WO2025199245A1 - Method of making and using secretome composition - Google Patents

Abstract

The present disclosure relates generally to methods of producing secretome from pluripotent stem cells, and compositions and methods for treating or preventing a disease using the secretome. The secretome may be produced by a method including: expanding mammalian pluripotent cells in a growth media, wherein the growth media comprises one or more growth factors to provide expanded cells; at least partially differentiating the expanded cells to provide partially differentiated cells; exposing the partially differentiated cells to a protein-free media; collecting the protein-free media that was exposed to the partially differentiated cells; adding arginine-HCl to the collected media; and concentrating the collected media to provide a concentrate comprising the secretome.

Description

METHOD OF MAKING AND USING SECRETOME COMPOSITION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Patent Application No. 63/567,351, filed March 19, 2024, the content of which is incorporated herein by reference in its entirety in the present disclosure.

FIELD

[0002] Provided herein are cell culture derived secretomes with metabolic modifying properties and their use of various medical conditions that include metabolic disorders.

BACKGROUND

[0003] Metabolic dysregulations are disorders that affect all tissue and organs in the body. The hallmark manifestation is an increase in body weight, particularly in the waist area, associated with elevated fasting glucose, elevated blood pressure and dyslipidemia, as defined in a “metabolic syndrome”. If not addressed, the metabolic syndrome slowly progresses to established chronic comorbidities including type II diabetes mellitus, cardiovascular disease and non-alcoholic fatty liver. Due to the complex nature of metabolic disorders, no targeted treatment is available in the early stages. The current weight loss medications interfere with the appetite or anabolic pathways that result in undesired adverse events such as muscle loss, liver, cardiac, and renal toxicity. Additional “enhancing” drugs including anabolizing steroids and amphetamines carry the same, or more serious toxicity.

[0004] The most accepted approach for weight loss is to increase physical activity, to increase the catabolic rate with demonstrated benefits to all organs and systems. However, the increased physical activity regimen has low compliance from patients for many reasons.

[0005] A treatment that can help increase the metabolism without excessive physical effort, or the use of drugs with proven toxicity, is highly desired. SUMMARY

[0006] Herein, we evidence that a combination of cell signaling included in a defined cell secretome can affect multiple metabolic pathways and can correct dysregulations that occur in a metabolic syndrome with additional benefits to all tissues and organs in the body.

[0007] In one aspect, presented herein is an effect of secretome on the metabolism by elevated oxygen consumption, reduction and browning of the white adipose tissue, increased lean body mass, increased bone density,

[0008] In one aspect, present herein is the effect of secretome on the immune system by improved bone marrow stroma to bone marrow adipocytes proportion, response to viral infections, enrichment of naive T-cells in aged animals, and anti-inflammatory effect. In addition, presented herein is the effect of the secretome on wound healing in a model of burn wounds. Also presented herein is the effect of the secretome on age associated cognitive decline. We present the effect of the secretome on various cell types intended as potency assay for the pharmaceutical compositions described herein.

[0009] In one aspect, provided herein is a method for producing a secretome, the method comprising: expanding mammalian pluripotent cells in a growth media, wherein the growth media comprises one or more growth factors to provide expanded cells; at least partially differentiating the expanded cells to provide partially differentiated cells; exposing the partially differentiated cells to a protein-free media; collecting the protein-free media that was exposed to the partially differentiated cells; adding arginine-HCl to the collected media; and concentrating the collected media to provide a concentrate comprising the secretome.

[0010] In some embodiments, said one or more growth factors comprise FGF2 and Activin A. In some embodiments, said one or more growth factors further comprise TGFbl. In some embodiments, the growth media further comprises one or more amino acids, peptides, lipids, and/or salts. In some embodiments, the growth media comprises one or more amino acids selected from the group consisting of arginine, leucine, and isoleucine. In some embodiments, the growth media comprises 0.01 to about 1.5 g/L of L-arginine, about 0.05 g/L to about 0.5 g/L of L-isoleucine, about 0.05 g/L to about 0.5 g/L of L-leucine. In some embodiments, the peptide is taurine. In some embodiments, the one or more lipids comprise ethanolamine. In some embodiments, the ethanolamine is included in the structure of other lipids. In some embodiments, the salt comprises selenium or selenite. In some embodiments, the growth media comprises arginine, isoleucine, leucine, ethanolamine, taurine, sodium selenite, FGF2, Activin A and trehalose. In some embodiments, the growth media comprises about 0.01 to about 1.5 g/L of L-arginine, about 0.05 g/L to about 0.5 g/L of L-isoleucine, about 0.05 g/L to about 0.5 g/L of L- leucinc, about 0.015 g/L to about 1 g/L of cthanolaminc, about 0.25 g/L to about 1.5 g/L of taurine, about 1 pg/L to about 100 pg/L of sodium selenite, about 1 ng/mL to about 100 ng/mL of FGF2, about 0.5 ng/mL to about 50 ng/mL of Activin A, and about 0.5 g/L to about 32 g/L of trehalose. In some embodiments, the growth media comprises about 0.35 g/L of L-arginine, about 0.12 g/L of L-isoleucine, about 0.12 g/L of L-leucine, about 0.015 g/L of ethanolamine, about 0.25 g/L of taurine, about 5 pg/L of sodium selenite, about 10 ng/mL of FGF2, about 10 ng/mL of Activin A, and about 16 g/L of trehalose. In some embodiments, the mammalian pluripotent cells are human pluripotent cells. In some embodiments, the partially differentiating comprises removing the growth factors from the growth media. In some embodiments, the partially differentiated cells comprise tri-germ embryonic markers, or bi- or single germ embryonic layer markers. In some embodiments, the protein-free media comprises trehalose and one or protein stabilizers. In some embodiments, the protein-free media that was exposed to the partially differentiated cells is collected daily for multiple days. In some embodiments, one or more protein stabilizers adjuvants are added with arginine-HCl to the collected media, wherein the protein stabilizers are selected from sugars, polyols, amino acids, and surfactants. In some embodiments, wherein concentrating the collected media comprises concentrating with TFF with a cutoff membrane to retain molecules above 3 kDa. In some embodiments, the method further comprises fractionating the concentrate for pre-defined molecular weights intervals. In some embodiments, the method further comprises purifying the protein from the concentrate. In some embodiments, wherein the protein is purified by ultracentrifugation, precipitation, dialysis, gel filtration, or chromatography.

[0011] In one aspect, provided herein is a composition comprising secretome obtained by the method described herein. F00121 In some embodiments, total protein concentration in the composition is 0.1 to 50 mg/mL. In some embodiments, the secretome is enriched for exosomes. In some embodiments, the secretome is depleted of exosomes. In some embodiments, the composition further comprises one or more growth factors, one or more small molecules, one or more antibodies, and/or one or more extracellular matrices. In some embodiments, the composition further comprises one or more therapeutic drugs. In some embodiments, the composition is included in a biodegradable slow-release matrix. In some embodiments, the composition comprises one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients may be selected from arginine, proline, lysine, glutamic acid, glycine, histidine, trehalose, dextrose, sucrose, glycerol, mannitol, sorbitol, EDTA, dextran, phosphate butter, citrate buffer, Tris buffer, HEPES buffer, and a combination thereof. In some embodiments, the composition is in a liquid form. In some embodiments, the composition is lyophilized. In some embodiments, the composition is packaged in a vial for injectable solutions or in a self-administering syringe. In some embodiments, the composition is suitable for intra-muscular, intravenous, subcutaneous, intrathecal or intracerebral administration.

[0013] In another aspect, provided herein is a method of preventing or treating a disease, comprising administering the composition of the preceding paragraphs to a patient in need thereof.

[0014] In some embodiments, the disease includes muscle atrophy or muscle strength loss; the disease includes a metabolic dysfunction; the disease includes an immune dysfunction; the disease includes a regenerative dysfunction; the disease involves a need for plastic surgery and/or cosmetic procedure; the disease is associated with cognitive decline; or the disease is associated with aging.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a graph showing exemplary exosome characterization by micro-RNA (miRNA) content. F00161 FIG. 2 is a graph showing GO enrichment display of target genes predicted by the top 10 miRNAs.

[0017] FIG. 3 is a graph showing GO enrichment display of verified target genes of Top 10 miRNAs.

[0018] FIG. 4 is a graph showing KEGG enrichment display of target genes predicted by the top 10 miRNAs.

[0019] FIG. 5 is a graph showing the KEGG enrichment display of verified target genes of top 10 miRNAs.

[0020] FIG. 6 is a graph showing the DO enrichment display of target genes predicted by the top 10 miRNAs.

[0021] FIG. 7 is a graph showing the DO enrichment display of verified target genes of top 10 miRNAs.

[0022] FIG. 8 is a graph showing the reactome enrichment display of target genes predicted by the top 10 miRNAs.

[0023] FIG. 9 is a graph showing the reactome enrichment display of verified target genes of top 10 miRNAs.

[0024] FIG. 10 is a graph showing the alterations in T cell responses in aged (22-24 month old) mice versus young (4-6 month old) mice in response to secretome administration.

[0025] FIGs. 11A-H are graphs showing whole-body metabolic profile of control and secretome administered mice.

[0026] FIGs. 12A-H are graphs showing whole body tissue and physical function changes for control and secretome-treated mice.

[0027] FIGs. 13A-J are graphs showing whole-body metabolic profile of secretome- treated mice and control at base line, week 4, and after withdrawal from treatment. F00281 FIGs. 14A-G are graphs and photograph showing skeletal muscle morphology of control and mice administered with secretome.

[0029] FIGs. 15A-F are graphs and photographs showing skeletal muscle remodeling after administration of secretome.

[0030] FIGs. 16A-G are graphs and plots showing skeletal muscle transcriptional and translational responses after administration of secretome.

[0031] FIGs. 17A-I are graphs and photograph showing adipose morphology and muscle lipid content after administration of secretome.

[0032] FIGs. 18A-H are plots, graphs and photograph showing cellular experiments and data from the experiments showing changes to myotubes and adipose.

[0033] FIG. 19 is a graph showing physical function by three-patient dose group throughout enrollment in study evaluating safety and efficacy of secretome in participants with muscle atrophy related to knee osteoarthritis (KOA).

[0034] FIG. 20 is a graph showing patient-reported WOMAC outcomes by dose group throughout enrollment in study evaluating safety and efficacy of secretome in participants with muscle atrophy related to KOA.

[0035] FIG. 21 is a graph showing patient reported knee pain by dose group throughout enrollment in study evaluating safety and efficacy of secretome in participants with muscle atrophy related to KOA.

[0036] FIG. 22 is a graph showing patient reported effect of knee pain on quality of life throughout enrollment in study evaluating safety and efficacy of secretome in participants with muscle atrophy related to KOA.

[0037] FIG. 23 is a graph showing patient-reported physical functional capabilities throughout enrollment in study evaluating safety and efficacy of secretome in participants with muscle atrophy related to KOA. DETAILED DESCRIPTION

Definitions

[0038] The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0039] As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0040] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In certain embodiments, the term “about” includes the indicated amount ± 10%. In other embodiments, the term “about” includes the indicated amount ± 5%. In certain other embodiments, the term “about” includes the indicated amount ± 1%. In certain other embodiments, the term “about” includes the indicated amount ± 0.05%. Also, to the term “about X” includes description of “X.”

[0041] Also, the singular forms “a” and “the” include plural references unless the context clearly dictates otherwise. Thus, e.g., reference to “the compound” includes a plurality of such compounds and reference to “the assay” includes reference to one or more assays and equivalents thereof known to those skilled in the art.

[0042] Provided are also pharmaceutically acceptable salts, stereoisomers, mixture of stereoisomers, hydrates, solvates, solid forms, and tautomeric forms of the compounds described herein.

