 The composition of alternative 112, wherein said exosomes possess MHC II.

[0281] 116. The composition of alternative 112, wherein said exosomes possess CD40.

[0282] 117. The composition of alternative 112, wherein said exosomes possess ICOS ligand.

[0283] 118. The composition of alternative 112, wherein said exosomes possess CD80.

[0284] 119. The composition of alternative 112, wherein said exosomes possess CD86.

[0285] 120. The composition of alternative 112, wherein said exosomes possess ability to induce antigen cross presentation.

[0286] 121. A cosmetic composition capable of inducing production of antiinflammatory cytokine levels which are sufficient to induce a regulatory T cell immunophenotype .

[0287] 122. The composition of alternative 121, wherein anti-inflammatory cytokine levels are sufficient to inhibit production of inflammatory cytokine by stimulated T cells by at least 20% relative to baseline.

[0288] 123. The composition of alternative 122, wherein said inflammatory cytokines are selected from IFN- gamma, IL-17A, IL-l-beta, IL-6, IL-33, HMGB1 and TNF- alpha.

[0289] 124. The composition of alternative 122, wherein said T cells are selected from CD8E+ T cells, CD4E+ T cells, gamma-delta T cells, and T regulatory cells.

[0290] 125. A cosmetic composition comprising conditioned media of a cell that is engineered to produce at least three anti-inflammatory cytokines at levels sufficient to inhibit an inflammatory response by at least 20% relative to a control.

[0291] 126. The composition of alternative 125, wherein said cell is engineered to express a homing molecule.

[0292] 127. The composition of alternative 126, wherein said engineering is performed by alteration of culture conditions.

[0293] 128. The composition of alternative 127, wherein said culture conditions comprises exposure to hypoxia.

[0294] 129. The composition of alternative 128, wherein said hypoxia is sufficient to induce translation of hypoxia inducible factor (HIF)-l.

[0295] 130. The composition of alternative 128, wherein said hypoxia consists of culture cell in 1-19% oxygen.

[0296] 131. The composition of alternative 127, wherein said alteration in culture condition is exposure to hyperthermia.

[0297] 132. The composition of alternative 127, wherein said alteration in culture condition is exposure to acidic conditions.

[0298] 133. The composition of alternative 127, wherein said alteration in culture condition is exposure to hypotonic conditions.

[0299] 134. The composition of alternative 126, wherein said homing molecule is anti-integrin alpha4,beta7.

[0300] 135 The composition of alternative 126, wherein said homing molecule is MAdCAM.

[0301] 136 The composition of alternative 126, wherein said homing molecule is CCR9.

[0302] 137. The composition of alternative 126, wherein said homing molecule is CXCR4.

[0303] 138. The composition of alternative 126, wherein said homing molecule is CXCR7.

[0304] 139 The composition of alternative 126, wherein said homing molecule is CCR2.

[0305] 140 The composition of alternative 126, wherein said homing molecule is GPR15.

[0306] 141. A cosmetic composition comprising conditioned media ofWharton jelly mononuclear cells re-programmed to possess a state of enhanced immaturity as compared to naturally residing Wharton jelly mononuclear cells.

[0307] 142. The composition of alternative 141, wherein said enhanced immaturity is increased differentiation efficacy.

[0308] 143. The composition of alternative 142, wherein said differentiation efficacy means ability to differentiate into other tissues and / or at a greater percentage of cells differentiating.

[0309] 144. The composition of alternative 143, wherein tissues in which said cells are capable of differentiating into are endodermal, ectodermal and mesodermal derived tissues.

[0310] 145. The composition of alternative 141, wherein said enhanced immaturity is increased ability to produce therapeutic factors.

[0311] 146. The composition of alternative 145, wherein said therapeutic factors are selected from: a) angiogenic factors; b) neurogenic factors; c) antiapoptotic factors and d) immune modulatory factors.

[0312] 147. The composition of alternative 146, wherein said angiogenic factors are selected from a group comprising of: a) VEGF; b) HGF-1; c) FGF-1; d) FGF-2; e) angiopoietin; and f) interleukin-20.

[0313] 148. The composition of alternative 146, wherein said neurogenic factors are selected from a group comprising of: a) NGF; b) BDNF; c) CNTF; and d) neurotrophin.

[0314] 149. The composition of alternative 146, wherein said antiapoptotic factors are selected from a group comprising of: a) molecules capable of increasing bcl-2 expression; b) molecules capable of decreasing BAD expression; c) molecules capable of increasing expression of bcl-2Xl; d) molecules capable of decreasing bcl-2Xs; e) molecules capable of decreasing expression of members of the caspase family; f) molecules capable of increasing survivin expression; and g) molecules capable of increasing livin expression.

[0315] 150. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting T cell activation.

[0316] 151. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting NK cell activation.

[0317] 152. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting NKT cell activation.

[0318] 153. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting gamma delta cell activation.

[0319] 154. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting dendritic cell maturation.

[0320] 155. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting complement activation.

[0321] 156. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of accelerating neutrophil apoptosis.

[0322] 157. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting mast cell activation.

[0323] 158. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting eosinophil activation.

[0324] 159. The composition of alternative 146 wherein said immunomodulatory factors are factors capable of inhibiting basophil activation.

[0325] 160. The composition of alternative 141, wherein said enhanced immaturity is obtained by culture with an agent associated with embryonic microenvironment.

[0326] 161. The composition of alternative 160, wherein said agent is leukemia inhibitory factor.

[0327] 162. The composition of alternative 161, wherein said leukemia inhibitory factor is administered to cells that have been synchronized in cell cycle.

[0328] 163. The composition of alternative 162, wherein said synchronization of cells in cell cycle is achieved by treatment with a mitotic inhibitor.

[0329] 164. The composition of alternative 163, wherein said mitotic inhibitor is mitomycin C.

[0330] 165. The composition of alternative 163, wherein synchronization is achieved by serum deprivation.

[0331] 166. The composition of alternative 161, wherein said leukemia inhibitory factor is administered at a concentration of 1 pg / ml to 100 ng / ml.

[0332] 167. The composition of alternative 161, wherein said leukemia inhibitory factor is administered at a concentration of 10 pg / ml to 10 ng / ml.

[0333] 168. The composition of alternative 161, wherein said leukemia inhibitory factor is administered at a concentration of 100 pg / ml to 5 ng / ml.

[0334] 169. The composition of alternative 161, wherein said leukemia inhibitory factor is administered together with a histone deacetylase inhibitor.

[0335] 170. The composition of alternative 169, wherein said histone deacetylase inhibitor is valproic acid.

[0336] 171. The composition of alternative 170, wherein said valproic acid is administered at a concentration of 1 pg / ml to 1 mg / ml.

[0337] 172. The composition of alternative 170, wherein said valproic acid is administered at a concentration of 100 pg / ml to 100 ng / ml.

[0338] 173. The composition of alternative 170, wherein said valproic acid is administered at a concentration of 1 ng / ml to 100 ng / ml.

[0339] 174. The composition of alternative 169, wherein said histone deacetylase inhibitor is vorinostat.

[0340] 175. The composition of alternative 169, wherein said histone deacetylase inhibitor is belinostat.

[0341] 176 The composition of alternative 169, wherein said histone deacetylase inhibitor is LAQ824.

[0342] 177. The composition of alternative 169, wherein said histone deacetylase inhibitor is trichostatin A.

[0343] 178. The composition of alternative 169, wherein said histone deacetylase inhibitor is Panobinostat.

[0344] 179. The composition of alternative 169, wherein said histone deacetylase inhibitor is entinostat.

[0345] 180. The composition of alternative 169, wherein said histone deacetylase inhibitor is CI994.

[0346] 181 The composition of alternative 169, wherein said histone deacetylase inhibitor is mocetinostat.

[0347] 182. The composition of alternative 169, wherein said histone deacetylase inhibitor is trapoxin B.

[0348] 183. The composition of alternative 169, wherein said histone deacetylase inhibitor is phenylbutyrate.

[0349] 184. The composition of alternative 169, wherein a DNA methyltransferase inhibitor is added to said combination leukemia inhibitory factor and said DNA histone deacetylase inhibitor.

[0350] 185. The composition of alternative 184, wherein said DNA methyltransferase inhibitory is a nucleic acid derivative.

[0351] 186. The composition of alternative 184, wherein said DNA methyltransferase inhibitory is decitabine.

[0352] 187. The composition of alternative 184, wherein said DNA methyltransferase inhibitory is 5- azacytabine.

[0353] 188. The composition of alternative 141, wherein said reprogramming is induced through suppression of the enzyme GSK-3.

[0354] 189. The composition of alternative 188, wherein said suppression of said enzyme GSK-3 is accomplished by treatment with lithium.

[0355] 190. The composition of alternative 189, wherein said lithium is administered at a concentration of 1 pg / ml to 1 mg / ml.

[0356] 191. The composition of alternative 189, wherein said lithium is administered at a concentration of 10 pg / ml to 100 ng / ml.

[0357] 192. The composition of alternative 189, wherein said lithium is administered at a concentration of 100 pg / ml to 10 ng / ml.

[0358] 193. The composition of alternative 141, wherein said reprogramming is induced through induction of expression of the gene OCT3 / 4.

[0359] 194. The composition of alternative 141, wherein said reprogramming is induced through induction of expression of the gene SOX2.

[0360] 195. The composition of alternative 141, wherein said reprogramming is induced through induction of expression of the gene KLF4.

[0361] 196. The composition of alternative 141, wherein said reprogramming is induced through induction of expression of the gene L-MYC.

[0362] 197. The composition of alternative 141, wherein said reprogramming is induced through induction of expression of the gene LIN28.

[0363] 198. The composition of alternative 141, wherein said reprogramming is induced through induction of expression of the gene BCL-xL.

[0364] 199. The composition of alternative 141 , wherein said reprogramming is induced through induction of expression of the gene BCL-xL.

[0365] 200. The composition of alternative 141, wherein said reprogramming is induced through inhibition of p53 expression.

[0366] 201. The composition of alternative 200, wherein p53 expression is suppression of p53 activity.

[0367] 202. The composition of alternative 201, wherein said suppression of p53 activity is mediated through administration of a small molecule inhibitor of p53.

[0368] 203. The composition of alternative 201, wherein said suppression of p53 activity is mediated through administration of decoy oligonucleotides.

[0369] 204. The composition of alternative 201, wherein said suppression of p53 activity is mediated through administration of decoy peptides.

[0370] 205. The composition of alternative 201, wherein said suppression of p53 expression is achieved through induction of RNA interference targeting p53.

[0371] 206. The composition of alternative 205, wherein said induction ofRNA interference targeting p53 is induced through administration of short interfering RNA.

[0372] 207. The composition of alternative 205, wherein said induction ofRNA interference targeting p53 is induced through administration of short hairpin RNA.

[0373] 208. The composition of alternative 201, wherein said suppression of p53 expression is achieved through administration of antisense oligonucleotides targeting p53.

[0374] 209. The composition of alternative 208, wherein said antisense oligonucleotides induce cleavage of nucleic acids through activation of RNAse H.

[0375] 210. The composition of alternative 201, wherein said suppression of p53 expression is achieved through administration of ribozymes targeting p53.