[0043] In many cases, the compounds of this disclosure are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. [00441 “Pharmaceutically acceptable” or “physiologically acceptable” refer to compounds, salts, compositions, dosage forms and other materials which are useful in preparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

[0045] The term “pharmaceutically acceptable salt” of a given compound refers to salts that retain the biological effectiveness and properties of the given compound, and which are not biologically or otherwise undesirable. “Pharmaceutically acceptable salts” or “physiologically acceptable salts” include, for example, salts with inorganic acids and salts with an organic acid. In addition, if the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare nontoxic pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like. Likewise, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines. Specific examples of suitable amines include, by way of example only, isopropyl amine, trimethyl amine, diethyl amine, tri(iso- propyl) amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0046] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0047] “Treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and/or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and/or preventing or delaying the spread (e.g., metastasis) of the disease or condition); and/or c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival).

[0048] ‘ ‘Prevention” or “preventing” means any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop. Compounds may, in some embodiments, be administered to a subject (including a human) who is at risk or has a family history of the disease or condition.

[0049] “Subject” or “patient” refers to an animal, such as a mammal (including a human), that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in human therapy and/or veterinary applications. In some embodiments, the subject or patient is a mammal. In some embodiments, the subject or patient is a human.

[0050] The term “therapeutically effective amount” or “effective amount” of a compound described herein means an amount sufficient to effect treatment when administered to a subject, to provide a therapeutic benefit such as amelioration of symptoms or slowing of disease progression. For example, a therapeutically effective amount may be an amount sufficient to decrease a symptom of a condition or disorder described herein. The therapeutically effective amount may vary depending on the subject, and disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, and the manner of administering, which can readily be determined by one of ordinary skill in the art.

[0051] As used herein, “secretome” or “secretomes” are the set of molecules and biological factors that arc secreted by cells, for example but not limited to pluripotent stem cells, into the extracellular space. “Secretome” or “secretomes” may refer to an individual molecule or a subset of such set of molecules and biological factors. Methods of preparing secretomes are also explained in International Patent Application Publication No. WO 2022/164854, published January 26, 2022, which is incorporated herein by reference in its entirety in the present disclosure.

[0052] The methods described herein may be applied to cell populations in vivo or ex vivo. “In vivo'’ means within a living individual, as within an animal or human. In this context, the methods described herein may be used therapeutically in an individual. “Ex vivo” means outside of a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples including fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the compounds and compositions described herein may be used for a variety of purposes, including therapeutic and experimental purposes. For example, the compounds and compositions described herein may be used ex vivo to determine the optimal schedule and/or dosing of administration of a compound of the present disclosure for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the compounds and compositions described herein may be suited are described below or will become apparent to those skilled in the art. The selected compounds may be further characterized to examine the safety or tolerance dosage in human or non-human subjects. Such properties may be examined using commonly known methods to those skilled in the art. Method of Producing Secretome

[0053] In one aspect, methods of producing secretome is provided. In some embodiments, a method of producing secretome includes: expanding pluripotent cells in a growth media, wherein the growth media comprises one or more growth factors to provide expanded cells; at least partially differentiating the expanded cells to provide partially differentiated cells; exposing the partially differentiated cells to a protein-free media; collecting the protein-free media that was exposed to the partially differentiated cells; adding argininc-HCl to the collected media; and concentrating the collected media to provide a protein concentrate comprising the secretome.

[0054] In a first step, the pluripotent stem cells may be expanded under any suitable conditions. In some embodiments, the mammalian pluripotent stem cells may be expended in a growth media.

[0055] Pluripotent stem cells can be of embryonic origin, extraembryonic teratocarcinoma origin or the result of induction (IPSCs). In some embodiments, the pluripotent stem cells are of animal origin. In some embodiments, the pluripotent stem cells are mammalian pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells.

[0056] In some embodiments, the cells are expanded for about 1 day to about 10 days, about 3 days to about 10 days, about 3 days to about 7 days, about 3 days to about 5 days, or about 1 day to about 7 days.

[0057] In some embodiments, the media may be serum free. In some embodiments, the media includes one or more growth factors. In some embodiments, the one or more growth factors include FGF2, Activin A, and/or TGFbl. In some embodiments, the media includes FGF2 and Activin A. In some embodiments, the media includes about 1-100 ng/mL of FGF2 and about 0.5-50 ng/mL of Activin A.

[0058] In some embodiments, the media may include at least one serum protein, an adherent substrate containing the laminin motif IKVAV (Ile-Lys-Val-Ala-Val), or aforementioned growth factors, such as FGF2 (bFGF) and/or Activin A. In some embodiments, the media may include proteins or biomolecules, such as albumin, transferrin, insulin, progesterone, thyroid hormones, laminin.

[0059] In some embodiments, the media includes one or more amino acids, arginine, leucine, and isoleucine. In some embodiments, the media includes about 0.01 to about 1.5 g/L of L-arginine, about 0.05 g/L to about 0.5 g/L of L-isoleucine, about 0.05 g/L to about 0.5 g/L of L- leucinc.

[0060] In some embodiments, the media includes a peptide, such as taurine. In some embodiments, the media includes about 0.25 g/L to about 1.5 g/L of taurine.

[0061] In some embodiments, the media includes one or more lipids, and the one or more lipids may include ethanolamine. The ethanolamine may be included in the structure of other lipids. In some embodiments, the media includes about 0.015 g/L to about 1 g/L of ethanolamine.

[0062] In some embodiments, the media includes one or more salts. The salt may include selenium. In some embodiments, the salt is selenite salt. In some embodiments, the salt is sodium selenite. In some embodiments, the media includes about 1 pg/L to about 100 pg/L of sodium selenite.

[0063] An exemplary basal media supplemented as described herein for stem cell expansion or differentiation can be a formulation such as DMEM-F12, RPMI or similar. In some embodiments, the media formulation contains amino acids. The amino acids may have the osmolarity adjusted to about 270 to about 300 mOsmol/L. The media may have a reduced amount of sodium salts and inclusion of ascorbic acid salt. The media may further include selenium, ethanolamine, and taurine. In some embodiments, the media may include variations of the concentration of amino acids.

[0064] In some embodiments, for successful expansion of the pluripotent stem cells, the combination of: selenium or a salt containing selenium such as sodium selenite; taurine; ethanolamine; and arginine at higher concentration is preferred. In some embodiments, the presence of Activin A, selenium and taurine in combination is preferred in the expansion media composition in addition to other well know components.

[0065] In some embodiments, the media includes about 0.35 g/L of L-arginine, about 0.12 g/L of L-isoleucine, about 0.12 g/L of L-leucine, about 0.015 g/L of ethanolamine, about 0.25 g/L of taurine, about 5 pg/L of sodium selenite, and about 10 ng/mL of FGF2, about 10 ng/mL of Activin A.

[0066] Exemplary components for the growth media is summarized in Table 1.

Table 1

[0067] A system comprising of a cell culture tank and automatic controls of dissolved oxygen and pH can be used to scale the production of large quantities of cells. Such bioreactors controlling essential parameters for cell culture are described elsewhere.

[0068] The expanded pluripotent stem cells can be partially differentiated. The originating cells for the secretome are partially differentiated pluripotent cells. For example, an exemplary secretome composition obtained from partial differentiated hiPSCs (human induced pluripotent stem cells) is shown in Table 2. The components are sorted for decreasing concentration and limited to top 400 components.

Table 2

[0069] The expanded pluripotent stem cells can be at least partially differentiated by changing the media composition, for example by removing and adding certain components. For example, the cells can be partially differentiated by removing certain growth factors from the media and exposing the cell to the modified media. In some embodiments, the partially differentiated cells comprise tri-germ embryonic markers, or bi- or single germ embryonic layer markers.

[0070] In some embodiments, the resulting secretome composition can be varied by differentiating cells into cells of different lineages, such as ectoderm, mesoderm or endoderm lineage. In some embodiments, a method to enhance the ectodermal secretome includes the exposure of pluripotent stem cells to neural differentiating signals known in the art. For example, the pluripotent stem cells may be exposed to a media composition that contains taurine, retinoic acid, selenium, ascorbic acid, thyroid hormone and insulin. In some embodiments, the media can include bFGF (FGF2) and noggin as additional morphogenic signals. The cell culture system may use a non-adherent hydrophobic substrate to encourage agglomeration of cells with homophilic adhesion molecules such as neural cell adhesion molecule (NCAM). The method may include enriching the composition for cells characterized by the presence of nestin, NCAM, vimentin and other neural lineage specific biomarkers.

[0071] Similarly, the secretome composition can be modified by preferentially differentiating into cells of the mesoderm lineage. In some embodiments, the mesoderm secretome may be prepared by isolating cells that are exposed to a substrate containing the RGD (arginine-glycine-aspartate) sequence and exposed to a media containing low amounts of bone morphogenic proteins that may be added separately, or as part of an animal serum supplement. In addition, the media may be supplemented with insulin, selenium, thyroid hormone, progesterone, FGF and optionally with EGF.

[0072] In some embodiments, an endodermic bias of pluripotent stem cell differentiation can be obtained by exposing the cells to a combination of a substrate containing the laminin motif IKV AV (Ile-Lys-Val-Ala-Val) and a media supplemented with Activin A, insulin, ascorbic acid FGF and optionally with retinoic acid and noggin.

[0073] Subsequent to the differentiation, the partially differentiated cells may be exposed to a protein-free media, such that the partially differentiated cells can produce secretome. In some embodiments, the protein-free media only includes components that are high purity and can be used for clinical human or veterinarian applications. For the production of secretome, the protein-free media herein may be supplemented with trehalose at a concentration of, for example, about 0.5 to about 32 g/L, about 1 to about 30 g/L, about 5 to 20 g/L or about 16 g/L, while the proteins added for the cell expansion described herein may be omitted. One or protein stabilizers may be further added.

[0074] After exposure to the protein-free media, the secretome may be collected. In some embodiments, the media exposed to the partially differentiated cells, which includes secretome, is collected daily for 1 to 10 days, after each overnight exposure to protein-free media. In some embodiments, the media may be collected in a batch feeding approach. In some embodiments, the media is collected while continuously feeding the cell cultures. In some embodiments, the collection may be performed in a closed system that limits environmental exposure of the secretome. In some embodiments, such system includes a refrigerated media reservoir, a media preconditioning system (warming, gas exchange), tubing, cell culture vessels, and/or refrigerated sterile lined collection tank. In some embodiments, the collection includes fractionating the concentrate for pre-defined molecular weights intervals.

[0075] In some embodiments, arginine (c.g., argininc-HCl) is added at a concentration of about 1 to about 10 g/L during, or at the end of collection, as a protein stabilizer to prevent agglomeration and denaturation. In some embodiments, the arginine-HCl is added at a concentration of about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, or about 10 g/L. In some embodiments, the protein stabilizers are selected from sugars, polyols, amino acids, and surfactants.

[0076] After collection, the collected media may concentrated with a suitable method. In some embodiments, the collected media may be concentrated tangential flow filtration (TFF) with a lower cutoff molecular weight. For example, the collected media may be concentrated with about 1 kDa to about 10 kDa filters, or with 3 kDa filters. In some embodiments, an upper cutoff filter can be also used to limit cell debris, and eliminate microbiological contamination. For example, a 0.1 pm filter may be used as an upper cutoff filter. During the TFF process, additional arginine-HCl can be added in a concentration of up to 100 g/L as a protein stabilizer. Additional protein stabilizers and excipients, including sugars, polyols, amino acids, and surfactants, can be introduced anytime during the downstream process to prevent aggregation. For example, glycerol, tris(hydroxymethyl)aminomethane hydrochloride, L-histidine, mannitol, L-proline Poloxamer 188, Sorbitol, glycine, sucrose and others, may be added alone or in combinations.

[0077] In some embodiments, the composition including secretome is enriched with exosomes produced by the same cell culture system. Additional exosome enrichment can be obtained by further concentrating the secretome by TFF or by centrifugation. Exosome purification can be obtained with a higher molecular weight cutoff TFF filter or by centrifugation. In an alternative embodiment an 100-200 kDa upper limit cut-off TFF filter can be used to produce a secretome that is substantially free of exosomes fractions.

[0078] In some embodiments, the concentrate is further purified, for example by ultracentrifugation, precipitation, dialysis, gel filtration, or chromatography, such that a purified secretome is provided.

[0079] Methods of preparing secretomes are also explained in International Patent Application Publication No. WO 2022/164854, published January 26, 2022, which is incorporated herein by reference in its entirety in the present disclosure.

Pharmaceutical Formulation

[0080] Provided herein are also pharmaceutical composition that include secretome, for example secretome produced by methods described herein. The pharmaceutical composition may be prepared for human or veterinarian use.

[0081] In some embodiments, the pharmaceutical composition includes components obtained by recombinant methods, such as growth factors, hormones, cytokines, and other biologically active molecules.

[0082] In some embodiments, the pharmaceutical composition includes other therapeutic drugs, for example a chemically synthesized drug. The secretome and the drug may show synergistic effects in combination.

[0083] In some embodiments, the pharmaceutical composition is included in a biodegradable slow-release matrix, to assist drug delivery.

[0084] In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable excipients, such as buffers, amino acids, stabilizers, antimicrobial preservatives and any other excipients. F00851 For examples, the pharmaceutical composition includes a buffer such as a phosphate buffer, citrate buffer, tris buffer, or HEPES, such that the composition can maintain pH in neutral range and prevent protein degradation.

[0086] In some embodiments, the pharmaceutical composition includes one or more amino acids, such as arginine, proline, lysine, glutamic acid, glycine, or histidine, which can prevent protein aggregation.

[0087] In some embodiments, the pharmaceutical composition includes one or more stabilizers, such as trehalose, dextrose, sucrose, glycerol, mannitol, sorbitol, or dextran, which has Kosmotropic effect and can stabilize the water phase and prevent protein misfolding.

[0088] In some embodiments, the pharmaceutical composition includes antimicrobial preservatives, such as benzyl alcohol, m-cresol, phenol, or 2-phenoxyethanol, to prevent microbial contamination during storage, or before or after administration.

[0089] In some embodiments, the pharmaceutical composition includes chelators such as EDTA, to prevent protein precipitation.

[0090] In some embodiments, the pharmaceutical composition includes one or more pharmaceutically acceptable excipients selected from arginine, proline, lysine, glutamic acid, glycine, histidine, trehalose, dextrose, sucrose, glycerol, mannitol, sorbitol, EDTA, dextran, phosphate butter, citrate buffer, Tris buffer, HEPES buffer, and a combination thereof.

[0091] In some embodiments, the pharmaceutical composition includes salts such as sodium chloride (e.g., 0.9%) for isotonic preparation. In some embodiments, the pharmaceutical composition includes cyclodcxtrins for lipid phase components, such as lipid modified proteins.

[0092] In some embodiments, the pharmaceutical composition includes arginine, trehalose, HEPES and phosphate buffer in 0.9% sodium chloride solution.

[0093] In some embodiments, the pharmaceutical composition is in liquid form. In some embodiments, the pharmaceutical composition is a lyophilized preparation. F00941 In some embodiments, the pharmaceutical composition is packaged in a suitable container. In some embodiments, the pharmaceutical composition is packaged in standard glass or polymeric vials for injection, or syringes (e.g., self-administering syringes). In some embodiments, the container may be a vial, jar, ampoule, preloaded syringe, and intravenous bag. In some embodiments, the pharmaceutical composition is formulated to be suitable for intramuscular, intravenous, subcutaneous, intrathecal or intracerebral administration.

[0095] In some embodiments, the pharmaceutical composition is formulated for prevention or treatment of the indications, including the diseases or conditions, described herein.

Treatment Methods and Uses

[0096] Provided herein are methods for preventing or treating disease or indications in a patient in need thereof, comprising administering the pharmaceutical composition including secretome as described herein. The secretome may be produced by methods described herein.

[0097] Exemplary applications target tissue regeneration, inflammation, and metabolic disfunctions resulting in a variety of diseases.

[0098] In some embodiments, the disease or indication may include muscle atrophy or muscle strength loss. In some embodiments, the disease or indication includes a metabolic dysfunction. In some embodiments, the disease or indication includes an immune dysfunction. In some embodiments, the disease or indication includes a regenerative dysfunction. In some embodiments, the disease or indication involves a need for plastic surgery and/or cosmetic procedure. In some embodiments, the disease or indication is associated with cognitive decline. In some embodiments, the disease or indication is associated with aging.

Dosing

[0099] The specific dose level of a compound of the present disclosure for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the subject undergoing therapy.

[0100] The compounds of the present disclosure or the compositions thereof may be administered once, twice, three, or four times daily, using any suitable mode described above. Also, administration or treatment with the compounds may be continued for a number of days; for example, commonly treatment would continue for at least 7 days, 14 days, or 28 days, for one cycle of treatment. Treatment cycles arc well known, and arc frequently alternated with resting periods of about 1 to 28 days, commonly about 7 days or about 14 days, between cycles. The treatment cycles, in other embodiments, may also be continuous.

EXAMPLES

[0101] The following examples are included to demonstrate specific embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques to function well in the practice of the disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1: Characterization of Exosome Content

[0102] Exemplary exosome content obtained in the concentrated secretome composition is presented in FIG. 1. FIG. 1 shows exemplary exosome characterization with dynamic light scattering (DLS) by micro-RNA content, where histogram of top 10 miRNA expression is shown.

[0103] The top 10 expressed miRNAs shown in FIG. 1 have functions associated with limiting inflammation, tumor suppression, suppressing adipogenesis. These targets were consistent with previously identified mechanisms of action for the secretome.

[0104] The predicted and experimentally verified target genes were subjected to functional enrichment analysis respectively. Specifically, it included Gene Ontology (GO), KEGG, disease ontology (DO), and rcactomc enrichment analysis. F01051 Gene Ontology (GO) is an internationally standardized gene function classification system that provides a set of dynamically updated standard vocabulary to comprehensively describe the properties of genes and gene products in organisms. GO has a total of three ontologies, which describe the molecular function (MF), cellular components (CC) and biological processes (BP) of genes, respectively. The basic unit of GO is term, and each term corresponds to an attribute. On the one hand, the GO function analysis gives the GO function classification annotation of the gene; on the other hand, it gives the GO function significance enrichment analysis of the gene. First, the gene to each term of the GO database (http://www.geneontology.org/) were mapped, and the number of genes in each term was calculated, so as to obtain the list of genes with a certain GO function and the statistics of the number of genes. Hypothesis testing was then applied to identify GO entries that were significantly enriched in genes compared to the whole genome background. The adjust p value (i.e. padj) was calculated by the Benjamini-Hochberg method (BH), and padj<0.05 was taken as the significant enrichment standard. FIG. 2 shows the GO enrichment display of target genes predicted by the top 10 miRNAs, and FIG. 3 shows the GO enrichment display of verified target genes of Top 10 miRNAs.

[0106] Pathway analysis helps to further understand the biological function of genes. KEGG is the primary public database on pathways. Pathway significant enrichment analysis takes KEGG Pathway as the unit, and applies super hypothesis test to find out the Pathways that are significantly enriched in genes compared with the whole genome background. The most important biochemical metabolic pathways and signal transduction pathways that genes participate in can be determined through the significant enrichment of pathway. The adjust p value (i.e. padj) was calculated with the Benjamini-Hochberg method (BH). FIG. 4 shows the KEGG enrichment display of target genes predicted by the top 10 miRNAs, and FIG. 5 shows the KEGG enrichment display of verified target genes of top 10 miRNAs.

[0107] The study of disease similarity plays an important role in the understanding of the pathogenesis of complex diseases, the early prevention and diagnosis of major diseases, the development of new drugs and the evaluation of drug safety. The enrichment analysis of disease ontology (DO) for differentially expressed genes has extremely high biological significance. FIG. 6 shows the DO enrichment display of target genes predicted by the top 10 miRNAs, and

FIG. 7 shows the DO enrichment display of verified target genes of top 10 miRNAs.

[0108] The reactome database is a collection of human reactions and biological pathways. Genes were mapped to each term of the Reactome database (https://reactome.org/), and calculated the number of genes in each term, so as to obtain a list of genes with a certain Reactome function and statistics on the number of genes. Hypothesis testing was then applied to identify Reactome pathways that were significantly enriched in genes compared to the whole genome background. FIG. 8 shows the reactome enrichment display of target genes predicted by the top 10 miRNAs, and FIG. 9 shows the reactome enrichment display of verified target genes of top 10 miRNAs.

[0109] In order to reduce the screening targets, the two target analysis results were intersected to obtain the common target genes of the two methods. In addition, the results of target genes verified by Luciferase reporter assay, qRT PCR, Northern blot, and EMSA experiments are generally considered to be more reliable.

Example 2: Hematopoietic Decline in Aging Adults

[0110] Secretome was administered to two-year-old aged male mice intramuscularly twice a week for four weeks. Peripheral stem and immune cell populations were measured by flowcytometry before the first administration of secretome and three days after the final administration. Stem and immune populations were then compared to baseline prior to treatment and against control mice treated with saline. Additionally immune populations were examined in the spleen and bone marrow which was also examined for changes in stem and progenitor populations in the stromal and hematopoietic lineages. Due to the systemic effects observed previously it is hypothesized that administration of secretome could lead to a remodeling of the stem and progenitor niche away from deposition of adipose potentially leading to an increase in hematopoietic output facilitated by an increase in stromal support for hematopoietic stem cells. This strategy could then be applied in the clinical setting to address hematopoietic decline in aging adults through secretome based remodeling of the hematopoietic niche and stromal compartment. [0111] Alterations in T cell responses in aged (22-24 month old) mice versus young (4-6 month old) mice were previously demonstrated. Specifically, aged animals exhibited an inverted CD4;CD8 ratio in blood and spleen, a shift in naive and memory T cells in old mice compared to young, and a trend towards an increase in a more “activated” phenotype e.g. CXCR3-expression and IFN-g expression in T cells in old mice compared to young mice.

[0112] Old mice were injected with the secretome 2x per week, for 3 weeks. As shown in FIG. 10, it demonstrated the decrease of the effector and memory T cells, while the total number of T cells was maintained 4 weeks post treatment. The CD4/CD8 ratio normalized (>1) from inverted status (<1), demonstrating reduced inflammation and improvement in immune system robustness

Example 3: Obesity and Metabolic Syndrome

[0113] The process of aging is accompanied by the progressive development of frailty, chronic diseases, and an impaired quality of life. While multifaceted, the severity of the aging phenotype is reported to be largely controlled by musculoskeletal and metabolic health, and their diverse cellular and molecular underpinnings. In fact, both skeletal mass and function as well as metabolic health are markedly impaired in older adults and associated to numerous negative health effects beneath the umbrella of aging. Aged skeletal muscle is often characterized by structural disorganization and fibrosis, neuromuscular degeneration, and blunted growth or impaired regenerative capacities. Similarly, adipose tissues exhibit metabolic dysregulation, adverse redistribution, and greater inflammatory profiles. Moreover, aged- induced muscle and adipose dysfunction, individually and combined (termed sarcopenic obesity), lead to generalized frailty, increased disease morbidity, and ultimately, mortality. Accordingly, there is great interest in the development of therapeutic strategies which combat the musculoskeletal and metabolic declines that coincide with increasing age.