[0376] 211. The composition of alternative 201 , wherein said suppression of p53 expression is achieved through gene editing.

[0377] 212. The composition of alternative 141, wherein said reprogramming is induced by culture of said cells in a liquid media containing ascorbic acid.

[0378] 213. The composition of alternative 141, wherein said reprogramming is induced by culture of said cells in a liquid media containing transferrin.

[0379] 214. The composition of alternative 141 , wherein said reprogramming is induced by culture of said cells in a liquid media containing sodium bicarbonate.

[0380] 215. The composition of alternative 141, wherein said reprogramming is induced by culture of said cells in a liquid media containing insulin.

[0381] 216. The composition of alternative 141, wherein said reprogramming is induced by culture of said cells in a liquid media containing sodium selenite.

[0382] 217. The composition of alternative 141, wherein said reprogramming is induced by culture of said cells in a hypoxic environment.

[0383] 218. The composition of alternative 207, wherein said hypoxic environment comprises of oxygen levels low enough to induce activation of hypoxia inducible factor (HIF)-l.

[0384] 219. The composition of alternative 217, wherein said hypoxic environment is culture of said cells in an environment less than 21% oxygen.

[0385] 220. The composition of alternative 217, wherein said hypoxic environment is culture of said cells in an environment less than 15% oxygen.

[0386] 221. The composition of alternative 217, wherein said hypoxic environment is culture of said cells in an environment less than 10% oxygen.

[0387] 222. The composition of alternative 217, wherein said hypoxic environment is culture of said cells in an environment containing approximately 5% oxygen.

[0388] 223. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing a MAP Kinase inhibitor.

[0389] 224. The composition of alternative 223, wherein said MAP kinase inhibitor is PD0325901.

[0390] 225. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing SB431542.

[0391] 226. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing CHIR99021.

[0392] 227. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing Y-27632.

[0393] 228. The composition of alternative 141 , wherein said reprogramming is induced by culture of cells in a liquid media containing Y- thiazovivin.

[0394] 229. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing FGF-1.

[0395] 230. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing FGF-2.

[0396] 231. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing FGF-5.

[0397] 232. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing sodium borate.

[0398] 233. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing erythropoietin (EPO).

[0399] 234. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin-3.

[0400] 235. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin-6.

[0401] 236. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin-8.

[0402] 237. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin-10.

[0403] 238. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin- 18.

[0404] 239. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin-20.

[0405] 240. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing interleukin-25.

[0406] 241. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing insulin-like growth factor- 1 (IGF-1).

[0407] 242. The composition of alternative 141 , wherein said reprogramming is induced by culture of cells in a liquid media containing dexamethasone.

[0408] 243. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing holo-transferrin.

[0409] 244. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing amino acids selected from a group comprising of Glycine, L- histidine, L-isoleucine, L-methionine, L- phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, and L- tyrosine, L-valine.

[0410] 245. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing vitamins and / or antioxidants selected from a group comprising of thiamine, reduced glutathione, ascorbic acid and 2-PO.sub.4.

[0411] 246. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing trace elements selected from a group comprising of AgE+, A1E3+, BaE2+, CdE2+, CoE2+, CrE3+, GeE4+, SeE4+, BrE-, IE-, FE-, MnE2+, SiE4+, VE5+, M0E6+, NiE2+, RbE+, SnE2+, and ZrE4+.

[0412] 247. The composition of alternative 141, wherein said reprogramming is induced by culture of cells in a liquid media containing cytoplasm of an undifferentiated cell.

[0413] 248. The composition of alternative 247, wherein said cell being reprogrammed has its membrane temporarily permeabilized.

[0414] 249. The composition of 248, wherein said temporary permeabilization allows for entry of cytoplasm of undifferentiated cell into cytoplasm of said cell to be reprogrammed.

[0415] 250. The composition of alternative 247, wherein said undifferentiated cell is syngeneic with the cell whose reprogramming is desired.

[0416] 251. The composition of alternative 247, wherein said undifferentiated cell is allogeneic with the cell whose reprogramming is desired.

[0417] 252. The composition of alternative 247, wherein said undifferentiated cell is xenogeneic with the cell whose reprogramming is desired.

[0418] 253. The composition of alternative 248, wherein said permeabilization is mediated by electroporation.

[0419] 254. The composition of alternative 248, wherein said permeabilization is mediated by Streptolysin O treatment,

[0420] 255. The composition of alternative 248, wherein said permeabilization is mediated by transient treatment with complement membrane attack complex.

[0421] 256. The composition of alternative 248, wherein said permeabilization is mediated by transient treatment with perforin.

[0422] 257. The composition of alternative 248, wherein said permeabilization is mediated by transient treatment with granzyme.

[0423] 258. The composition of alternative 247, wherein said undifferentiated cell is an oocyte.

[0424] 259. The composition of alternative 258, wherein said oocyte is programmed to be at G0 / G1 of cell cycle.

[0425] 260. The composition of alternative 259, wherein said programming to be at G0 / G1 of cell cycle is accomplished by exposure to mitomycin C.

[0426] 261. The composition of alternative 259, wherein said programming to be at G0 / G1 of cell cycle is accomplished by exposure to serum starvation.

[0427] 262. The composition of alternative 247, wherein said undifferentiated cell is an inducible pluripotent stem cell.

[0428] 263. The composition of alternative 247, wherein said undifferentiated cell is a parthenogenic derived stem cell.

[0429] 264. The composition of alternativewherein said undifferentiated cell is an embryonic stem cell.

[0430] 265. The composition of alternative 247, wherein said undifferentiated cell is a somatic cell nuclear transfer derived stem cell.

[0431] 267. The composition of alternative 247, wherein said undifferentiated cell is a cytoplasmically reprogrammed stem cell.

[0432] 268. The composition of alternative 247, wherein said undifferentiated cell is a cell obtained by fusion of an adult cell with a pluripotent stem cell.

[0433] 269 The composition of alternative 268, wherein said fusion is accomplished by the use of polyethylene glycol.

[0434] 270. The composition of alternative 268, wherein said fusion is accomplished by the use of electrically mediated fusion.

[0435] 271. A cosmetic composition capable of suppressing skin inflammation by generating T regulatory cells comprising the steps of: obtaining a population of naive T cells; contacting said naive T cells with the composition of mesenchymal stem cell conditioned media in a manner capable of eliciting immune modulation; and providing conditions so as to enable differentiation of naive T cells into T regulatory cells.

[0436] 272. The composition of alternative 271, wherein said naive T cells are CD4 T cells.

[0437] 273 The composition of alternative 271, wherein said naive T cells are CD8 T cells.

[0438] 274. The composition of alternative 271, wherein said naive T cells are CD45RO T cells.

[0439] 275. The composition of alternative 271, wherein said naive T cells are antigenically naive.

[0440] 276. The composition of alternative 271, wherein said naive T cells express IL-2 receptor alpha chain.

[0441] 277. The composition of alternative 271, wherein said cells of composition of alternative 1 are capable of immune modulation have been cultured in interferon gamma.

[0442] 278. The composition of alternative 271, wherein said wherein said cells of composition of alternative 1 are capable of immune modulation have been cultured in interferon gamma.

[0443] 279. The composition of alternative 271, wherein said wherein said cells of composition of alternative 1 are capable of immune modulation have been cultured under hypoxic conditions.

[0444] 280. The composition of alternative 271, wherein said wherein said cells of composition of alternative 1 are capable of immune modulation have been transfected with cytoplasm from immature dendritic cells.

[0445] 281 The composition of alternative 280, wherein said immature dendritic cells lack substantial expression of CD40.

[0446] 282. The composition of alternative 280, wherein said immature dendritic cells lack substantial expression of CD80.

[0447] 283. The composition of alternative 280, wherein said immature dendritic cells lack substantial expression of CD86.

[0448] 284. The composition of alternative 280, wherein said immature dendritic cells lack substantial expression of HLA II.

[0449] 285. The composition of alternative 280, wherein said immature dendritic cells possess PD-1L.

[0450] 286. The composition of alternative 280, wherein said immature dendritic cells possess ILT-3.

[0451] 287. The composition of alternative 280, wherein said immature dendritic cells secrete IL- 10.

[0452] 288 The composition of alternative 280, wherein said immature dendritic cells are derived from a cell line.

[0453] 289 The composition of alternative 280, wherein said immature dendritic cells are derived from primary donors.

[0454] 290. The composition of alternative 271, wherein said wherein said cells of composition of alternative 1 are capable of immune modulation have been cultured in platelet rich plasma.

[0455] 291. The composition of alternative 271, wherein said wherein said cells of composition of alternative 1 are capable of immune modulation have been genetically modified to express an immune suppressive protein.

[0456] 292. The composition of alternative 291, wherein said immune suppressive protein is IL- 10.

[0457] 293. The composition of alternative 291, wherein said immune suppressive protein is TGF-beta.

[0458] 294. The composition of alternative 291, wherein said immune suppressive protein is IL-32.

[0459] 295. The composition of alternative 291, wherein said immune suppressive protein is IL-35.

[0460] 296. The composition of alternative 291, wherein said immune suppressive protein is IL-12p40 homodimers.

[0461] 297. The composition of alternative 291, wherein said immune suppressive protein is HLA-G.

[0462] 298. The composition of alternative 291, wherein said immune suppressive protein is ILT-3.

[0463] 299. The composition of alternative 291, wherein said immune suppressive protein is indolamide 2,3 deoxygenase.

[0464] 300. The composition of alternative 291, wherein said immune suppressive protein is indolamide 2,3 deoxygenase.

[0465] 301. A method of treating an inflammatory skin condition such as the condition of aging, comprising the steps of: a) obtaining an umbilical cord derived mesenchymal stem cell population; b) culturing said umbilical cord derived mesenchymal stem cell population in conditions to allow for augmentation of an immune modulating effect; and c) administering said cell population into a patient in need of treatment.

[0466] 302. The method of alternative 301, wherein said inflammatory condition is an autoimmune condition.

[0467] 303. The method of alternative 302, wherein said autoimmune condition is a state in which immune cells of the patient recognize and attack tissue of said patient.

[0468] 304 The method of alternative 303, wherein said immune cells are T cells.

[0469] 305 The method of alternative 303, wherein said immune cells are B cells.

[0470] 306. The method of alternative 303, wherein said immune cells areNK cells.

[0471] 307. The method of alternative 301, wherein said inflammatory condition is characterized by increased production of inflammatory cytokines as compared to an age matched patient not suffering from said inflammatory condition.

[0472] 308 The method of alternative 307, wherein said inflammatory cytokine is TNF-alpha.

[0473] 309. The method of alternative 307, wherein said inflammatory cytokine is IL-1.

[0474] 310. The method of alternative 307, wherein said inflammatory cytokine is IL-6.

[0475] 311. The method of alternative 307, wherein said inflammatory cytokine is IL-11.

[0476] 312. The method of alternative 307, wherein said inflammatory cytokine is IL-12.

[0477] 313. The method of alternative 307, wherein said inflammatory cytokine is IL- 17.

[0478] 314. The method of alternative 307, wherein said inflammatory cytokine is IL-18.

[0479] 315 The method of alternative 307, wherein said inflammatory cytokine is IL-21.

[0480] 316 The method of alternative 307, wherein said inflammatory cytokine is IL-33.

[0481] 317. The method of alternative 301, wherein said inflammatory condition is characterized by increased activation of the complement system as compared to an age matched patient not suffering from said inflammatory condition.

[0482] 318. The method of alternative 307, wherein said umbilical cord stem cell populations are cultured in interferon gamma at a concentration and duration sufficient to induce anti-inflammatory properties of said umbilical cord stem cell.

[0483] 319. The method of alternative 318, wherein said anti-inflammatory properties are selected from a group consisting of: a) suppression of ongoing mixed lymphocyte reaction; b) suppression of inflammatory cytokine production; and c) stimulation of T regulatory cells.

[0484] 320. The method of alternative 318, wherein said umbilical cord stem cell populations are cultured in the presence of interferon gamma at a concentration of 1-100 lU / ml.

[0485] 321. The method of alternative 320, wherein said interferon gamma is used to treat umbilical cord stem cells at a concentration of 10-75 lU / ml.

[0486] 322. The method of alternative 321, wherein said interferon gamma is used to treat umbilical cord stem cells at a concentration of 25-50 lU / ml.

[0487] 323. The method of alternative 318, wherein said interferon gamma is used to treat umbilical cord stem cells for a period of time ranging from 1 hour to 14 days.

[0488] 324. The method of alternative 318, wherein said interferon gamma is used to treat umbilical cord stem cells for a period of time ranging from 1 to 7 days.

[0489] 325. The method of alternative 318, wherein said interferon gamma is used to treat umbilical cord stem cells for a period of time ranging from 1 to 7 days.

[0490] 326. The method of alternative 318, wherein said biological response modifier is platelet rich plasma.

[0491] 327. The method of alternative 301, wherein platelet rich plasma used to treat umbilical cord stem cell at a concentration of 1-50% volume by volume in tissue culture media in which said umbilical cord stem cell are cultured.

[0492] 328. The method of alternative 327, wherein said platelet rich plasma used to treat umbilical cord stem cells at a concentration of 5-20% volume by volume in tissue culture media in which said umbilical cord stem cells are cultured.

[0493] 329. The method of alternative 327, wherein said platelet rich plasma used to treat umbilical cord stem cells at a concentration of 5-10% volume by volume in tissue culture media in which said umbilical cord stem cells are cultured.

[0494] 330. The method of alternative 327, wherein said platelet rich plasma is used to treat umbilical cord stem cells for a period of time ranging from 1 hour to 14 days.

[0495] 331. The method of alternative 327, wherein said platelet rich plasma is used to treat umbilical cord stem cells for a period of time ranging from 1 to 7 days.

[0496] 332. The method of alternative 327, wherein said platelet rich plasma is used to treat umbilical cord stem cells for a period of time ranging from 1 to 7 days.

[0497] 333. The method of alternative 301, wherein said umbilical cord stem cells are cultured in a media selected from a group comprising of: a) Roswell Park Memorial Institute (RPMI-1640); b) Dublecco's Modified Essential Media (DMEM); c) Eagle's Modified Essential Media (EMEM); d) Optimem; and e) Iscove's Media.

[0498] 334. The method of alternative 301, wherein said umbilical cord stem cells are selected for expression of CD73.

[0499] 335. The method of alternative 334, wherein selection for CD73 is performed during isolation of umbilical cord mononuclear cells from said umbilical cord tissue.

[0500] 336. The method of alternative 335, wherein said isolation of said umbilical cord mononuclear cells is performed from said umbilical cord tissue by use of enzymatic digestion.

[0501] 337. The method of alternative 335, wherein said isolation of said umbilical cord mononuclear cells is performed from said umbilical cord tissue by use of mechanical dissociation.

[0502] 338. The method of alternative 335, wherein said isolation of said umbilical cord mononuclear cells is performed from said umbilical cord tissue by use of mechanical dissociation and enzymatic digestion.

[0503] 339. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of interleukin-7 receptor.

[0504] 340. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of interleukin-3 receptor.

[0505] 341. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of Receptor for Advanced Glycation End Products (RAGE).

[0506] 342. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TNF-alpha receptor p55.

[0507] 343. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TNF-alpha receptor p75.

[0508] 344. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of stem cell factor receptor.

[0509] 345. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of GM-CSF receptor alpha.

[0510] 346. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of VPR-Binding Protein.

[0511] 347. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of transferrin receptor.

[0512] 348. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of Tmc5 protein.

[0513] 349. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-9.

[0514] 350. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of zinc transporter 9.

[0515] 351. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of seminal vesicle antigen-like 3.

[0516] 352. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of sarcoma antigen NY-SAR-41.

[0517] 353. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of cell surface vimentin.

[0518] 354. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of fibrosin-1.

[0519] 355. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of IL-1 receptor.

[0520] 356. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of IL-3 receptor.

[0521] 357. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of IL-6 receptor.

[0522] 358. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of HGF receptor.

[0523] 359. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of thrombopoietin receptor.

[0524] 360. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of prolactin receptor.

[0525] 361. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of IGF-1 receptor.

[0526] 362. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of PDGF-BB receptor.

[0527] 363. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of angiopoietin receptor.

[0528] 364. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of VEGF receptor.

[0529] 365. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-2.

[0530] 366. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-3.

[0531] 367. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-4.

[0532] 368. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-5.

[0533] 369. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-7.

[0534] 370. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of TLR-8.

[0535] 371. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of oxysterol-binding protein 1.

[0536] 372. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of nesprin-2.

[0537] 373. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of myomesin-3.

[0538] 374. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of mucin-2.

[0539] 375. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of FRA SI -related extracellular matrix protein 3.

[0540] 376. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of C-C chemokine receptor type 10.

[0541] 377. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of CXCR4.

[0542] 378. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of CCR5

[0543] 379. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of cartilage intermediate layer protein 2.

[0544] 380. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of HLA-DR.

[0545] 381. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of oxytocin receptor.

[0546] 382. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of CD77.

[0547] 383. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of CD56.

[0548] 384. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of poliovirus receptor.

[0549] 385. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of plexin A2.

[0550] 386. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of plexin A4.

[0551] 387. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of HLA-G.

[0552] 388. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of membrane bound TGF-beta.

[0553] 389. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of membrane bound TNF-alpha.

[0554] 390. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of plasticity-related protein 2.

[0555] 391. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of BDNF receptor.

[0556] 392. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of occludin.

[0557] 393. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of neuronal pentraxin receptor.

[0558] 394. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of neuropilin-1.

[0559] 395. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of netrin 2-like.

[0560] 396. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of mucolipin 1.

[0561] 397. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of MEGF10 protein.

[0562] 398. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of mannose binding lectin (A).

[0563] 399. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of Leukemia Inhibitory Factor Receptor.

[0564] 400. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of lipocalin 3.

[0565] 401. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of lipocalin 12.

[0566] 402. The method of alternative 335, wherein said CD73 expressing cells are further selected for expression of lipocalin 13.

[0567] 403. A method of generating cosmetic composition comprising isolating a population of CD105 expressing monocytes from umbilical cord blood.EXAMPLES

[0568] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned and those objects, ends, and advantages inherent herein. Changes therein and other uses which are encompassed within thespirit of the disclosure, as defined by the scope of the claims, will occur to those skilled in the art. Examples of some embodiments containing specific biomaterials, manufacturing methods these embodiments, and testing these embodiments for desired physical properties and device efficacy are illustrative and not intended to limit the present disclosure in any fashion.Example 1 : Growing / Expanding Isolated Cells

[0569] Bone marrow MSCs can be generated through any means known in the literature and to those skilled in the art. In this example, BM-MSCs were generated as follows:

[0570] 1. A 500 mL Isolation Buffer was prepared (PBS+2% FBS+2 mM EDTA) using sterile components or filtering Isolation Buffer through a 0.2 micron filter. Once made, the Isolation Buffer was stored at about 2 to about 8 degrees Celsius.

[0571] 2 The total number of nucleated cells in the BM sample was counted by taking 10 uL BM and diluting it 1 / 50-1 / 100 with 3% Acetic Acid with Methylene Blue (STEMCELL Catalog #07060). Cells were counted using a hemacytometer

[0572] 3 50 mL Isolation Buffer was warmed to room temperature for 20 minutes prior to use. Bone marrow was diluted 5 / 14 final dilution with room temperature Isolation Buffer (e.g. 25 mL BM was diluted with 45 mL Isolation Buffer for a total volume of 70 mL).

[0573] 4. 17 mL FicolLPaque.TM. PLUS (Catalog #07907 / 07957) was pipetted into three 50 mL conical tubes (BD Catalog #352070) (51 mL total). About 23 mL of the diluted BM from step 3 was carefully layered on top of the FicolLPaque.TM. PLUS in each tube.

[0574] 5 The tubes were centrifuged at room temperature (15-25 °C) for 30 minutes at 300 x g in a bench top centrifuge with the brake off.

[0575] 6. The upper plasma layer was removed and discarded without disturbing the plasma:Ficoll-Paque.TM. PLUS interface. The mononuclear cells located at the interface layer were carefully removed and placed in a new 50 mL conical tube. Mononuclear cells were resuspended with 40 mL cold (about 2 to about 8 degrees Celsius) Isolation Buffer and mixed gently by pipetting.

[0576] 7. Cells were centrifuged at 300.times.g for 10 minutes at room temperature in a bench top centrifuge with the brake on. The supernatant was removed and the cell pellet resuspended in 1-2 mL cold Isolation Buffer.

[0577] 8. Cells were diluted 1 / 50 in 3% Acetic Acid with Methylene Blue and the total number of nucleated cells counted using a hemacytometer.

[0578] 9. Cells were diluted in Complete Human MesenCult.RTM. -Proliferation medium (STEMCELL catalog #05411) at a final concentration of 1.times.10E6 cells / mL.

[0579] 10. BM-derived cells were ready for expansion and CFU-F assays in the presence of GW2580, which were then used for specific applications.

[0580] 11. MSCs were also generated according to protocols previously utilized for treatment of patients utilizing bone marrow derived MSC. Specifically, bone marrow was aspirated (10-30 ml) under local anesthesia (with or without sedation) from the posterior iliac crest, collected into sodium heparin containing tubes and transferred to a Good Manufacturing Practices (GMP) clean room. Bone marrow cells are washed with a washing solution such as Dulbecco's phosphate- buffered saline (DPBS), RPMI, or PBS supplemented with autologous patient plasma and layered on to 25 ml of Percoll (1.073 g / ml) at a concentration of approximately 1-2 x 107 cells / ml. Subsequently the cells were centrifuged at 900 g for approximately 30 min or a time period sufficient to achieve separation of mononuclear cells from debris and erythrocytes. Said cells were then washed with PBS and plated at a density of approximately 1 x 106 cells per ml in 175 cm2 tissue culture flasks in DMEM with 10% FCS with flasks subsequently being loaded with a minimum of 30 million bone marrow mononuclear cells. The MSCs were allowed to adhere for 72 h followed by media changes every 3-4 days. Adherent cells were removed with 0.05% trypsin-EDTA and replated at a density of 1 x 106 per 175 cm2.