[0114] It is well known that musculoskeletal, metabolic health, and adiposity can be improved by physical activity and exercise, as well as nutritional interventions. Despite the remarkable plasticity of skeletal muscle and adipose tissues aging is accompanied by anabolic resistance which diminishes the effectiveness of exercise and dietary interventions. This is exacerbated by sarcopenia, obesity and sarcopenic obesity which culminate to impair skeletal muscle and metabolic function. Moreover, older adults are often afflicted by conditions (e.g., pain following surgery) that limit their ability to maintain physically active lifestyles. Therefore, treatments which target specific age-related deficits in musculoskeletal health and adiposity that result in improved physical activity and physical function levels are of great interest.

[0115] Stem cells exist along a spectrum of differentiation with zygote formed and artificially induced pluripotent stem cells possessing boundless cellular fates. Stem cell therapies show promise in regenerative medicine, yet as direct (in-vivo) application can induce dysfunction, secondary techniques have arisen including the enrichment of culture media with stem cell secretory factors. These secretomes include numerous soluble and encapsulated (extracellular vesicles) signaling molecules (cytokines, growth factors) that exert cellular and tissue adaptive effects. Suitably, secretome products from various stem cell origins have been utilized to enhance muscular outcomes and combat the effects of aging in mice. Importantly, most research has utilized these products in a regenerative capacity, following muscular atrophy or damage. Conversely, it has been reported that intravenous treatment with extracellular vesicles from adipocyte derived stem cells improves muscular strength and function while decreasing frailty in old mice without previous intervention. Ostensibly, it is contemplated that stem cell secretome treatments may combat the effects of aging in skeletal muscle, yet the secondary whole-body effects including changes in adipose tissues and body composition, as well as metabolic function are unknown.

[0116] Therefore, the purpose of this study was to investigate the effects of twice weekly intramuscular treatment for 4 weeks with a pluripotent stem cell secretome product in aged mice on whole-body metabolism (energy expenditure, tissue composition, activity levels), physical function, as well as skeletal muscle and adipose tissue remodeling. Furthermore, we examined the acute muscular transcriptional responses to secretome treatment as well as the direct effects of the secretome product on adipocytes in vitro. We hypothesized that chronic intramuscular secretome treatment would ameliorate muscle aging (decrease muscle fibrosis, increase capillarity, stem cells, and myofiber hypertrophy) and adiposity while improving whole body energy metabolism and physical function capacity in aged mice. Moreover, we hypothesized that acute secretome treatment would directly reduce lipid accumulation in-vitro. Animals and Experimental Design

[0117] Male C57BL/6 mice (obtained from the National Institute of Aging rodent colony) began experiments at 22-24 months and finished at 23-25 months. Mice were maintained in a temperature controlled (22-23°C) facility on a 12:12-h light/dark cycle and housed with ad libitum food and water access. After one week or later of acclimating to their cages mice were assigned to either secretome treatment or saline control groups (n=16/group) in a matched fashion (body, fat, and lean mass). After the intervention, a subset of mice (n=6/group) underwent metabolic chamber measurements (detailed below). These same mice were utilized to examine the effects of secretome/s aline treatment and withdrawal for two weeks after the final injections. Most of the remaining mice (n=8/group) were fasted for ~4h and euthanized under isoflurane followed by cervical dislocation. Tissues (quadriceps, gastrocnemius, inguinal: I- WAT and epididymal: E-WAT fat pads, heart, liver) were dissected and weighed and placed in paraformaldehyde, frozen in liquid nitrogen, or frozen in optimal cutting temperature (OCT) (Fisher Scientific 23-730-571, Waltham, MA, USA) in isopentane, and stored at -80°C based on the specific analysis. All animal procedures were conducted in agreement with standards set by the University of Utah Institutional Animal Care and Use Committee.

Secretome Treatment

[0118] The secretome treatment was derived from partially differentiated pluripotent human embryonic stem cells (CSC14, CVCL_B918) using a method described herein. Cultured media collected from these cells was pooled, sterile filtered, concentrated, and prepared as a USP grade cell-free stem cell-based secretome product. Fifty microliters of secretome or saline (0.9% USP) were delivered to the right quadricep muscle via intramuscular injection under sterile conditions twice per week for 4 weeks (8 total injections/mouse). Secretome treatment was injected at a 0.4% concentration in saline based on pilot data and previous research.

[0119] The secretome product contains a host of soluble signaling molecules with prominent proteins regulating cellular growth, remodeling, and immunomodulation, as shown in Table 2. Extracellular vesicles from the secretome product were examined via nanoparticle tracking followed by microRNA isolation and next generation sequencing by an independent party (Creative Biolabs, Inc.). Extracellular vesicles had an average particle size of 118 (nm) and concentration 2.8¹² (particles/mL), while microRNA concentration was 23 ng/uL with a quality score of 30 (99.9%). The top 10 identified microRNAs and their validated target genes are presented in FIG. 1.

Body Tissue Composition and Physical Function Testing

[0120] A nuclear magnetic resonance (NMR) instrument (Bruker Minispec MQ20 NMR analyzer, Rheinstetten, German) was used to assess whole body tissue composition. In addition, whole-body strength, plus balance and coordination were assessed by grip strength and rotarod instrument, respectively, as we have conducted previously. NMR and grip strength were assessed weekly while rotarod was tested before treatment and then repeated after the 4- week intervention. Whole-body grip strength was assessed using a grip strength meter with a mesh wire attachment (Columbus Instruments, Columbus, OH, USA). After acclimation testing a week prior, mice were placed on the mesh wire and pulled by the base of their tail, parallel to the mesh wire. Peak force was recorded and an average of three trials was recorded. Balance and coordination were assessed using rotarod testing on a Rotamex-5 (Columbus Instruments, Columbus, OH). The speed began at O.lrpm/s and increased by 0.3rpm/s increments with the final recorded time when mice fell off the rotating bar. Each mouse performed the test three times, and an average time was recorded. Mice were acclimated on the rotarod two days prior to testing. All physical function tests were conducted by the same research personnel. To give indication regarding frailty, mice were categorized by percentiles at the four-week timepoint compared to values based on a modified murine frailty index including weight loss, weakness (grip strength), and walking speed (rotarod speed).

Metabolic Measurements

[0121] To measure whole-body metabolic parameters, a subset of mice was placed in metabolic cages (CLAMS; Columbus Instruments Comprehensive Lab Animal Monitoring System (Columbus, OH, USA, serial# 180072)) for 72h at the end of the 4- week experimental intervention. Mice were single-housed and acclimated for 48h with the final 24h data used for analysis. Respiratory exchange ratio (RER) was calculated from VCO2 production and VO2 consumption and energy expenditure was calculated by dividing heat production (kcal/hr) by body weight. Ambulatory activity was calculated by summing ambulatory beam breaks in the x, y, and z directions. Food intake was calculated from a food scale inside the CLAMS unit.

[0122] Prior to tissue collection following the 4- week experiments, separate mice from above performed a 120 min glucose tolerance test (10% glucose solution injected at Ig/kg) with tail blood sampling including assessment of fasting and 30 min fed insulin levels via Ultra- Sensitive Mouse ELISA kit (Crystal Chcm, Elk Grove Village, II, USA) per manufacturer recommendations .

Immunohistochemistry

[0123] Frozen OCT-embedded quadriceps (cut in a longitudinal plane) were sectioned at a thickness of 10pm using a Leica cryostat (CM186O, Lecia Biosystems, Wetzlar, DE) and maintained at -20°C until stained. Muscle sections were stained to assess myofiber cross- sectional area (CSA) and myosin heavy chain (MyHC) fiber type, satellite/muscle stem cell content (Pax7+), capillarization (CD31+), and collagen IV turnover as described previously. Detailed methodologies and reagents are reported in the Supplemental Methods.

Fat and Liver Histology

[0124] After dissection, a portion of each fat pad and the left lobe of the liver was placed in 4% paraformaldehyde for 24h, after which it was stored in 70% ethanol until analysis. Samples were submitted to Associated Regional and University Pathologists (ARUP) laboratories at the University of Utah and the Department of Pathology for hematoxylin and eosin (H&E) (fat pads) while livers were stained for H&E and Masson’s trichome. Briefly, samples were embedded in paraffin, sectioned at 5pm thickness then H&E stained to visualize lipid droplets, and trichrome stained to visualize tissue fibrosis. Slides were imaged on a Zeiss Slide Scanner Axio Scan.Zl (Carl Zeiss Inc.) with a lOx (fat pads, liver trichrome) or 20x (liver H&E) objective lens. Fat pad images were analyzed using a Fiji plugin Adiposoft as described by others to determine average adipocyte diameter for each sample across 3 randomly selected fields. Liver lipid accumulation was assessed from H&E images in Fiji, briefly, by thresholding to identify lipid droplets, analyzing particles, and removing erroneous areas from analysis. Liver fibrosis was analyzed using the Automated Fibrosis Analysis Toolkit plugin for Fiji as described elsewhere.

RNA Sequencing and Western Blotting

[0125] In a separate experiment to determine the acute effects of the secretome treatment on muscle, we delivered a single intramuscular treatment of secretome or saline to C57BL/6 mice (26-28 months old) in a fasted (4h) state. Three hours after injection, mice were euthanized and injected quadriceps were collected for bulk RNA sequencing, qPCR and immunoblotting. Hallmark, KEGG, and REACTOME pathways were identified using the fast gene set enrichment analysis in MSigDB using a 5% FDR. Data can be found on the Gene Expression Omnibus (GSE242211).

[0126] Injected quadriceps muscle from the acute and 4-week experiments as well as adipose pads from the 4-week investigation were additionally used to isolate RNA and/or protein for downstream real-time PCR and western blotting for anabolic, catabolic, and metabolic targets. Additional details on methodology can be found in the Supplemental Methods.

Lipidomics

[0127] A portion of injected quadricep muscle (-15 mg) from the 4-week experimental study was used to extract lipids and prepare samples for liquid chromatography mass spectrometry (LC-MS) metabolomic analysis as described elsewhere. Processed lipid metabolites including triglycerides, ceramides, and diacylglycerides with relative standard deviation less than 30% of quality controls were examined individually and as population totals. Detailed methods for all analyses can be found in the Supplemental Methods.

Adipocyte and Muscle Cell Culture Experiments

[0128] C2C12 myoblasts were grown to confluence then differentiated for -4 days under standard conditions in 6-well dishes. Media was replaced every 48 hours during growth and differentiation. Cultured myotubes were treated with 4% secretome product replacement for 24 hours based on previous experiments and analyzed for changes to myotube area and fusion index (n=6). Separate groups of control and treated (4% secretome) myotubes were used to produce culture media for 3 hours (n=6). Culture media was then placed on fully differentiated myotubes for 24 hours to assess autocrine and paracrine effects of cultured media followed by the same measurements described above (n=4). Cultured media replicates (n=7) and undiluted secretome product (n=3) were additionally tested for IL-6 concentration using a Mouse Quantikine ELISA Kit per manufacturer recommendations (R&D Systems, Minneapolis, MN, USA) Detailed methodologies and reagents can be found in the Supplemental Methods.