[0581] In order to determine the quality of MSC cultures, flow cytometry was performed on all cultures for surface expression of SH-2, SH-3, SH-4 MSC markers and lack of contaminating CD14- and CD-45 positive cells. Cells were detached with 0.05% trypsin- EDTA , washed with DPBS + 2% bovine albumin, fixed in 1% paraformaldehyde, blocked in 10% serum, incubated separately with primary SH-2, SH-3 and SH-4 antibodies followed by PE-conjugated anti-mouse IgG(H+L) antibody. Confluent MSC in 175 cm2 flasks were washed with Tyrode's salt solution, incubated with medium 199 (Ml 99) for 60 min, and detached with 0.05% trypsin-EDTA (Gibco). Cells from 10 flasks were detached at a time and MSCs were resuspended in 40 ml of M199 + 1% human serum albumin (HSA; American Red Cross, Washington DC, USA). MSCs harvested from each 10-flask set were stored for up to 4h at 4°C and combined at the end of the harvest. A total of 2-10 x 106 MSC / kg were resuspended in M199 + 1% HSA and centrifuged at 460 g for 10 min at 20°C. Cell pellets were resuspended in fresh M199 + 1% HSA media and centrifuged at 460 g for 10 min at 20°C for three additional times. Total harvest time was 2-4 h based on MSC yield per flask and the target dose. Harvested MSCs were cryopreserved in Cryocyte (Baxter, Deerfield, IL, USA) freezing bags using a rate controlled freezer at a final concentration of 10% DMSO (Research Industries, Salt Lake City, UT, USA) and 5% HSA.

[0582] On the day of infusion, cryopreserved units were thawed at the bedside in an about 37°C water bath and transferred into 60 ml syringes within 5 min and infused intravenously into patients over 10-15 min. Patients were premedicated with 325-650 mg acetaminophen and 12.5-25 mg of diphenhydramine orally. Blood pressure, pulse, respiratory rate, temperature and oxygen saturation were monitored at the time of infusion and every 15 min thereafter for 3 h followed by every 2 h for 6 h.Example 2: Stimulation of Collagen Synthesis by Activated Mesenchymal Stem Cells

[0583] Umbilical cord mesenchymal stem cells were isolated based on plastic adherence and expression of CD73. Isolation based on CD73 was performed using MACS. Cells were plated at 70% confluence and cultured with: a) saline (control); b) CpG DNA (20 ng / ml); c) beta glucan (100 ng / ml) or d) the combination (FIG. 1). Cells were cultured for the indicated times, conditioned media collected, and added to cultures of keratinocytes. Production of collagen was assessed by ELISA.Example 3: Stimulation of Exosome Production by CpG and Valproic Acid Treatment of Umbilical Cord Blood Mesenchymal Stem Cells

[0584] In order to generate cellular materials, umbilical cord mesenchymal stem cells (UC-MSCs) were isolated from fresh umbilical cord tissue obtained with informed consent from healthy donors. The tissue was minced and enzymatically digested using collagenase and dispase, followed by centrifugation to collect the cell pellet. The isolated UC- MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and maintained at 37°C in a 5% CO2 incubator. Cells were expanded to passage 3 to ensure sufficient cell numbers and purity, withviability confirmed above 95% using trypan blue exclusion. Four experimental groups, each containing 10 independent cultures, were established to investigate exosome production: a) control UC-MSCs cultured in media alone, b) UC-MSCs treated with 1 mM valproic acid (VP A), c) UC-MSCs treated with 5 pg / mL of ODN 2006 CpG DNA (a commercially available synthetic oligodeoxynucleotide with the sequence 5'-TCCATGACGTTCCTGATGCT-3', phosphorothioate-modified for stability), and d) UC-MSCs treated with both 1 mM VPA and 5 pg / mL ODN 2006 CpG DNA.

[0585] For each group, UC-MSCs were seeded at a density of 1 x 105cells / mL in 6-well plates and cultured for 48 hours to allow adherence. After this period, the respective treatments were applied: control cultures received fresh DMEM with 10% exosome-depleted FBS, while experimental groups received the same media supplemented with either VPA, ODN 2006 CpG DNA, or both. Cultures were maintained for an additional 72 hours, with media changes every 24 hours to ensure consistent nutrient availability and reagent exposure. Supernatants were collected at the end of the 72-hour period and processed for exosome isolation using differential ultracentrifugation. Briefly, supernatants were centrifuged at 2,000 x g for 10 minutes to remove debris, followed by 10,000 x g for 30 minutes to eliminate larger vesicles. Exosomes were pelleted at 100,000 x g for 70 minutes, washed in phosphate-buffered saline (PBS), and repelleted. Exosome quantification was performed using nanoparticle tracking analysis (NTA) to measure particle concentration, with exosome identity confirmed by the presence of CD63, CD81, and TSG101 markers via Western blot.

[0586] Exosome production varied significantly across groups. The control group (media alone) yielded an average of 1.2 x io9exosomes / mL (standard deviation [SD] ± 0.15 x 109). The VPA-treated group showed a 120% increase in exosome production, with an average of 2.64 x 109exosomes / mL (SD ± 0.28 x io9), consistent with VPA’s role in enhancing exosome biogenesis through histone deacetylase inhibition. The ODN 2006 CpG DNA-treated group exhibited a 37% increase over the control, averaging 1.64 x io9exosomes / mL (SD ± 0.19 x io9), likely due to CpG-induced activation of toll-like receptor 9 signaling pathways. The combination group, treated with both VPA and ODN 2006 CpG DNA, demonstrated a synergistic effect, producing a 584% increase over the control with an average of 8.21 x io9exosomes / mL (SD ± 0.92 x io9). Statistical analysis using one-way ANOVA followed by Tukey’s post-hoc test confirmed significant differences (p < 0.05) between allgroups, with the combination treatment showing the most pronounced effect. Cell viability across all groups remained above 90%, as assessed by MTT assay, indicating that the treatments did not induce significant cytotoxicity. The synergistic effect in the combination group suggests that VPA and ODN 2006 CpG DNA may target complementary pathways to enhance exosome release, potentially involving epigenetic modifications and immune activation. These findings described in Table 1 align with previous reports on VPA and CpG DNA as exosome production enhancers, though the magnitude of the combination effect exceeds typical additive outcomes. Further characterization of exosome cargo and functional properties is warranted to understand the therapeutic potential of these enhanced yields, particularly for applications in regenerative medicine and drug delivery.Table 1Example 4: Stimulation of Exosome Mesenchymal Stem Cell Stimulatory Activity by CpG and Valproic Acid Treatment of Umbilical Cord Blood Mesenchymal Stem Cells

[0587] Umbilical cord mesenchymal stem cells (UC-MSCs) were isolated from fresh umbilical cord tissue obtained with informed consent from healthy donors. The tissue was minced and enzymatically digested using collagenase and dispase, followed by centrifugation to collect the cell pellet. The isolated UC-MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and maintained at 37°C in a 5% CO2 incubator. Cells were expanded to passage 3 to ensure sufficient cell numbers and purity, with viability confirmed above 95% using trypan blue exclusion. Four experimental groups, each containing 10 independent cultures, were established to generate exosomes: a) control UC-MSCs cultured in media alone,b) UC-MSCs treated with 1 mM valproic acid (VP A), c) UC-MSCs treated with 5 pg / mL of ODN 2006 CpG DNA (a commercially available synthetic oligodeoxynucleotide with the sequence 5'-TCCATGACGTTCCTGATGCT-3', phosphorothioate-modified for stability), and d) UC-MSCs treated with both 1 mM VPA and 5 pg / mL ODN 2006 CpG DNA. These concentrations were selected based on prior studies demonstrating their efficacy in modulating MSC behavior without compromising cell viability.

[0588] For exosome production, UC-MSCs were seeded at a density of 1x105cells / mL in 6-well plates and cultured for 48 hours to allow adherence. After this period, treatments were applied: control cultures received fresh DMEM with 10% exosome-depleted FBS, while experimental groups received the same media supplemented with either VPA, ODN 2006 CpG DNA, or both. Cultures were maintained for an additional 72 hours, with media changes every 24 hours. Supernatants were collected and processed for exosome isolation using differential ultracentrifugation (2,000 x g for 10 minutes, 10,000zg for 30 minutes, and 100,000 x g for 70 minutes, followed by a PBS wash and repelleting). Exosome identity was confirmed by the presence of CD63, CD81, and TSG101 markers via Western blot. Exosomes from each group were quantified using nanoparticle tracking analysis (NTA) and diluted to a standardized concentration of 1 x 108exosomes / mL in DMEM with 10% exosome-depleted FBS to ensure equivalent dosing across groups.

[0589] To assess the effect of these exosomes on MSC proliferation, fresh UC- MSCs (passage 3) from the same donor pool were seeded at 5 x 103cells / well in 96-well plates and cultured in DMEM with 10% FBS for 24 hours. The media was then replaced with media containing exosomes (1x108exosomes / mL) from each of the four groups. After 48 hours of co-culture, cell proliferation was measured using a [3 H] -thymidine incorporation assay. Briefly, 1 pCi of [3H]-thymidine was added to each well for the final 18 hours of culture, and incorporated radioactivity was measured using a scintillation counter, with results expressed as counts per minute (CPM). Each group consisted of 10 replicates, and the experiment was designed to evaluate whether exosomes from differentially treated UC-MSCs influenced MSC proliferation differently despite being normalized to the same concentration. Proliferation results showed distinct effects across groups. The control group (exosomes from untreated UC- MSCs) yielded an average proliferation rate of 12,500 CPM (standard deviation [SD] ± 1,200). Exosomes from VPA-treated UC-MSCs increased proliferation by approximately 12%,averaging 14,000 CPM (SD ± 1 ,400), likely due to subtle changes in exosome cargo enhancing growth signaling. Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 23% increase over control, averaging 15,375 CPM (SD ± 1,600), possibly reflecting immune- modulatory cargo alterations. The combination group (exosomes from VPA + ODN 2006 CpG DNA-treated UC-MSCs) showed the highest proliferation, with a 324% increase over control, averaging 53,000 CPM (SD ± 5,800), suggesting a synergistic effect on exosome content that potently stimulates MSC proliferation. One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups. Cell viability remained above 90% across all groups, as assessed by MTT assay, indicating no cytotoxic effects from exosome treatment. These findings as described in Table 2 suggest that the molecular cargo of exosomes, influenced by VPA and CpG DNA treatments, significantly impacts their proliferative effects on UC-MSCs, with potential implications for optimizing MSC-based therapies.Table 2Example 5: Stimulation of Angiogenesis by Stimulated Umbilical Cord MSC Release.

[0590] Umbilical cord mesenchymal stem cells (UC-MSCs) were isolated from fresh umbilical cord tissue obtained with informed consent from healthy donors. The tissue was minced and enzymatically digested using collagenase and dispase, followed by centrifugation to collect the cell pellet. The isolated UC-MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and maintained at 37°C in a 5% CO2 incubator. Cells were expanded to passage 3 to ensure sufficient cell numbers and purity, with viability confirmed above 95%using trypan blue exclusion. Four experimental groups, each containing 10 independent cultures, were established to generate exosomes: a) control UC-MSCs cultured in media alone, b) UC-MSCs treated with 1 mM valproic acid (VP A), c) UC-MSCs treated with 5 pg / mL of ODN 2006 CpG DNA (a commercially available synthetic oligodeoxynucleotide with the sequence 5'-TCCATGACGTTCCTGATGCT-3', phosphorothioate-modified for stability), and d) UC-MSCs treated with both 1 mM VPA and 5 pg / mL ODN 2006 CpG DNA. These concentrations were selected based on prior studies demonstrating their efficacy in modulating MSC behavior without compromising cell viability.

[0591] For exosome production, UC-MSCs were seeded at a density of 1 x 103cells / mL in 6-well plates and cultured for 48 hours to allow adherence. After this period, treatments were applied: control cultures received fresh DMEM with 10% exosome-depleted FBS, while experimental groups received the same media supplemented with either VPA, ODN 2006 CpG DNA, or both. Cultures were maintained for an additional 72 hours, with media changes every 24 hours. Supernatants were collected and processed for exosome isolation using differential ultracentrifugation (2,000 x g for 10 minutes, 10,000 x g for 30 minutes, and 100,000 x g for 70 minutes, followed by a PBS wash and repelleting). Exosome identity was confirmed by the presence of CD63, CD81, and TSG101 markers via Western blot. Exosomes from each group were quantified using nanoparticle tracking analysis (NTA) and diluted to a standardized concentration of 1x108exosomes / mL in endothelial cell growth medium (EGM-2) to ensure equivalent dosing across groups. To assess the effect of these exosomes on human umbilical vein endothelial cell (HUVEC) proliferation, HUVECs (passage 3-5) were seeded at 5 x io3cells / well in 96-well plates coated with 1% gelatin and cultured in EGM-2 supplemented with 2% FBS for 24 hours. The media was then replaced with EGM-2 containing exosomes (1x108exosomes / mL) from each of the four groups. After 48 hours of co-culture, cell proliferation was measured using a [3H]-thymidine incorporation assay. Briefly, 1 pCi of [3H]-thymidine was added to each well for the final 18 hours of culture, and incorporated radioactivity was measured using a scintillation counter, with results expressed as counts per minute (CPM). Each group consisted of 10 replicates, and the experiment was designed to evaluate whether exosomes from differentially treated UC-MSCs influenced HUVEC proliferation differently despite being normalized to the same concentration.-I l l-

[0592] Proliferation results showed distinct effects across groups. The control group (exosomes from untreated UC-MSCs) yielded an average proliferation rate of 10,000 CPM (standard deviation [SD] ± 1,000). Exosomes from VPA-treated UC-MSCs increased HUVEC proliferation by approximately 24%, averaging 12,400 CPM (SD ± 1,200), likely due to exosome cargo enriched with pro-angiogenic factors. Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 47% increase over control, averaging 14,700 CPM (SD ± 1,500), possibly reflecting immune-modulatory or growth-promoting cargo alterations. The combination group (exosomes from VPA + ODN 2006 CpG DNA-treated UC-MSCs) showed the highest proliferation, with a 324% increase over control, averaging 42,400 CPM (SD ± 4,600), suggesting a synergistic effect on exosome content that potently stimulates HUVEC proliferation. One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups. Cell viability remained above 90% across all groups, as assessed by MTT assay, indicating no cytotoxic effects from exosome treatment. These findings as evidenced by Table 3 suggest that the molecular cargo of exosomes, influenced by VPA and CpG DNA treatments, significantly impacts their proliferative effects on HUVECs, with potential implications for optimizing exosome-based therapies in angiogenesis and vascular repair.Table 3Example 6: Enhancement of Fibroblast Biological Activities Relevant to Aging by Exosomes Isolated from Valproic Acid + CpG DNA Treated Mesenchymal Stem Cells

[0593] Umbilical cord mesenchymal stem cells (UC-MSCs) were isolated from fresh umbilical cord tissue obtained with informed consent from healthy donors. The tissuewas minced and enzymatically digested using collagenase and dispase, followed by centrifugation to collect the cell pellet. The isolated UC-MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and maintained at 37°C in a 5% CO2 incubator. Cells were expanded to passage 3 to ensure sufficient cell numbers and purity, with viability confirmed above 95% using trypan blue exclusion. Four experimental groups, each containing 10 independent cultures, were established to generate exosomes: a) control UC-MSCs cultured in media alone, b) UC-MSCs treated with 1 mM valproic acid (VP A), c) UC-MSCs treated with 5 pg / mL of ODN 2006 CpG DNA (a commercially available synthetic oligodeoxynucleotide with the sequence 5'-TCCATGACGTTCCTGATGCT-3', phosphorothioate-modified for stability), and d) UC-MSCs treated with both 1 mM VPA and 5 pg / mL ODN 2006 CpG DNA. These concentrations were selected based on prior studies demonstrating their efficacy in modulating MSC behavior without compromising cell viability. For exosome production, UC-MSCs were seeded at a density of 1 x 105cells / mL in 6-well plates and cultured for 48 hours to allow adherence. After this period, treatments were applied: control cultures received fresh DMEM with 10% exosome-depleted FBS, while experimental groups received the same media supplemented with either VPA, ODN 2006 CpG DNA, or both. Cultures were maintained for an additional 72 hours, with media changes every 24 hours. Supernatants were collected and processed for exosome isolation using differential ultracentrifugation (2,000 x g for 10 minutes, 10,000 x g for 30 minutes, and 100,000 x g for 70 minutes, followed by a PBS wash and repelleting). Exosome identity was confirmed by the presence of CD63, CD81, and TSG101 markers via Western blot. Exosomes from each group were quantified using nanoparticle tracking analysis (NTA) and diluted to a standardized concentration of 1 x 108exosomes / mL in DMEM with 10% FBS for fibroblast proliferation assays or in serum -free DMEM for collagen production assays to minimize background collagen signals.

[0594] Fibroblast Proliferation Assay

[0595] To assess the effect of these exosomes on human dermal fibroblast proliferation, fibroblasts (passage 3-5) were seeded at 5 x 103cells / well in 96-well plates and cultured in DMEM with 10% FBS for 24 hours. The media was then replaced with DMEM containing exosomes (1x108exosomes / mL) from each of the four groups. After 48 hours of co-culture, cell proliferation was measured using a [3 H] -thymidine incorporation assay.Briefly, 1 piCi of [3H]-thymidine was added to each well for the final 18 hours of culture, and incorporated radioactivity was measured using a scintillation counter, with results expressed as counts per minute (CPM). Each group consisted of 10 replicates. The control group (exosomes from untreated UC-MSCs) yielded an average proliferation rate of 8,000 CPM (standard deviation [SD] ± 800). Exosomes from VPA-treated UC-MSCs increased fibroblast proliferation by approximately 34%, averaging 10,720 CPM (SD ± 1, 100). Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 76% increase over control, averaging 14,080 CPM (SD ± 1,400). The combination group (exosomes from VPA + ODN 2006 CpG DNA-treated UC-MSCs) showed the highest proliferation, with a 583% increase over control, averaging 54,640 CPM (SD ± 5,900). One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups.

[0596] Type I Collagen Production Assay

[0597] To evaluate the effect of exosomes on type I collagen production, fibroblasts were seeded at 1 * IO3cells / well in 6-well plates and cultured in DMEM with 10% FBS for 24 hours. The media was replaced with serum-free DMEM containing exosomes (l x 108exosomes / mL) from each group. After 72 hours, supernatants were collected, and type I collagen levels were quantified using a human type I collagen ELISA kit, with results expressed as ng / mL. Each group consisted of 10 replicates. The control group yielded an average type I collagen production of 200 ng / mL (SD ± 20). Exosomes from VPA-treated UC- MSCs increased type I collagen production by approximately 5%, averaging 210 ng / mL (SD ± 22). Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 24% increase, averaging 248 ng / mL (SD ± 25). The combination group showed a 75% increase, averaging 350 ng / mL (SD ± 38). Statistical analysis confirmed significant differences (p < 0.05) between all groups.

[0598] Type III Collagen Production Assay

[0599] To assess type III collagen production, the same experimental setup was used as for type I collagen, with fibroblasts cultured in serum-free DMEM containing exosomes (1x108exosomes / mL) for 72 hours. Type III collagen levels were quantified using a human type III collagen ELISA kit, with results expressed as ng / mL. Each group consisted of 10 replicates. The control group yielded an average type III collagen production of 150 ng / mL (SD ± 15). Exosomes from VPA-treated UC-MSCs increased type III collagenproduction by approximately 54%, averaging 231 ng / mL (SD ± 24). Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 52% increase, averaging 228 ng / mL (SD ± 23). The combination group showed a 324% increase, averaging 636 ng / mL (SD ± 69). Statistical analysis confirmed significant differences (p < 0.05) between all groups. Cell viability remained above 90% across all assays, as assessed by MTT assay, indicating no cytotoxic effects from exosome treatment. These findings as provided for in Table 4 suggests that exosomes from VPA- and CpG-treated UC-MSCs significantly enhance fibroblast proliferation and collagen production, with the combination treatment exhibiting synergistic effects, potentially valuable for wound healing and tissue repair applications.Table 4Example 7: Enhancement of Fibroblast Biological Activities Relevant to Aging by Exosomes Isolated from hCG + CpG DNA Treated Mesenchymal Stem Cells

[0600] Umbilical cord mesenchymal stem cells (UC-MSCs) were isolated from fresh umbilical cord tissue obtained with informed consent from healthy donors. The tissue was minced and enzymatically digested using collagenase and dispase, followed by centrifugation to collect the cell pellet. The isolated UC-MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and maintained at 37°C in a 5% CO2 incubator. Cells were expanded to passage 3 to ensure sufficient cell numbers and purity, with viability confirmed above 95% using trypan blue exclusion. Four experimental groups, each containing 10 independent cultures, were established to generate exosomes: a) control UC-MSCs cultured in media alone, b) UC-MSCs treated with 100 lU / mL human chorionic gonadotropin (hCG), c) UC-MSCs treated with 5 pg / mL of ODN 2006 CpG DNA (a commercially available synthetic oligodeoxynucleotide with the sequence 5'-TCCATGACGTTCCTGATGCT-3', phosphorothioate-modified for stability), and d) UC-MSCs treated with both 100 lU / mL hCG and 5 pg / mL ODN 2006 CpG DNA. These concentrations were selected based on prior studies demonstrating their efficacy in modulating MSC behavior without compromising cell viability.

[0601] For exosome production, UC-MSCs were seeded at a density of 1 x IO3cells / mL in 6-well plates and cultured for 48 hours to allow adherence. After this period, treatments were applied: control cultures received fresh DMEM with 10% exosome-depleted FBS, while experimental groups received the same media supplemented with either hCG, ODN 2006 CpG DNA, or both. Cultures were maintained for an additional 72 hours, with media changes every 24 hours. Supernatants were collected and processed for exosome isolation using differential ultracentrifugation (2,000 x g for 10 minutes, 10,000 x g for 30 minutes, and 100,000 x g for 70 minutes, followed by a PBS wash and repelleting). Exosome identity was confirmed by the presence of CD63, CD81, and TSG101 markers via Western blot. Exosomesfrom each group were quantified using nanoparticle tracking analysis (NTA) and diluted to a standardized concentration of 1 x 108exosomes / mL in DMEM with 10% FBS for fibroblast proliferation assays or in serum-free DMEM for collagen production assays to minimize background collagen signals.