[0129] 3T3-L1 preadipocytes were prepared according to manufacturer recommendations then differentiated into adipocytes in 8-well chamber slides for 5-7 days until confluent, followed by 3 days of differentiation, and finally cellular maintenance and treatment. Media was replaced every 48-72 hours during growth and differentiation. Replicate wells of cultured adipocytes (n=7) were treated via 5% or 20% secretome product media replacement for 24 hours and compared to untreated controls. Adipocytes were stained and assessed for content as described elsewhere and in the Supplemental Methods. Separate groups (n=6) of control and treated adipocytes (20% secretome) with and without insulin treatment (100 nM) were then analyzed for insulin sensitivity via western blot (Akt phosphorylation, Ser473). Finally, groups of differentiated 3T3-L1 adipocytes (n=4) were treated with 20% media replacement using the C2C12 culture mediums above and assessed as previously described.

Statistical Analyses

[0130] All data are shown as mean ± SD. Where appropriate one-way or two-way ANOVAs were used with Holm-Bonferroni or Dunnett’s multiple comparisons test. If missing datapoints were present or groups unbalanced, mixed effects analysis were used in place of ANOVA. When comparing two groups at a single time point, unpaired and paired t-test were used where appropriate. Data sets were assessed visually for normality via Q-Q plots, skewedness and kurtosis, and tested with Shapiro-Wilks test if necessary. Statistical significance was set to p<0.05. GraphPad Prism (vlO.0.1, La Jolla, CA, USA) was utilized for all statistical analyses and figure assembly. Secretome treatment enhanced whole-body metabolic rate and physical activity levels in aged mice

[0131] To explore the whole-body metabolic effects of secretome treatment on aged mice, we performed 24 hours of metabolic profiling following 4-weeks of secretome treatment vs saline using CLAMS. As shown in FIGs. 11A and 11B, Oxygen consumption (VO2 - ml/kg/hr) and carbon dioxide production (VCO2 - ml/kg/hr) across 24 hours in comprehensive lab animal monitoring system (CLAMS) for control and sccrctomc-trcatcd mice. 24-hour average VO₂ (FIG. 11C) and VCO2 (FIG. 11D), 12-hour average dark cycle respiratory exchange ratio (RER - VCO2/VO2) (FIG. HE), 24-hour average energy expenditure (FIG. HF; kcal/kg/hr), activity (FIG. 11G; movement - X+Y+Z), and food intake (g) (FIG. 11H). All data presented as mean ± SD, white bars represent controls while blue squares represent secretome- treated mice, n-6 for both groups. * indicates significant difference between conditions for the indicated timepoints with * = p<0.05, ** = p<0.01, and *** = pcO.OOL

[0132] It was found that secretome-treated mice displayed greater oxygen consumption (FIG. 11C; 3347 ± 353 vs 2957 ± 233 ml/kg/hr, p<0.01), CO₂ production (FIG. 1 ID; 3237 ± 440 vs 2832 ± 312 ml/kg/hr, p<0.01) and dark cycle respiratory exchange ratio (FIG. 1 IE; p=0.02) compared to control mice. Accordingly, 24-hour energy expenditure (FIG. 1 IF; 0.016 ± 0.0 vs 0.014 ± 0.0, kcal/kg/hr p<0.01), physical activity levels (FIG. 11G; 1377 ± 603 vs 1041 ± 369 X±Y±Z, p<0.01), and food intake (FIG. 11H; 6.6 ± 1.3 vs 5.6 ± 1.2 g, p<0.01) were significantly elevated in secretome treated compared to control mice. Following 4-weeks of secretome treatment, mice did not have different fasting glucose (83.1 + 9.5 vs 90.0 ± 10.0 mg/dL, p=0.70) or responses to glucose stimulation following an i.p. glucose tolerance test measured by area under the curve analysis (150.3 ± 52.8 vs 143.8 ± 41.7 au, p=0.81) compared to control mice. Similarly, insulin levels were not different at fasting (0.55 ± 0.13 vs 0.46 ± 0.06 ng/mL, p=0.20) or following glucose injection (0.97 ± 0.16 vs 0.98 ± 0.07 ng/mE, p=0.96) comparing secretome treated and control mice. Moreover, HOMA-IR (fasting insulin x fasting glucose) was not different between the groups (2.0 ± 0.6 vs 1.8 ± 0.2 au, p=0.46).

Secretome treatment increased lean mass and physical function and reduced whole body fat mass in aged mice F01331 To identify the whole-body metabolic effects of the secretome treatment, we analyzed the weekly effects of the secretome treatment on whole-body tissue content via NMR spectroscopy, the impact on physical function (grip strength and rotarod performance) at baseline and at the end of the 4-weeks treatment, and localized effects on specific tissues (quadriceps, I- WAT, E-WAT). Specifically, weekly changes lean mass (FIG. 12A), fat mass (FIG. 12B), body fat % (FIG. 12C), and body mass (FIG. 12D) for control and secretome-treated mice. Weekly whole-body grip strength (FIG. 12E) and change (A) in rotarod performance time from baseline (FIG. 12F). Quadriceps mass (FIG. 12G) and fat mass (FIG. 12H) following 4-wccks. Control mice are represented by black circles and bars while secretome-treated mice are noted by blue squares and bars. All data presented as mean ± SEM. A-H: control mice (n=15), secretome- treated mice (n=16). Weekly changes analyzed via mixed-effects model with Holm-Bonferroni multiple comparisons plus planned comparison t-tests at the 4-week timepoint. # indicates significant difference (p<0.05) from baseline for respective group. * indicates significant difference (p<0.05) between groups at the indicated time point.

[0134] It was discovered that the secretome treatment progressively increased body mass and decreased body fat percentage over baseline levels, which were driven by consistent increases in lean mass and decreases in fat mass (FIGs. 12A-D). Conversely, aged mice treated with saline showed a decrease in body mass compared to baseline (FIG. 12D), likely as result of modest decrements in lean mass (FIG. 12A) by week 4. Group comparison after 4 weeks treatment demonstrated greater lean mass (24.8 ± 1.3 vs 23.6 ± 1.1 g, p=0.02), lower fat mass (2.9 ± 1.3 vs 4.3 ± 1.4 g, p=0.02) and lower body fat percentage (12.5 ± 3.7 vs 8.4 ± 3.7 %, p<0.01) in secretome-treated mice relative to control mice (FIGs. 12A-C). Assessment of strength, balance and coordination demonstrated that 1) whole-body grip strength was increased over baseline strength at 3 and 4 weeks in secretome-treated mice, 2) secretome-treated mice were significantly stronger than control mice at the 4-week timepoint (2.5 ± 0.2 vs 2.3 ± 0.3 N, p=0.03) (FIG. 2E), and 3) secretome-treated mice, but not saline-treated controls, had increased rotarod performance time compared to baseline performance (A 9.5 ± 14.3 sec, p=0.03 (FIG. 12F). Individual values for all described measures at baseline and Week 4 are presented in FIGs. 13A-D. Finally, though frailty index was not able to be fully characterized, we noted a greater proportion of control versus the secretome-treated mice were below or equal to the 50^th (62.5 vs 33.3 %) and 25^th (37.5 vs 6.7%) percentiles of the combined population considering the weight loss, weakness, and walking speed categories (data not shown).

[0135] To assess persistence of the described whole-body tissue composition and physical function effects, in a separate group of mice, we ceased the secretome treatment for two weeks (withdrawal) following the 4-week treatment period and compared this response to the 4- week treatment timepoint for whole body tissue composition outcomes, grip strength, rotarod performance, and tissue weights. In FIGs. 13A-J, baseline, 4-wcck, and withdrawal from treatment values were shown: baseline and week 4 lean mass (FIG. 13A), fat mass (FIG. 13B), body fat % (FIG. 13C), and body mass (FIG. 13D) for control and secretome treated mice; body (FIG. 13E), lean (FIG. 13F), and fat mass (FIG. 13G), body fat % (FIG. 13H), grip strength (FIG. 131; Newtons), and change (A) in rotarod performance time (FIG. 13 J) comparing posttreatment to two weeks withdrawal after 4 weeks of secretome treatment. All data presented as mean ± SD. FIGs. 13A-D: n=15-16 for each group, FIGs. 13E-J: n=7 for each group. Analyzed via t-tests or two-way ANOVAs with Holm-Bonferroni comparison. # indicates significant difference (p<0.05) compared to baseline for respective group. * indicates significant difference between conditions with * = p<0.05, ** = p<0.01, and *** = pcO.OOl. We found that following two weeks of withdrawal in previously secretome-treated mice, that the prior gains in body mass (FIG. 13E) and lean mass (FIG. 13F) were lost. However, secretome-mediated adaptations in fat mass, % body fat, grip strength, and rotarod performance (FIG. 13G-J) were maintained.

[0136] Next, the localized effects of secretome treatment on specific tissues (quadriceps, I-WAT, E-WAT) were assessed. After 4 weeks, secretome-treated mice had greater right (190.8 ± 18.9 vs 165.3 ± 16.7 mg, p<0.01) and left (191.0 ± 15.9 vs 169.2 ± 23.7 mg, p=0.02) quadriceps mass compared to control mice (FIG. 12G). Furthermore, secretome-treated mice had lower I-WAT (462.0 ± 103.4 vs 591 .7 ± 152.9 mg, p=0.03) compared to control mice, while E- WAT did not differ (FIG. 12H). Finally, gastrocnemius, liver, and heart weights were not different between groups at 4-weeks (p>0.05), as shown in Table 3. Table 3 shows tissue and organ weights following 4-weeks for control and secretome treated mice corrected to body mass. Muscle tissues include: right, left, and combined ratios for quadriceps and gastrocnemius. Adipose tissues include I-WAT, E-WAT, and combined depots. Secondary tissues include heart and liver weights. All data presented as mean ± SD. n=8 . Analyzed via t-tests. * indicates significant difference between conditions.

Table 3

Secretome treatment increased myofiber size and promoted myofiber remodeling and transcriptional reprogramming in aged mice

[0137] We next used immunohistochemistry to analyze the effects of 4 weeks secretome treatment on quadricep muscle morphology (CSA, feret diameter, fiber type) and cellular remodeling events (satellite cells, capillarization, collagen IV turnover), physiological parameters that are notably disrupted in muscle aging. Skeletal muscle morphology after 4 weeks of secretome treatment are shown in FIGs. 14A-14G: average quadriceps fiber cross sectional area pm2 (FIG. 14A); minimum feret diameter p m2 (FIG. 14B); fiber type proportion (FIG. 14C; MyHC - Ila/IIb %) for control (n=8) and secretome treated (n=8) mice; and size distribution (%) for total (FIG. 14E), Ila (FIG. 14F) and lib (FIG. 14G) fiber types across 500 p m2 increments. Representative histochemical image of fiber type including cell border with laminin, MyHC Ila, MyHC lib, and scale bar of 50 pm is shown in FIG. 14D. All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome-treated mice. n=8 for each group. Analyzed via t-tests and mixed effects models with Holm-Bonferroni comparisons. * indicates significant difference between groups at indicated category with * = p<0.05, ** = P<0.01, *** = PcO.OOl.

[0138] After 4- weeks of secretome treatment, mice had greater average myofiber CSA (FIG. 14A; 2514 ± 186.9 vs 2248 ± 245.4 pm², p=0.03) and minimum feret diameter (FIG. 14B; 48.7 ± 2.3 vs 45.4 ± 2.2 pm², p=0.01 ) compared to saline-treated controls. Fiber type % (MyHC Ila and lib) was not different between groups (FIG. 14C) nor was fiber size altered based on fiber type (data not shown). However, secretome-treated quadriceps displayed a rightward shift in the size distribution of total, Ila and lib fibers (FIG. 14E-G). Notably, analysis by 500 pm² increments demonstrated that the proportion of total fibers (FIG. 14E) >500-1500 pm²were lower while fibers >3500-4000 pm² were greater comparing secretome-treated to control mice. Additionally, there were less Ila fibers (FIG. 14F) between >500-1000 pm² and more fibers >1000-1500 pm² in secretome-treated muscles. Similarly, secretome-treated mice displayed a lower proportion of lib fibers (FIG. 14G) >500-1500 pm² and a higher proportion >2500-4000 pm² compared to controls. Representative histochemical images of fiber type are shown in FIG. 14D.