[0602] Fibroblast Proliferation Assay

[0603] To assess the effect of these exosomes on human dermal fibroblast proliferation, fibroblasts (passage 3-5) were seeded at 5 x 103cells / well in 96-well plates and cultured in DMEM with 10% FBS for 24 hours. The media was then replaced with DMEM containing exosomes (1x108exosomes / mL) from each of the four groups. After 48 hours of co-culture, cell proliferation was measured using a [3H]-thymidine incorporation assay. Briefly, 1 pCi of [3H]-thymidine was added to each well for the final 18 hours of culture, and incorporated radioactivity was measured using a scintillation counter, with results expressed as counts per minute (CPM). Each group consisted of 10 replicates. The control group (exosomes from untreated UC-MSCs) yielded an average proliferation rate of 8,000 CPM (standard deviation [SD] ± 800). Exosomes from hCG-treated UC-MSCs increased fibroblast proliferation by approximately 5%, averaging 8,400 CPM (SD ± 850). Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 45% increase over control, averaging 11,600 CPM (SD ± 1,200). The combination group (exosomes from hCG + ODN 2006 CpG DNA- treated UC-MSCs) showed a 185% increase over control, averaging 22,800 CPM (SD ± 2,500). One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups.

[0604] Type I Collagen Production Assay

[0605] To evaluate the effect of exosomes on type I collagen production, fibroblasts were seeded at 1x105cells / well in 6-well plates and cultured in DMEM with 10% FBS for 24 hours. The media was replaced with serum-free DMEM containing exosomes (1 x 108exosomes / mL) from each group. After 72 hours, supernatants were collected, and type I collagen levels were quantified using a human type I collagen ELISA kit, with results expressed as ng / mL. Each group consisted of 10 replicates. The control group yielded an average type I collagen production of 200 ng / mL (SD ± 20). Exosomes from hCG-treated UC- MSCs increased type I collagen production by approximately 55%, averaging 310 ng / mL (SD ± 32). Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 57% increase,averaging 314 ng / mL (SD ± 33). The combination group showed a 542% increase, averaging 1,284 ng / mL (SD ± 140). Statistical analysis confirmed significant differences (p < 0.05) between all groups.

[0606] Type III Collagen Production Assay

[0607] To assess type III collagen production, the same experimental setup was used as for type I collagen, with fibroblasts cultured in serum-free DMEM containing exosomes (1x108exosomes / mL) for 72 hours. Type III collagen levels were quantified using a human type III collagen ELISA kit, with results expressed as ng / mL. Each group consisted of 10 replicates. The control group yielded an average type III collagen production of 150 ng / mL (SD ± 15). Exosomes from hCG-treated UC-MSCs increased type III collagen production by approximately 75%, averaging 262.5 ng / mL (SD ± 27). Exosomes from ODN 2006 CpG DNA-treated UC-MSCs resulted in a 75% increase, averaging 262.5 ng / mL (SD ± 26). The combination group showed a 753% increase, averaging 1,279.5 ng / mL (SD ± 140).Statistical analysis confirmed significant differences (p < 0.05) between all groups. Cell viability remained above 90% across all assays, as assessed by MTT assay, indicating no cytotoxic effects from exosome treatment. These findings as described in Table 5 suggest that exosomes from hCG- and CpG-treated UC-MSCs significantly enhance fibroblast proliferation and collagen production, with the combination treatment exhibiting synergistic effects, potentially valuable for wound healing and tissue repair applications.Table 5Example 8: Stimulation of Anti -Aging Associated Cytokines by Ex Vivo Activation of Adipose Derived Mesenchymal Stem Cells.

[0608] Adipose-derived mesenchymal stem cells (AD-MSCs) were isolated from human adipose tissue obtained through liposuction with informed consent from healthy donors. The tissue was washed with phosphate-buffered saline (PBS) containing 1% penicillinstreptomycin, minced, and digested with 0.1% collagenase type I at 37°C for 1 hour with gentle agitation. The digested tissue was centrifuged at 1,200 x g for 10 minutes to obtain the stromal vascular fraction (SVF), which was resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The SVF was plated and cultured at 37°C in a 5% CO2 incubator, with non-adherent cells removed after 24 hours. AD-MSCs were expanded to passage 3, with cell viability confirmed above 95% using trypan blue exclusion. For the experiment, AD-MSCs were seeded at 1x105cells / mL in 6-well plates and cultured for 48 hours to ensure adherence. Twelve groups were established: a control group (media alone) and 11 groups treated with one of 10 toll-like receptor (TLR) agonists at concentrations based on prior studies: Pam3CSK4 (TLR1 / 2, 100 ng / mL), Poly(I:C) (TLR3, 10 pg / mL), LPS (TLR4, 100 ng / mL), Flagellin (TLR5, 100 ng / mL),FSL-1 (TLR6 / 2, lOO ng / mL), Imiquimod (TLR7, 1 pg / mL), ssRNA40 (TLR8, 1 pg / mL), ODN 2006 CpGDNA (TLR9, 5 pg / mL), Zymosan (TLR2 / 6, 10 pg / mL), and Gardiquimod (TLR7 / 8, 1 pg / mL). An additional combination group was treated with all 10 TLR agonists simultaneously at the same concentrations. Each group consisted of 10 replicates. After 48 hours of treatment, conditioned media (CM) was collected from each group by centrifuging supernatants at 2,000 x g for 10 minutes to remove debris. The CM was stored at -80°C until use. Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using CD14+ magnetic bead selection, achieving >90% purity. Monocytes were seeded at 1 x 106cells / mL in 24-well plates in RPMI 1640 medium with 10% FBS and cultured for 24 hours. The media was then replaced with CM from each AD-MSC group, diluted 1 : 1 with fresh RPMI 1640. After 48 hours of co-culture, monocyte supernatants were collected and analyzed for IL-10, fibroblast growth factor-2 (FGF-2), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) using specific ELISA kits, with concentrations expressed as pg / mL. The experiment was designed to assess how AD-MSC- derived CM, modulated by TLR agonists, influences monocyte cytokine production. Cell viability of monocytes remained above 90%, as assessed by MTT assay, indicating no cytotoxicity from the CM. The results showed varied cytokine production across groups. For IL- 10, the control CM group yielded an average of 100 pg / mL (SD ± 10). Pam3CSK4, Poly(LC), and LPS increased IL-10 production significantly, with averages of 250 pg / mL (SD ± 25), 300 pg / mL (SD ± 30), and 280 pg / mL (SD ± 28), respectively, while the combination group reached 600 pg / mL (SD ± 65). For FGF-2, the control group averaged 50 pg / mL (SD ± 5), with Flagellin and Zymosan showing notable increases at 150 pg / mL (SD ± 15) and 180 pg / mL (SD ± 18), and the combination group at 400 pg / mL (SD ± 45). KGF production in the control group averaged 30 pg / mL (SD ± 3), with Imiquimod and Gardiquimod increasing to 100 pg / mL (SD ± 10) and 120 pg / mL (SD ± 12), and the combination group at 250 pg / mL (SD ± 27). HGF production averaged 80 pg / mL (SD ± 8) in the control, with ssRNA40 and ODN 2006 CpG DNA at 200 pg / mL (SD ± 20) and 220 pg / mL (SD ± 22), and the combination group at 500 pg / mL (SD ± 55). One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups for each cytokine. These findings suggest in Tables 6A and 6B that TLR agonist-treated AD-MSC CM differentially modulates monocyte cytokineproduction, with synergistic effects in the combination group, potentially enhancing antiinflammatory and regenerative responses in therapeutic applications.Table 6ATable 6BExample 9: Valproic Acid Enhances Cytokine Secretion After TLR Activation

[0609] Adipose-derived mesenchymal stem cells (AD-MSCs) were isolated from human adipose tissue obtained through liposuction with informed consent from healthy donors. The tissue was washed with phosphate-buffered saline (PBS) containing 1% penicillinstreptomycin, minced, and digested with 0.1% collagenase type I at 37°C for 1 hour with gentle agitation. The digested tissue was centrifuged at 1,200 x g for 10 minutes to isolate the stromal vascular fraction (SVF), which was resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The SVF was plated and cultured at 37°C in a 5% CO2 incubator, with non-adherent cells removed after 24 hours. AD-MSCs were expanded to passage 3, ensuring sufficient cell numbers and purity, with viability confirmed above 95% using trypan blue exclusion. Eight experimental groups, each with 20 replicates, were established: a) control (media alone), b) valproic acid (VP A, 1 mM), c) Pam3CSK4 (TLR1 / 2, 100 ng / mL), d) Poly(I:C) (TLR3, 10 pg / mL), e) Flagellin (TLR5, 100 ng / mL), f) VPA (1 mM) + Pam3CSK4 (100 ng / mL), g) VPA (1 mM) + Poly(LC) (10 pg / mL), and h) VPA (1 mM) + Flagellin (100 ng / mL). Concentrations were based on prior studies demonstrating efficacy in modulating MSC behavior. AD-MSCs were seeded at 1 x 105cells / mL in 6-well plates, cultured for 48 hours to ensure adherence, and then treated for an additional 48 hours with media changes every 24 hours.

[0610] Conditioned media (CM) was collected from each group by centrifuging supernatants at 2,000 x g for 10 minutes to remove debris and stored at -80°C. Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using CD14+ magnetic bead selection, achieving >90% purity. Monocytes were seeded at 1 x 106cells / mL in 24-well plates in RPMI 1640 medium with 10% FBS and cultured for 24 hours. The media was then replaced with CM from each AD-MSC group, diluted 1 : 1 with fresh RPMI 1640. After 48 hours of co-culture, monocyte supernatants were collected and analyzed for IL-10, fibroblast growth factor-2 (FGF-2), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) using specific ELISA kits, with concentrations expressed as pg / mL. Monocyte viability remained above 90%, as assessed by MTT assay, indicating no cytotoxicity from the CM. One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups for each cytokine, with VPA-containing treatments (VPA alone and VPA + TLR agonists) showing at least 230% higher cytokine production compared to the media-only control, highlighting VPA’s potentiating effect. The results demonstrated significant cytokine induction, particularly with VPA-containing treatments. For IL- 10, the control group averaged 100 pg / mL (SD ± 10), VPA alone at 350 pg / mL (SD ± 35), Pam3CSK4 at 200 pg / mL (SD ± 20), Poly(LC) at 250 pg / mL (SD ± 25), Flagellin at 220 pg / mL (SD ± 22), VPA + Pam3CSK4 at 600 pg / mL (SD ± 65), VPA + Poly(LC) at 700 pg / mL (SD ± 75), and VPA + Flagellin at 650 pg / mL (SD ± 70). For FGF-2, the control averaged 50 pg / mL (SD ± 5), VPA at 180 pg / mL (SD ± 18), Pam3CSK4 at 100 pg / mL (SD ± 10), Poly(LC) at 120 pg / mL (SD ± 12), Flagellin at 110 pg / mL (SD ± 11), VPA + Pam3CSK4 at 350 pg / mL (SD ± 38), VPA + Poly(I:C) at 400 pg / mL (SD ± 44), and VPA + Flagellin at 380 pg / mL (SD ± 41). KGF production averaged 30 pg / mL (SD ± 3) in the control, VPA at 100 pg / mL (SD ± 10), Pam3CSK4 at 60 pg / mL (SD ± 6), Poly(LC) at 70 pg / mL (SD ± 7), Flagellin at 65 pg / mL (SD ± 7), VPA + Pam3CSK4 at 230 pg / mL (SD ± 25), VPA + Poly(LC) at 250 pg / mL (SD ± 27), and VPA + Flagellin at 240 pg / mL (SD ± 26). HGF production averaged 80 pg / mL (SD ± 8) in the control, VPA at 280 pg / mL (SD ± 28), Pam3CSK4 at 160 pg / mL (SD ± 16), Poly(LC) at 180 pg / mL (SD ± 18), Flagellin at 170 pg / mL (SD ± 17), VPA + Pam3CSK4 at 500 pg / mL (SD ± 54), VPA + Poly(LC) at 550 pg / mL (SD ± 60), and VPA + Flagellin at 520 pg / mL (SD ± 56). These results as presented in Table 7 underscore VPA’s ability to significantly enhance TLR agonist-induced cytokine production, suggesting synergistic potential for immunomodulatory therapies.Table 7Example 10: Human Chorionic Gonadotropin Enhances Cytokine Secretion After TLR Activation

[0611] Adipose-derived mesenchymal stem cells (AD-MSCs) were isolated from human adipose tissue obtained through liposuction with informed consent from healthy donors. The tissue was washed with phosphate-buffered saline (PBS) containing 1% penicillinstreptomycin, minced, and digested with 0.1% collagenase type I at 37°C for 1 hour with gentle agitation. The digested tissue was centrifuged at 1,200 * g for 10 minutes to isolate the stromalvascular fraction (SVF), which was resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The SVF was plated and cultured at 37°C in a 5% CO2 incubator, with non-adherent cells removed after 24 hours. AD-MSCs were expanded to passage 3, ensuring sufficient cell numbers and purity, with viability confirmed above 95% using trypan blue exclusion. Eight experimental groups, each with 20 replicates, were established: a) control (media alone), b) human chorionic gonadotropin (hCG, 100 lU / mL), c) Pam3CSK4 (TLR1 / 2, 100 ng / mL), d) Poly(I:C) (TLR3, 10 pg / mL), e) Flagellin (TLR5, 100 ng / mL), f) hCG (100 lU / mL) + Pam3CSK4 (100 ng / mL), g) hCG (100 lU / mL) + Poly(LC) (10 pg / mL), and h) hCG (100 lU / mL) + Flagellin (100 ng / mL). Concentrations were based on prior studies demonstrating efficacy in modulating MSC behavior. AD-MSCs were seeded at 1 x 105cells / mL in 6-well plates, cultured for 48 hours to ensure adherence, and then treated for an additional 48 hours with media changes every 24 hours.

[0612] Conditioned media (CM) was collected from each group by centrifuging supernatants at 2,000 x g for 10 minutes to remove debris and stored at -80°C. Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using CD14+ magnetic bead selection, achieving >90% purity. Monocytes were seeded at 1 x 106cells / mL in 24-well plates in RPMI 1640 medium with 10% FBS and cultured for 24 hours. The media was then replaced with CM from each AD-MSC group, diluted 1 : 1 with fresh RPMI 1640. After 48 hours of co-culture, monocyte supernatants were collected and analyzed for IL- 10, fibroblast growth factor-2 (FGF-2), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) using specific ELISA kits, with concentrations expressed as pg / mL. Monocyte viability remained above 90%, as assessed by MTT assay, indicating no cytotoxicity from the CM. One-way ANOVA with Tukey’s post-hoc test confirmed significant differences (p < 0.05) between all groups for each cytokine, with hCG alone increasing cytokine production by 14-79% over the control and hCG combined with TLR agonists showing synergistic increases of 200-2500% over the control, highlighting hCG’s potentiating effect.The results demonstrated significant cytokine induction, particularly with hCG and TLR agonist combinations. For IL-10, the control group averaged 100 pg / mL (SD ± 10), hCG alone at 140 pg / mL (SD ± 14, 40% increase), Pam3CSK4 at 200 pg / mL (SD ± 20), Poly(I:C) at 250 pg / mL (SD ± 25), Flagellin at 220 pg / mL (SD ± 22), hCG + Pam3CSK4 at 600 pg / mL (SD ±65, 500% increase), hCG + Poly(T:C) at 2,600 pg / mL (SD ± 280, 2500% increase), and hCG + Flagellin at 1,100 pg / mL (SD ± 120, 1000% increase). For FGF-2, the control averaged 50 pg / mL (SD ± 5), hCG at 80 pg / mL (SD ± 8, 60% increase), Pam3CSK4 at 100 pg / mL (SD ± 10), Poly(LC) at 120 pg / mL (SD ± 12), Flagellin at 110 pg / mL (SD ± 11), hCG + Pam3CSK4 at 300 pg / mL (SD ± 33, 500% increase), hCG + Poly(I:C) at 1,300 pg / mL (SD ± 140, 2500% increase), and hCG + Flagellin at 550 pg / mL (SD ± 60, 1000% increase). KGF production averaged 30 pg / mL (SD ± 3) in the control, hCG at 45 pg / mL (SD ± 5, 50% increase), Pam3CSK4 at 60 pg / mL (SD ± 6), Poly(I:C) at 70 pg / mL (SD ± 7), Flagellin at 65 pg / mL (SD ± 7), hCG + Pam3CSK4 at 150 pg / mL (SD ± 16, 400% increase), hCG + Poly(LC) at 630 pg / mL (SD ± 68, 2000% increase), and hCG + Flagellin at 270 pg / mL (SD ± 29, 800% increase). HGF production averaged 80 pg / mL (SD ± 8) in the control, hCG at 90 pg / mL (SD ± 9, 14% increase), Pam3CSK4 at 160 pg / mL (SD ± 16), Poly(LC) at 180 pg / mL (SD ± 18), Flagellin at 170 pg / mL (SD ± 17), hCG + Pam3CSK4 at 240 pg / mL (SD ± 26, 200% increase), hCG + Poly(I:C) at 1,280 pg / mL (SD ± 140, 1500% increase), and hCG + Flagellin at 480 pg / mL (SD ± 52, 500% increase). These results as illustrated in Table 8 highlight hCG’s synergistic enhancement of TLR agonist-induced cytokine production, suggesting potential for tailored immunomodulatory therapies.Table 8Example 10: Lithium Enhances Cytokine Secretion After TLR Activation

[0613] Adipose-derived mesenchymal stem cells (AD-MSCs) were isolated from human adipose tissue obtained through liposuction with informed consent from healthy donors. The tissue was washed with phosphate-buffered saline (PBS) containing 1% penicillinstreptomycin, minced, and digested with 0.1% collagenase type I at 37°C for 1 hour with gentle agitation. The digested tissue was centrifuged at 1,200 x g for 10 minutes to isolate the stromal vascular fraction (SVF), which was resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The SVF was plated and cultured at 37°C in a 5% CO2 incubator, with non-adherent cells removed after 24 hours. AD-MSCs were expanded to passage 3, ensuring sufficient cell numbers and purity, with viability confirmed above 95% using trypan blue exclusion. Eight experimental groups, each with 20 replicates, were established: a) control (media alone), b)lithium chloride (LiCl, 1 mM), c) Pam3CSK4 (TLR1 / 2, 100 ng / mL), d) Poly(I:C) (TLR3, 10 pg / mL), e) Flagellin (TLR5, 100 ng / mL), f) LiCl (1 mM) + Pam3CSK4 (100 ng / mL), g) LiCl (1 mM) + Poly(LC) (10 pg / mL), and h) LiCl (1 mM) + Flagellin (100 ng / mL). Concentrations were based on prior studies demonstrating efficacy in modulating ...

Claims

1. WHAT IS CLAIMED IS:

1. A method for making an anti-inflammatory composition comprising an at least one regenerative factor, the method comprising: a) extracting at least one cell from a tissue; b) exposing the at least one cell to conditions allowing for production of at least one regenerative factor; c) isolating the at least one regenerative factor; and d) admixing the at least one regenerative factor into an anti-inflammatory composition, wherein the at least one cell comprises a mesenchymal stem cell (MSC) and / or a cell derived from a mesenchymal stem cell.

2. The method of claim 1, wherein the tissue is selected from the group consisting of adipose, nail cuticle, deciduous tooth, hair follicle, skin, bone marrow, placenta, umbilical cord blood, mobilized peripheral blood, peripheral blood, urine, breast milk, menstrual blood, fallopian tube, or a combination thereof.

3. The method of claim 1 or 2, wherein the at least one regenerative factor comprises an exosome.

4. The method of claim 3, wherein the exosome possesses at least one of: a) a diameter of between 2 nm and 200 nm; b) a molecular weight of at least about 100 kDa; c) a hydrodynamic radius of at most about 100 nm; or d) any combination of (a)-(c) thereof.

5. The method of any one of claims 1 to 4, wherein the conditions allowing for production of an at least one regenerative factor comprises exposing the at least one cell to an agent that resembles cellular injury or cellular damage.

6. The method of claim 5, wherein the agent that resembles cellular injury or cellular damage is at least one toll-like receptor agonist, valproic acid (VP A), human chorionic gonadotropin, ODN 2006 CpG DNA with the sequence 5'-TCCATGACGTTCCTGATGCT- 3', or a combination thereof.

7. The method of claim 6, wherein the at least one toll like receptor agonist is selected from a group consisting of Lipopolysaccharide (LPS), Pam3CSK4, lipoteichoic acid(LTA), zymosan, flagellin, polyinosinic:polycytidylic acid (Poly(FC)), imiquimod, resiquimod (R848), loxoribine, gardiquimod, CL075, CL097, isatoribine (ANA-245), vesatolimod (GS-9620), monophosphoryl lipid A (MPL), lipid IVa, eritoran (primarily an antagonist but with agonist-like effects in some contexts), CpG oligodeoxynucleotides (CpG- ODN), SM360320, CU-T12-9, GS-9688, R07020531, JNJ-4964, CBLB612, ISA-201, OPN- 305 (primarily antagonist but with agonist potential in trials), TMX-101, DSP-0509, IMO- 2055 (Amplivax), IC-31, E-6020, PF-4878691, RO6870868, RO6864018, high mobility group box 1 (HMGB1), peptidoglycan, bacterial DNA, fibrinogen, fibronectin, extracellular matrix proteins, 852A, VTX-2337, Bacillus Calmette-Guerin (BCG), Poly ICLC, synthetic imidazoquinoline, small-molecule selective TLR8 agonist, mannose-capped lipoarabinomannan (ManLAM), phenol-soluble modulin, inulin acetate, retrocyclin, CL429, CpG-ODN: AG-OVA, P-glucans (general), Sparassis crispa P-glucan, Phellinus linteus P- glucan, Platycodon grandiflorum P-glucan, Cordyceps militaris P-glucan, Angelica gigas Nakai P-glucan, Saccharomyces cerevisiae P-glucan, Laminarin (P-glucan), Curdlan (P- glucan), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shock protein 22 (HSP22), heat shock protein gp96, or a combination thereof.