[0139] In parallel with changes to muscle morphology, secretome treatment improved cellular remodeling events in the injected quadricep muscle, affecting satellite cell content, capillarization, and collagen IV turnover. FIGS. 15A-15F shows skeletal muscle remodeling events: muscle satellite cell content (Pax7⁺/DAPI⁺ - co-localization) corrected to number of fibers (FIG. 15A); representative image of satellite cell localization with PAX7⁺ in off-red/pink, laminin fiber borders in green, DAPI in blue, satellite cells indicate by white arrows, and scale bar of 50 pm² (FIG. 15B); fiber capillarization (CD31⁺) corrected to number of muscle fibers (FIG. 15C). Representative image of capillarization with CD31⁺, laminin, and DAPI is shown in FIG. 15D. Ratio of B-CHP to COL-IV is shown in FIG. 15E and representative image with B- CHP and COL-IV is shown in FIG. 15F. All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome-treated mice. Control mice (n=8), secretome-treated mice (n=7-8), right (injected) quadriceps assessed. Analyzed via t-tests. * indicates significant difference between conditions for the indicated timepoints with * = p<0.05, ** = p<0.01. Specifically, compared to saline treatment, secretome-treated quadriceps showed greater satellite cell content per fiber (Pax7⁺; 0.09 ± 0.06 vs 0.02 ± 0.02 cells/fiber, p=0.02) (FIG. 15 A) and greater absolute satellite cell count (corrected to total fiber area) (data not shown). In addition, secretome-treated muscles displayed greater capillary to fiber content (number of CD31⁺per muscle fibers) (FIG. 15C; 4.7 ± 1.2 vs 3.1 ± 0.5 CD31⁺/fiber, p<0.01) as well as greater total CD31⁺ area and CD31⁺area: fiber ratio (data not shown). Finally, using the ratio of Collagen hybridizing peptide (B-CHP) to collagen IV (COL-IV) as an indicator of collagen turnover, secretome-treated muscles had greater Col IV turnover (FIG. 15E; 1.21 ± 0.1 vs 0.99 ± 0.1, p<0.01). This effect was the result of greater B-CHP and lower COL-IV content in secretome-treated muscles, which were not significantly different on their own.

[0140] As a follow up to the 4-week treatment, we performed a single unilateral right quadriceps injection with the secretome product or saline in a subset (n=10) of aged male C57BL/6 mice (26-28 months old) for analysis of acute transcriptional responses in muscle. After three hours, injected quadriceps were harvested for bulk RNA sequencing and pathway analysis (Hallmark, KEGG, Reactome). FIG. 16A shows a volcano plot identifying transcriptional divergence following acute (3-hour) intramuscular (quadriceps) treatment with secretome product compared (log2 fold change) to saline treated controls. Significant gene transcripts (log 10 adjusted p<1.3=p<0.05) identified as positive and negative directionality. Top 10 significantly regulated (positive and negative) genes comparing conditions are shown in FIG. 16B. Top 10 significantly regulated (enrichment) transcriptional pathways across Hallmark, KEGG, and REACTOME databases is shown in FIG. 16C. n = 10 for each group, * indicates significant difference between conditions with * = p<0.05, ** = pcO.Ol. Consistent with the above observed whole body and cellular remodeling events, muscle transcriptomic analysis revealed increases in hypertrophy and mitochondrial-related pathway enrichment as well as downregulation in inflammatory and immune-related responses following acute secretome injection in comparison to controls.

[0141] We additionally conducted western blotting on notable anabolic and catabolic protein phosphorylation events following acute injection of the secretome and found that secretome-treated mice had increased 4E-BP1 phosphorylation compared to control mice (1.13 ± 0.09 vs 1.0 ± 0.12, p=0.01). However, other relevant protein targets (p-mTOR, p-rps6k, p- SMAD2/3, p-ERKl/2, p-FOXO3a, PGC-l a) were not significantly different between groups. Skeletal muscle protein phosphorylation status for acute secretome treated mice and controls are shown in FIG. 16D and representative western blot images are shown in FIG. 16E. Skeletal muscle gene expression following acute secretome and control treatment is shown in FIG. 16F (n = 2-3 per group) and 4-weeks secretome and control treatment are shown in FIG. 16G (n = 4 per group). * indicates significant difference between conditions with * = p<0.05, ** = pcO.Ol. Assessment of a series of proteolytic and atrophy-related gene targets as reported elsewhere by qPCR revealed lower expression levels of E3 ubiquitin ligases (MUSA1, Traf6, FBXO32) in the secretome treated group. Interestingly, at the 4-week timepoint these transcriptional responses were not different between groups suggesting a transient activation following secretome treatment.

Intramuscular secretome treatment reduced adiposity in a depot-specific manner in old mice

[0142] As our analysis revealed robust secretome treatment effects on whole body adiposity, we next investigated the localized effects on adipose compartments, namely I-WAT and E-WAT depots following 4-weeks of treatment. FIGs. 17A-17I shows adipose morphology and muscle lipid content: average cellular diameter pm of 1-WAT (FIG. 17A) and E-WAT (FIG. 17B) depots; size distribution (%) of I-WAT (FIG. 17C) and E-WAT (FIG. 17D) cells across 5 pm increments; and average liver lipid droplet (FIG. 17E) and fibrosis (FIG. 17F; trichrome staining) area (%) as assessed with H&E. Representative image of I-WAT and E-WAT depots as well as liver H&E and trichrome staining with scale bar 50 pm is shown in FIG. 17G. Protein phosphorylation status for protein kinase B (Akt) and hormone sensitive lipase (HSL) for I-WAT and E-WAT depots and representative western blot image is shown in FIG. 17H. Muscle lipid content including total triglycerides (TAGs) diglycerides (DAGs), ceramides (Cer), and C18:0 ceramide (C18:0 Cer) is show in FIG. 171. All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome treated groups. FIGs. 17A-17G: n=8 for each group, FIG. 17H: n=7 for control, n=6 for secretome, FIG. 171: n=6 for control, n=8 for secretome. Analyzed via t-tests (FIGs. 17A, B, E, F, H, I) or two-way ANOVA with Holm-Bonferroni comparison (FIGs. 17C, D). * indicates significant difference between groups at indicated category with * = p<0.05, ** = p<0.01.

[0143] Wc found that average I-WAT diameter (FIG. 17A; 13.8 ± 2.8 vs 19.8 ± 4.3 pm, p<0.01) were lower in secretome-treated mice while average E-WAT diameter was not different between groups (FIG. 17B). Assessment of adipocyte size distribution in I-WAT and E-WAT depots demonstrated a leftward shift in secretome-treated mice compared to controls. Specifically, there were a greater proportion of smaller adipocytes in the I-WAT and E-WAT depots such that I-WAT cells (FIG. 17C) sized 1-10 pm (33.5 ± 8.7 vs 13.8 ± 8.4 %, p<0.01) and 10-15 m (31.9 ± 9.7 vs 20.6 ± 11.4 %, p=0.02) and the proportion of E-WAT cells (FIG, 17D) <15 pm (34.3 ± 10.6 vs 11.3 ± 11.3 %, p=0.03) was higher in secretome-treated mice compared to control mice. We further assessed protein phosphorylation in both adipose depots and found that secretome-treated mice had increased I-WAT Akt phosphorylation (2.7 ± 2.1 vs 1.0 ± 0.40, p=0.04) and E-WAT hormone sensitive lipase (HSL) (2.3 ± 1.1 vs 1.0 ± 0.7, p=0.03) phosphorylation compared to controls (FIG. 17H). There were no differences in liver lipid accumulation (droplet area) or fibrosis (trichrome staining) between groups (FIGs. 17E and 17F). Finally, lipidomic analysis of 4-wcck treated quadriceps muscle tissue revealed a significant decrease in total intramuscular triglycerides (1.0xl0⁵± 5.5xl0⁵ vs 5.5xl0⁵ ± 4.9xl0⁵ pmol/mg, p=0.04) and ceramides (519.1 ± 189.5 vs 1152 ± 782.8 pmol/mg, p=0.04) as well as the muscle- enriched C18:0 ceramide (334.7 ± 128.6 vs 813.3 ± 591.9 pmol/mg, p=0.04) in secretome- treated muscle. Diglyceride content was not different (3494 ± 1693 vs 6164 ± 3651 pmol/mg, p=0.09) following secretome-treatment compared to control mice (FIG. 171).

Secretome treatment altered muscle and adipocyte cells in-vitro through direct and indirect mechanisms

[0144] To explore the direct and indirect effects of the secretome product on muscle and adipose tissues, a series of experiments using myotubes and 3T3-L1 preadipocytes in-vitro. Based on our previous experiments, we first confirmed that 4% media replacement with secretome product for 24h increased myotube area (31.8 ± 3.8 vs 19.2 ± 3.5 %, p=0.01) and myonuclear fusion index (0.46 ± 0.04 vs 0.32 ± 0.09 au, p=0.03) compared to untreated controls. Average myotube area (%) is shown in FIG. 18B and myonuclear fusion index (au) is shown in FIG. 18C, for control, secretome treated, control CM, and secretome CM conditions in differentiated C2C12 myotubes.

[0145] To further examine the indirect effects of muscle secretome treatment, we treated a separate group of differentiated myotubes with 4% secretome replacement for 24h, then allowed them to produce fresh culture media for 3h. This cultured media was tested for IL-6 content, then the media was used to treat both fully differentiated myotubes and adipocytes for 24h followed by the same analyses as described previously. The cell culture design for media replacement and cultured media (CM) experiments is shown in FIG. 18 A. F01461 Interleukin 6 (IL-6) content in the culture media collected from secretome treated

(4%) and control C2C12 myotubes (Fold change) is shown in FIG. 18D. IL-6 was present in control culture media (132.08 ± 7.5 pg/mL) but was over three times as concentrated in the culture media generated from secretome-treated myotubes (424.02 ± 29.29 pg/mL, p<0.01). We additionally tested undiluted secretome product (n=3) and found considerably lower IL-6 concentrations (9.86 ± 2.26 pg/mL), confirming the increased IL-6 in secretome-treated culture media originated from muscle cells. Compared to cultured media from untreated cells, culture media from sccrctomc-trcatcd C2C12 increased myotubc growth (FIG. 18B; 45.93 ± 10.1 vs 29.06 ± 7.7 %, p<0.01) but not fusion index (Fig. 6C; 0.52 ± 0.08 vs 0.40 ± 0.08 au, p=0.16). Additionally, myotube area was greater for secretome cultured media treated cells than standard control (p<0.01) and direct secretome-treated (p=0.01) conditions (FIGs. 18B-C).

[0147] Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3-L1 adipocytes for control and 20% media replacement with culture media from control and secretome treated C2C12 cells (G). Representative images of myotubes (H) and adipocytes treated with secretome (I) and culture media (J). B-G: n=4-7 per group or replicate. Analyzed via one-way ANOVA with Holm-Bonferroni comparison (B-C, E, G) or t-tests (D, F). * indicates significant difference between groups as indicated with * = p<0.05, ** = p<0.01, *** = p<0.001.