8. The method of any one of claims 1 to 7, wherein exposing the at least one cell to conditions allows for production of the at least one regenerative factor comprise exposing the at least one cell to at least one stimuli selected from the group consisting of hypoxia, hyperthermia, hypotonic challenge, oxidative stress, inflammatory stimuli, toll-like receptors, or any combination thereof.

9. The method of any one of claims 1 to 7, wherein exposing the at least one cell to conditions allowing for production of at least one regenerative factor comprises exposing the at least one cell to a histone deacetylase inhibitor, an amino acid, a leukemia inhibitory factor, a DNA methyltransferase inhibitor, ascorbic acid, insulin, lithium, transferrin, sodium bicarbonate, sodium selenite, a MAPK inhibitor, SB431542, CHIR99021, Y-27632, Y- thiazovivin, FGF-1, FGF-2, FGF-5, sodium borate, erythropoietin, IL-3, IL-6, IL-8, IL-10, IL- 18, IL-20, IL-25, IGF-1, dexamethasome, holo-transferrin, interferon gamma, cytoplasm of an undifferentiated cell, or any combination thereof.

10. The method of any one of claims 1 to 9, wherein the at least one cell expresses CD73, and further expresses at least one of TLR9, CD4, CD8, CD25, CD34, CD39, CD56,CD77, CD105, CTLA-4, GITR, lipocalin 3, lipocalin 12, lipocalin 13, IL-1, IL-3, IL-6, IL-7, IL-10, IL-17, IL-32, IL-35, TLR-2, TLR-3, TLR-4, TLR-5, TLR-7, TLR-8, angiopoietin receptor, BDNF receptor, cartilage intermediate layer protein 2, C-C chemokine receptor type 10, CCR5, CXCR4, RAGE, TNFa, TNFb, TNF-alpha receptor p55, TNF-alpha receptor p75, stem cell factor receptor, FRASl-related extracellular matrix protein 3, GM-CSF receptor alpha, HGF receptor, HLA-DR, HLA-G, IGF-1 receptor, IL12-p40, ILT-3, indolamide 2,3 deoxygenase, Leukemia Inhibitory Factor Receptor, mannose binding lectin, MEGF10, mucin- 2, mucolipin 1, nesprin-2, netrin 2-like, neuropilin- 1, neuronal pentraxin receptor, occludin, oxytocin receptor, oxy sterol -binding protein 1, PDGF-BB receptor, plasticity -related protein 2, plexin A2, plexin A4, poliovirus receptor, prolactin receptor, VPR-Binding Protein, TGF-b, thrombopoietin receptor, transferrin receptor, Tmc5, zinc transporter 9, seminal vesicle antigen-like 3, sarcoma antigen NY-SAR-41, vimentin, VEGF receptor, fibrosin 1, or any combination thereof; wherein the at least one cell does not express CD 14 or CD45.

11. A composition for topical skin application, comprising: a conditioned medium comprising endogenous progenitor / stem cells, wherein said endogenous progenitor / stem cells are stimulated with at least one regenerative factor; and a dermatologically acceptable carrier.

12. The composition of claim 11, wherein the endogenous progenitor / stem cells are mesenchymal stem cells.

13. The composition of claim 11 or 12, wherein the at least one regenerative factor is selected from the group consisting of at least one toll-like receptor agonist, valproic acid (VPA), human chorionic gonadotropin, ODN 2006 CpG DNA with a sequence 5'- TCCATGACGTTCCTGATGCT-3', LiCl or a combination thereof.

14. The composition of claim 13, wherein the at least one toll like receptor agonist is selected from a group consisting of Lipopolysaccharide (LPS), Pam3CSK4, lipoteichoic acid (LTA), zymosan, flagellin, polyinosinic:polycytidylic acid (Poly(I:C)), imiquimod, resiquimod (R848), loxoribine, gardiquimod, CL075, CL097, isatoribine (ANA-245), vesatolimod (GS-9620), monophosphoryl lipid A (MPL), lipid IVa, eritoran (primarily an antagonist but with agonist-like effects in some contexts), CpG oligodeoxynucleotides (CpG-ODN), SM360320, CU-T12-9, GS-9688, R07020531, JNJ-4964, CBLB612, ISA-201, OPN- 305 (primarily antagonist but with agonist potential in trials), TMX-101, DSP-0509, IMO- 2055 (Amplivax), IC-31, E-6020, PF-4878691, RO6870868, RO6864018, high mobility group box 1 (HMGB1), peptidoglycan, bacterial DNA, fibrinogen, fibronectin, extracellular matrix proteins, 852A, VTX-2337, Bacillus Calmette-Guerin (BCG), Poly ICLC, synthetic imidazoquinoline, small-molecule selective TLR8 agonist, mannose-capped lipoarabinomannan (ManLAM), phenol-soluble modulin, inulin acetate, retrocyclin, CL429, CpG-ODN: AG-OVA, P-glucans (general), Sparassis crispa P-glucan, Phellinus linteus P- glucan, Platycodon grandiflorum P-glucan, Cordyceps militaris P-glucan, Angelica gigas Nakai P-glucan, Saccharomyces cerevisiae P-glucan, Laminarin (P-glucan), Curdlan (P- glucan), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shock protein 22 (HSP22), heat shock protein gp96, or a combination thereof15. The composition of any one of claims 11 to 13, wherein the endogenous progenitor / stem cells expresses CD73 and TLR9.

16. The composition of any one of claims 11 to 15, wherein the endogenous progenitor / stem cells resemble cellular injury or cell damage by a toll-like receptor agonist.

17. A topical anti-inflammatory composition, comprising: activator of endogenous progenitor / stem cells.

18. The composition of claim 17, further comprising: an angiogenic factor; a neurogenic factor; an antiapoptotic factors; and an immune modulatory factors.

19. The composition of claim 18, further comprising at least one of: platelet rich plasma, human chorionic gonadotropin, one or more antioxidants, one or more antiseptic agents, one or more anti-inflammatory agents, one o r more delivery vehicles, one or more known composition agents, or any combination thereof.

20. The composition of claim 19, wherein the one or more delivery vehicle is selected from the group consisting of phospholipids, palmityl myristate, DMSO, a polymer, liposomes, Trojan peptides, chariot peptides, small elastic vesicles, microspheres, chitosan suspensions, a matrix, nanoparticle, or a combination thereof.21 . The composition of claim 17 or 18, wherein the one or more known composition agents is selected from the group consisting of beta-glucan, propylene glycol, butylene glycol, polyethylene glycol, olive oil, dimethyl isosorbide, dimethylformamide, methyl salicylate, long chain oleic acids, lactic acid, senolytic agent, senomorphic agent, or a combination thereof.

22. The composition of claim 17 or 18, wherein the one or more known composition agents is selected from BCL2, BCL2-XL, survivin, or any combination thereof.

23. The composition of any one of claims 17 to 22, wherein the angiogenic factor is selected from the group consisting of VEGF, HGF-1, FGF-1, FGF-2, angiopoietin, interleukin- 20, or any combination thereof.

24. The composition of any one of claims 12 to 17, wherein the neurogenic factor is selected from the group consisting of NGF, BDNF, CNTF, neurotrophins, or any combination thereof.

25. The composition of any one of claims 12 to 18, wherein the antiapoptotic factor is selected from the group consisting of molecules capable of increasing bcl-2 expression, molecules capable of decreasing BAD expression, molecules capable of increasing expression of bcl-2Xl, molecules capable of decreasing bcl-2Xs, molecules capable of decreasing expression of members of the caspase family, molecules capable of increasing surviving expression, molecules capable of increasing living expression; or any combination thereof.

26. A method for enhancing exosome production, the method comprising: treating a population of mesenchymal stem cells with an effective amount of a CpG and valproic acid.

27. The method of claim 26, wherein the CpG is ODN 2006 CpG DNA with a sequence 5'-TCCATGACGTTCCTGATGCT-3'.

28. The method of claim 26 or 27, wherein the method further comprises culturing a population of mesenchymal stem cells in a culture medium.

29. The method of claim 28, wherein the method further comprises isolating exosomes from the culture medium.

30. The method of any one of claims 26 to 29, wherein the exosomes comprise a molecular cargo that stimulates the proliferation of the exosomes from the mesenchymal stem cells.-MS-31. A method for stimulating angiogenesis, comprising administering to a subject or target tissue with a composition from any one of claims 11 to 25.

32. The method of claim 31, wherein exosomes from mesenchymal stem cells.

33. The method of claim 31 or 32, wherein stimulating angiogenesis promotes vascular repair.

34. The method of any one of claims 31 to 33, wherein the composition comprises valproic acid and ODN 2006 CpG DNA with a sequence 5'-TCC ATGACGTTCCTGATGCT- 3'.

35. A method for enhancing biological activity of a fibroblast, comprising contacting the fibroblast with an exosome isolated from a mesenchymal stem cell, wherein the mesenchymal stem cell was treated with the composition from any one of claims 11 to 25.

36. The method of claim 35, wherein the biological activity is fibroblast proliferation.

37. The method of claim 35, wherein the biological activity is collagen production.

38. The method of claim 35, wherein the contacting step results in a synergistic enhancement of the biological activity.

39. The method of any one of claims 35 to 38, wherein the composition comprises valproic acid and ODN 2006 CpG DNA with a sequence 5'-TCCATGACGTTCCTGATGCT- 3'.

40. A method for promoting wound healing or tissue repair, comprising administering to a target site a composition comprising exosomes isolated from mesenchymal stem cells treated with valproic acid and ODN 2006 CpG DNA with the sequence 5'- TCC ATGACGTTC CTGATGCT-31.

41. A method for enhancing a biological activity of a fibroblast, comprising contacting the fibroblast with an exosome isolated from mesenchymal stem cells, wherein the mesenchymal stem cells were treated with a combination of hyperthermia and ODN 2006 CpG DNA with a sequence 5 -TCCATGACGTTCCTGATGCT-3'.

42. The method of claim 41, wherein the biological activity is fibroblast proliferation or collagen production.

43. A method for modulating monocyte cytokine production, comprising:activating a population of adipose derived mesenchymal stem cells ex vivo with a toll-like receptor agonist; and contacting a population of monocytes with the activated adipose derived mesenchymal stem cells or a secretion product thereof.

44. The method of claim 43, wherein the modulation of monocyte cytokine production enhances an anti-inflammatory or regenerative response.

45. A method for enhancing cytokine production in mesenchymal stem cells, comprising: contacting mesenchymal stem cells with the composition from any one of claims 11 to 25.

46. The method of claim 45, wherein the composition comprises valproic acid, and gonadotropin, and the mesenchymal stem cells are further exposed to hyperthermia.

47. A method for synergistically enhancing cytokine production in mesenchymal stem cells, comprising contacting the mesenchymal stem cells with a toll-life receptor agonist and Li Cl.

48. A method for suppressing cytokine production in mesenchymal stem cells, comprising contacting the mesenchymal stem cells with LiCl in the absence of a toll-life receptor agonist.

49. A method for synergistically amplifying cytokine production in mesenchymal stem cells, comprising contacting the mesenchymal stem cells with the composition from any one of claims 11 to 25.

50. The method of claim 49, wherein the composition comprises a toll-life receptor agonist and the mesenchymal stem cells are further exposed to hyperthermia.

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