[0148] Next, we performed 24h secretome treatment in 3T3-L1 cells using a low (5%) and high (20%) proportion of media replacement. Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3-L1 adipocytes for control, 5 and 20% secretome product media replacement is shown in FIG. 18E. 3T3-L1 adipocytes treated with 5% secretome replacement were not different while 20% replacement robustly decreased the amount of area occupied by lipids corrected for the area of DAPI (0.24 ± 0.13 fold change, p<0.01) compared to untreated controls. Similarly, when corrected to the area occupied by cell body or number of DAPI, the reported effects were the same (data not shown). Furthermore, 20% secretome replacement decreased the number of lipid droplets per cell, corrected to DAPI count, (0.31 ± 0.11 fold change, p<0.01) compared to untreated controls. Notably, the average size of lipid droplets was additionally lower for 20% secretome replacement (1.9 ± 0.5 vs 2.7 ± 0.6 pm, p=0.02) compared to control cells while 5% replacement was not different. Additionally, phosphorylated corrected to total Akt protein (fold change) for 20% secretome treated and control 3T3-L1 adipocytes following overnight fast and insulin (100 nM) stimulation is hsown in FIG. 18F. 3T3-L1 cells treated with 20% secretome replacement displayed enhanced insulin sensitivity as indicated by greater insulin-stimulated Akt phosphorylation compared to untreated controls (3.58 ± 0.67 vs 1.00 ± 0.10 fold change, p<0.01).

[0149] Finally, we tested the indirect effects of secretome treatment in 3T3-L1 adipocytes by replacing 20% of media with the previously described cultured mediums. Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3-L1 adipocytes for control and 20% media replacement with culture media from control and secretome treated C2C12 cells is shown in FIG. 18G. Culture media from secretome treated C2C12 reduced lipid area (0.63 ± 0.5 fold change, p<0.01) while untreated C2C12 culture media did not (0.82 ± 0.11 fold change, p=0.11). Secretome treated C2C12 culture media did not significantly reduce the number of lipid droplets (0.84 ± 0.15 fold change, p=0.15). Notably, preliminary testing with 1% and 5% culture media replacement had no effect on 3T3-L1 outcomes regardless of condition (data not shown).

[0150] Representative images for myotubes are presented in FIG. 18H. Representative images for direct and indirect 3T3-L1 experiments are presented in FIGs. 181- 18 J, respectively.

[0151] Here we examined the effects of bi-weekly intramuscular treatment in aged mice for 4 weeks with a stem-cell derived secretome product that is enriched with extracellular vesicles, and matrix, immunomodulatory, and growth factors. We found that, in 4 weeks, secretome treatment increased lean mass and enhanced local muscle cellular remodeling including reductions in intramuscular lipid content. Remarkably, the secretome treatment enhanced whole body energy expenditure, physical activity, and physical function while reducing whole-body and local adiposity. Moreover, direct secretome treatment in-vitro robustly enhanced muscle cell growth and reduced lipid droplet content. Finally, cultured media from secretome-treated muscle cells displayed autocrine and paracrine functions stimulating muscle cell growth and lipid droplet reduction in naive cells. Together, 4-weeks administration of a stem-cell secretome ameliorated several hallmarks of aging in mice resulting in improvements in physical function, metabolic rate, adiposity, and muscle cellular remodeling. F01521 A major finding of this current investigation was that intramuscular secretome treatment in aged mice decreased whole-body adiposity, reduced fat depot mass and adipocyte size, increased insulin sensitivity and lipolysis (Akt and HSL phosphorylation) in adipose tissues and improved whole-body metabolic rate. As adverse body fat accumulates with age, the reduction of fat mass following the secretome treatment and the maintenance of this loss after treatment cessation are impactful. Excessive adiposity with age contributes to reduced capacity to perform or adapt to physical activity, which was also improved following secretome treatment. Previous research demonstrated that 4-wccks of daily treadmill running modestly increased metabolic rate (VCO2 and RER) in old mice while decreasing body fat to levels in young counterparts. Accordingly, the robust increases in metabolic outcomes (VO2, VCO2, RER, activity levels) and reductions in whole-body and localized fat depots (I-WAT, E-WAT) with secretome treatment alone across a similar timeframe as exercise are very promising.

[0153] The increased activity was reflecting a food seeking behavior with systematic inspection of the food distribution area and identification of activities around the cages. The behavior was suggestive for improved spatial navigation, object recognition and associative learning, characteristics of improved cognitive process.

[0154] We also report increased whole-body lean mass, as well as greater quadriceps mass and myofiber size, with an emphasis on type II fiber growth, following 4-weeks of unilateral intramuscular secretome treatment. Additionally, grip strength progressively increased over time and persisted with two-weeks of withdrawal independent of lean mass maintenance. These responses were underpinned by robust muscular remodeling including greater collagen IV turnover, capillarization, and muscle stem (satellite) cell content which are known to enhance muscular function, regeneration, and recovery from disuse atrophy. Furthermore, the adaptations to muscle mass and fiber type with secretome treatment are important for maintaining body composition, metabolism, and muscle function with age and perhaps lends some evidence to the observed improvements to whole-body metabolic and grip strength. Cumulatively, the rapid increase of muscle size, strength, and remodeling following secretome-based treatment could be relevant for at risk aging populations including those affected by health conditions including metabolic dysfunction and sarcopenia. F01551 Consistent with reduced adiposity and enhanced leanness, we observed lower muscle triglycerides and ceramides following 4- weeks of secretome treatment. The accumulation of skeletal muscle lipids including triglycerides and ceramides are associated with impaired metabolic health and the development of metabolic diseases. Accordingly, increased intramuscular triglyceride turnover and reductions in storage have positive effects on the health of humans and animals. Improved triglyceride handling is further associated with decreases in muscle ceramides, particularly ceramide Cl 8:0, which is linked to insulin resistance and the development of type 2 diabetes. In fact, reducing C18:0 ceramide content in the skeletal muscle of mice improves whole-body metabolic health. Moreover, muscle ceramides have been shown to promote muscle atrophy while genetic ablation of ceramides enhanced mitochondrial function and proteostasis in aged mice. Therefore, reductions in muscle lipids, specifically muscle ceramides, may be a possible mechanism for the improved muscle and physical function following secretome treatment.

[0156] The secretome product also enhanced skeletal muscle remodeling in aging noted by higher levels collagen IV turnover, capillarization, and muscle stem cell (satellite cell) content. Interestingly, excessive collagen deposition not only impairs contractile function, but also the ability for satellite cells to proliferate and infiltrate the extracellular muscle environment. Similarly, greater muscle capillarization, and thereby perfusion, enhances satellite cell dynamics and promotes muscular recovery following damage which is diminished, yet, reversable in aging. Accordingly, the robust increase in muscle satellite cell expansion observed here and what we have reported previously, could be partially driven by enhanced cell migration triggered by enhance perfusion and increased collagen turnover. Noting that satellite cell content and function are limited in aging, an enhanced satellite cell pool would be beneficial to promote muscle regrowth following disuse atrophy and/or muscle regeneration following injury.

[0157] The simultaneous adaptations observed for whole-body and site-specific lean and fat tissues, as well as whole-body metabolic rate in the current study are encouraging and warrant discussion. Lean mass is the primary determinant of metabolic rate and is the main site of glucose uptake, which may partly explain the greater energy expenditure and glucose utilization (indicated by RER) following secretome treatment in aged mice. Under this premise, a subsequent decrease in fat mass (due to increased lipolysis) may be partially explained by increases in lean mass (and metabolic rate) to mobilize the energy demands of muscle tissue. It is possible that increased physical activity levels in the secretome-treated mice may further accelerate this cycle. Further, the magnitude and rapidity of effects on body composition observed in the present study are notable and suggestive of direct as well as secondary effects across tissues following secretome treatment.

[0158] Appropriately, the secretome product contains a host of bioactivc signaling factors capable of stimulating growth, metabolism, and remodeling in both muscle and adipose tissue. Moreover, we report the presence of extracellular vesicles containing microRNAs within the secretome product which can have pronounced effects on skeletal muscle, immune cells, and adipose tissues. Though we arc not yet able to identify the specific factors driving the direct effects of secretome product treatment, the responses in skeletal muscle and adipose tissue have application to metabolic and musculoskeletal diseases and conditions. Furthermore, while it is unknown if the product directly reaches tissues secondary to the site of injection, we have identified direct and indirect treatment effects in muscle cells and adipocytes with muscle derived IL-6 as a possible mediator for the secondary actions. Both skeletal muscle and adipose tissue release extracellular vesicles, myokines, and adipokines upon adequate stimulation, such as exercise. Here, we have shown that prior secretome treatment enhances the production of IL-6 in myotube culture media which subsequently stimulates myotube growth and lipid reduction in adipocytes. In agreement, muscle derived IL-6 can mediate adaptations including hypertrophy and lipolysis in skeletal muscle as well as lipolysis and metabolic reprograming in adipose tissue. Accordingly, we postulate that secretome treatment may have autocrine and paracrine effects on muscle and fat tissue through the release of IL-6. Notably, the secondary treatment responses in adipocytes were lower than direct treatment and it is unclear to what degree they individually or cumulatively drive the observed effects in-vivo. Finally, it is unknown if the indirect treatment effects were driven by increases in muscle cell mass or changes in phenotype, and if other bioactive factors are produced by muscle or other cell types following secretome treatment. Future investigations including exploration of tissue secreted factors and cross-talk following secretome treatment are justified. F01591 In summary, the results from this experiment show that 4- weeks of bi-weekly intramuscular treatment with a stem-cell derived secretome product enhanced metabolic rate while reducing whole -body and site-specific adiposity in aged mice. Moreover, the secretome treatment promoted skeletal muscle hypertrophy, cellular remodeling, and greater physical function in aged mice. Finally, secretome treatment in-vitro demonstrates both direct and indirect effects on myotube growth and adipocyte lipolysis. Together these results suggest that as little as 4 weeks of secretome treatment ameliorated many hallmarks of aging in mice. While currently unknown, the translatability of these results to humans is a topic of interest and is under investigation in a phase l/2a clinical trial (NCT05211986) currently underway. Considering the beneficial pre-clinical effects reported here and elsewhere, human investigations using stem cell- derived secretome products are warranted.

Example 4: Wound Healing after Burns

[0160] Secretome was administered to 8-10 week-old male and female mice intradermally at four timepoints over two weeks in a full thickness burn wound model. Secretome administration was performed on immediately following burn wound and 2, 5 and 10 days post wound induction. Full thickness bum wounds were generated by submerging the depilated backs of the mice in 65 °C water bath for 20 seconds, accounting for roughly 10% of their total body surface area. Wounds were photographed daily, and wound areas were quantified using ImageJ. Additionally, re-epithelialization and wound morphology by H&E and Masson’s trichrome, collagen deposition by picrosirius red staining and immune cell subsets and neoangiogenesis by immunohistochemistry were assessed.

Example 5: Vaccination Response and Post-viral Infection Inflammation

[0161] Age related decline in immune responses including responses to vaccination is well documented and a serious health care issue for the aged populace. This impaired response is also linked to extended infections, increased recovery periods and a prolonged elevated inflammatory states including those described “long covid”. Enhancement of the immune system through administration of secretome could drastically improve public health by increasing the protection provided by immunization in the aged population as well as reducing the prolonged inflammatory events associated with viral infections in the elderly. In this study mice are injected with 4 priming doses of secretome over two-week period prior to initial immunization and then immunized. This would translate in humans to an initial dosing period of secretome prior to immunization and/or boosting to enhance the overall responsiveness of the immune system including but not limited to enhance cell-mediated response and increased antibody production.

Example 6: Fertility Window Extension

[0162] Secretome is administered to enhance oocyte production and uterine morphology and functionality to maintain or extend fertility. Unlike hormonal products, the secretome is not required to be administered in cycles, for example to stimulate ovulation, the mechanism mostly addressing the trophicity of reproductive tissue. Similarly, the secretome is administered over the course of several weeks to enhance spermatogenesis. In previous studies male mice treated with secretome demonstrated enhanced spermatogenesis, while in rat females slightly increased the ovary and uterine weights with enhanced endometrial thickness.

Example 7: Arterial Stiffens and Perfused Micro vascular Density

[0163] Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide and advancing age is an independent risk factor for the development of CVD. Likewise, aging is associated with impairments in metabolic function that can also contribute to CVD as well metabolic disease. Identifying novel therapeutics to combat multiple comorbidities of aging may improve health outcomes and reduce health care costs in the face of our increasingly older population. Mouse models of aging have proven to be faithful models of human aging, demonstrating vascular dysfunction such as large artery stiffening and microvascular dysfunction. Here, we demonstrate that the use of a stem cell derived product can ameliorate/reverse age-related vascular decline in mice. To do so, young (4-6 mo) and old (22-24 mo) mice were treated with secretome for 2 (N=6/age group) or 4 (N=6/age group) weeks and measures of vascular and metabolic function were assessed. In vivo vascular measures include assessments of arterial stiffness by pulse wave velocity and a terminal measure of microvascular function in the mesenteric circulation using the intravital microscopy prior to blood and tissue collection for histology and flow cytometry on the spleen to determine the impact of treatment on vascular morphology and immune cell populations.

[0164] The results show that the treatment with secretome dampened aging-associated increase in large artery stiffness within 2 weeks after treatment cessation and increases perfused microvasculature diameter and density. The changes in arterial stiffness and micro vascular perfusion result in decreased peripheral resistance and reduction of systolic and diastolic blood pressure.

Example 8: In-human Clinical Experience

[0165] As the pre-clinical data supports the secretome as an attractive candidate therapeutic in conditions associated with reduced mobility and disuse-associated muscle atrophy such as occurs in knee osteoarthritis (KOA), the investigational drug was tested for safety and preliminary indications of efficacy in an open label US FDA approved Phase l/2a clinical trial. The trial was designed as an open-label, 3+3 dose escalation study. Up to 18 participants were planned to receive twice weekly intramuscular (IM) administration of the secretome pharmaceutical preparation for 4 weeks in 3 dose cohorts of about 250 pg, 500 pg; and 1 mg total protein per single dose.

[0166] The data from the phase I clinical trial for muscle atrophy associated with knee osteoarthritis is suggestive that the product is well tolerated and may have systemic benefits as measured by muscle performance and quality of life questionnaires. Thus, we will continue to explore the efficacy of the secretome in conditions that involve muscle weakness (sarcopenia), and later with a follow-up placebo-controlled trial in knee osteoarthritis, in a separate trial.

[0167] The next stage of clinical development is a placebo-controlled, dose expansion study that begins with 4 parallel dosing groups and one placebo control group (phase Ila). The treatment period and follow up will last 24 weeks. An interim analysis will be performed 12 weeks after treatment completion will define the parameters for the second phase of the study (lib). The study will evaluate safety, body composition, and muscle strength and function. Additional metabolic, inflammatory, and muscle-adipose crosstalk markers will be monitored. F01681 The treatment consists of four weeks of twice weekly intramuscular (IM) injections with 2-3 day intervals between injections. The dosing groups vary by the secretome total protein and will be adjusted for identical volumes or equivalent saline volume for placebo. The proposed doses will be 0.5 mg administered twice per week; 1 mg once per week (alternating with a placebo given on the second visit of the week); 1 mg twice per week; and 2 mg once per week (also alternating with placebo).

Safety in humans

[0169] Investigators and safety monitoring board members assessed a multitude of safety data points for each subject during the trial and found no safety concerns. No adverse events of grade 2 of higher (CTCAE v5.0 standard) or serious adverse events were reported throughout the clinical trial, thus limiting the enrolled participants to 3 per dose group per protocol design.

Physical function of the knee

[0170] Physical function of the affected knee was measured using the 6-minute walk test (6MWT). At eight study visits, the distance ambulated by participants within 6 minutes was recorded to assess overall functional mobility. FIG. 19 illustrates the available data for physical function by three-patient dose group throughout enrollment in study, suggesting a benefit to quality of life following four weeks of treatment twice per week with the investigational product in patients with muscle atrophy related to knee osteoarthritis. Patients also completed patient- reported outcome (PRO) questionnaires at six visits throughout enrollment.

Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)

[0171] A standardized questionnaire completed by patients at six visits throughout trial enrollment measured the condition of patient quality of life through the metrics: pain, stiffness, and physical function. FIG. 20 displays the reported and trended data collected from the first complete cohort (Group A, B, and C) and suggests a decrease in pain, stiffness, and physical limitation throughout trial participation in the treatment period and continued improvement throughout the safety follow-up period (visits 10-13). Pain Visual Assessment Scale (VAS)

[0172] Participants were presented with a blank horizontal line measuring 100mm in length and asked to mark their current level of knee pain between 0 (no pain) and 100 (worst imaginable pain). FIG. 21 shows collected patient reported data and indicates a continued decline in pain throughout trial participation.

PROMIS-PI (Pain Interference)

[0173] PROMIS-PI measured the consequences of knee pain’s impact on daily activities considering the previous seven days. FIG. 22 displays reported and trended patient answers throughout the trial and shows a decrease in how pain impacted daily activities.

PROMIS-PF (Physical Function)

[0174] PROMIS-PF measures the self-reported capability to perform physical activities, such as dexterity, mobility and completion of activities of daily living. FIG. 23 shows patient- reported physical functional capabilities throughout enrollment in study evaluating safety and efficacy of secretome in participants with muscle atrophy related to KOA, showing increase in physical activities score.

* * *

[0175] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0176] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0177] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0178] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Claims

WHAT IS CLAIMED IS:

1. A method for producing a secretome, the method comprising: expanding mammalian pluripotent cells in a growth media, wherein the growth media comprises one or more growth factors to provide expanded cells; at least partially differentiating the expanded cells to provide partially differentiated cells; exposing the partially differentiated cells to a protein-free media; collecting the protein-free media that was exposed to the partially differentiated cells; adding arginine-HCl to the collected media; and concentrating the collected media to provide a concentrate comprising the secretome.

2. The method of claim 1, wherein said one or more growth factors comprise FGF2 and Activin A.

3. The method of claim 2, wherein said one or more growth factors further comprise TGFbl.

4. The method of any one of claims 1-3, wherein the growth media further comprises one or more amino acids, peptides, lipids, and/or salts.

5. The method of claim 4, wherein the growth media comprises one or more amino acids selected from the group consisting of arginine, leucine, and isoleucine.

6. The method of claim 5, wherein the growth media comprises about 0.01 to about 1.5 g/L of L-arginine, about 0.05 g/L to about 0.5 g/L of L-isoleucine, about 0.05 g/L to about 0.5 g/L of L-leucine.

7. The method of claim 4, wherein the peptide is taurine.

8. The method of claim 7, wherein the one or more lipids comprise ethanolamine.

9. The method of claim 8, wherein the ethanolamine is included in the structure of other lipids.

10. The method of claim 4, wherein the salt comprises selenium or selenite.

11. The method of claim 1, wherein the growth media comprises arginine, isoleucine, leucine, ethanolamine, taurine, sodium selenite, FGF2, Activin A.

12. The method of claim 1, wherein the growth media comprises about 0.01 to about 1.5 g/L of L- arginine, about 0.05 g/L to about 0.5 g/L of L-isoleucine, about 0.05 g/L to about 0.5 g/L of L-leucine, about 0.015 g/L to about 1 g/L of ethanolamine, about 0.25 g/L to about 1.5 g/L of taurine, about 1 pg/L to about 100 pg/L of sodium selenite, about 1 ng/mL to about 100 ng/mL of FGF2, about 0.5 ng/mL to about 50 ng/mL of Activin A.

13. The method of claim 1, wherein the growth media comprises about 0.35 g/L of L- arginine, about 0.12 g/L of L-isoleucine, about 0.12 g/L of L-leucine, about 0.015 g/L of ethanolamine, about 0.25 g/L of taurine, about 5 pg/L of sodium selenite, and about 10 ng/mL of FGF2, about 10 ng/mL of Activin A.

14. The method of any one of claims 1-13, wherein the mammalian pluripotent cells are human pluripotent cells.

15. The method of any one of claims 1-14, wherein the partially differentiating comprises removing the growth factors from the growth media.

16. The method of any one of claims 1-15, wherein the partially differentiated cells comprise tri-germ embryonic markers, or bi- or single germ embryonic layer markers.

17. The method of claim any one of claims 1-16, wherein the protein-free media comprises trehalose and one or protein stabilizers.

18. The method of claim any one of claims 1-17, wherein the protein-free media that was exposed to the partially differentiated cells is collected daily for multiple days.

19. The method of claim any one of claims 1-18, wherein one or more protein stabilizers adjuvants arc added with arginine-HCl to the collected media, wherein the protein stabilizers are selected from sugars, polyols, amino acids, and surfactants.

20. The method of claim any one of claims 1-19, wherein concentrating the collected media comprising concentrating with TFF with a cutoff membrane to retain molecules above 3 kDa.

21. The method of claim any one of claims 1-20, further comprising fractionating the concentrate for pre-defined molecular weights intervals.

22. The method of claim any one of claims 1-21, further comprising purifying the protein from the concentrate.

23. The method of claim 22, wherein the protein is purified by ultracentrifugation, precipitation, dialysis, gel filtration, or chromatography.

24. A composition comprising secretome obtained by the method of any one of claims 1-23.

25. The composition of claim 24, wherein total protein concentration is 0.1 to 50 mg/mL.

26. The composition of claim 24 or 25, wherein the secretome is enriched for exosomes.

27. The composition of claim 24 or 25, wherein the secretome is depleted of exosomes.

28. The composition of any one of claims 24-27, further comprising one or more growth factors, one or more small molecules, one or more antibodies, and/or one or more extracellular matrices.

29. The composition of any one of claims 24-28, further comprising one or more therapeutic drugs.

30. The composition of any one of claims 24-29, wherein the composition is included in a biodegradable slow-release matrix.

31. The composition of claim any one of claims 24-30, further comprising one or more pharmaceutically acceptable excipients.

32. The composition of claim 31, wherein the pharmaceutically acceptable excipients are selected from arginine, proline, lysine, glutamic acid, glycine, histidine, trehalose, dextrose, sucrose, glycerol, mannitol, sorbitol, EDTA, dextran, phosphate butter, citrate buffer, Tris buffer, HEPES buffer, and a combination thereof.

33. The composition of any one of claims 24-32, wherein the composition is in a liquid form.

34. The composition of claim any one of claims 24-33, wherein the composition is lyophilized.

35. The composition of any one of claims 24-34, wherein the composition is packaged in a vial for injectable solutions or in a self-administering syringe.

36. The composition of claim any one of claims 24-35, wherein the composition is suitable for intra-muscular, intravenous, subcutaneous, intrathecal or intracerebral administration.

37. A method of preventing or treating a disease, comprising administering the composition of any one of claims 1-36 to a patient in need thereof.

38. The composition of any one of claims 1-36 for use in preventing or treating a disease.

39. Use of the composition of any one of claims 1-36, for the manufacture of a medicament for preventing or treating a disease.

40. The method of claim 37, the composition for use of claim 38, or the use of claim 39, wherein: the disease includes muscle atrophy or muscle strength loss; the disease includes a metabolic dysfunction; the disease includes an immune dysfunction; the disease includes a regenerative dysfunction; the disease involves a need for plastic surgery and/or cosmetic procedure; the disease is associated with cognitive decline; or the disease is associated with aging.