Generation of secretome-containing compositions, and methods of using and analyzing the same

Abstract

The present disclosure provides methods for generating and / or purifying secretomes, extracellular vesicles, and fractions thereof, from progenitor cells; and provides compositions containing such generated secretomes, extracellular vesicles, and fractions thereof. The present disclosure further provides methods for analyzing activities, and the functionality and potency, of such secretomes, extracellular vesicles, and fractions thereof. The present disclosure also relates to the therapeutic use of secretomes, extracellular vesicles, and fractions thereof. The present disclosure further relates to good manufacturing practices (GMP)-ready, scalable, culture protocol for the release of clinic-ready secretomes.

Description

GENERATION OF SECRETOME-CONTAINING COMPOSITIONS, AND METHODS OF USING AND ANALYZING THE SAMECROSS REFERENCE TO RELATED APPLICATIONThis application claims priority from U.S. Provisional Patent Application No. 63 / 625,899 filed January 26, 2024, the entire disclosures of which are incorporated herein by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTINGThe content of the electronically ssuubbmmiitttteedd sequence listing, file name: A304651_Sequence listing as filed.xml; size: 14,869 bytes; date of creation: January 24, 2025, filed herewith, is incorporated herein by reference in its entirety.FIELD OF THE INVENTIONThe present disclosure relates generally to the generation, purification, isolation, and / or enrichment, of secretomes from cells (such as, but not limited to, progenitor cells); secretomecontaining compositions containing such generated, purified, isolated, and / or enriched, secretomes; and to methods for analyzing one or more activities, properties, and / or characteristics, of such secretome-containing compositions. The present disclosure also relates to the therapeutic use of secretome-containing compositions containing secreted bioactive molecules, produced, purified, isolated, and / or enriched, by a method or methods disclosed herein. The present disclosure further relates to good manufacturing practices (GMP)-ready, scalable, culture protocols for the release, purification, isolation, and / or enrichment, of clinic-ready secretomes, compositions and use thereof.BACKGROUNDCells, including those in in vitro or ex vivo culture, secrete a large variety of molecules and biological factors (collectively known as a secretome) into the extracellular space. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). As part of the secretome, various bioactive molecules are secreted from cells within membrane-bound extracellular vesicles, such as exosomes. Extracellular vesicles are capable of altering the biology of other cells through signaling, or by thedelivery of their cargo (including, for example, proteins, lipids, and nucleic acids). The cargo of extracellular vesicles is encased in a membrane which, amongst others, allows for specific targeting (e.g., to target cells) via specific markers on the membrane; and increased stability during transport in biological fluids, such as through the bloodstream or across the blood-brain-barrier (BBB).Exosomes exert a broad array of important physiological functions, e.g., by acting as molecular messengers that traffic information between different cell types. For example, exosomes deliver proteins, lipids and soluble factors including RNA and microRNAs which, depending on their source, participate in signaling pathways that can influence apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, immunity by directing T cells towards immune activation, immune suppression, growth, division, survival, differentiation, stress responses, apoptosis, and the like. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). Extracellular vesicles may contain a combination of molecules that may act in concert to exert particular biological effects. Exosomes incorporate a wide range of cytosolic and membrane components that reflect the properties of the parent cell. Therefore, the terminology applied to the originating cell can in some instances be used as a simple reference for the secreted exosomes.Progenitor cells have proliferative capacity and can differentiate into mature cells, making progenitor cells attractive for therapeutic applications such as regenerative medicine, e.g., in treating myocardial infarction and congestive heart failure. It has been reported that extracellular vesicles secreted by human embyonic stem cell-derived cardiovascular progenitor cells produce similar therapeutic effects to their secreting cells in a mouse model of chronic heart failure, see Kervadec et al. (J. Heart Lung Transplant, 2016; 35:795-807), suggesting that a significant mechanism of action of transplanted progenitor cells is in the release of biological factors following transplantation (e.g., which stimulate endogenous regeneration or repair pathways). This raises the possibility of effective, cell-free therapies (with benefits such as improved convenience, stability, and operator handling). However, there currently is a need for improved production methods for generating, purifying, isolating, and / or enriching, extracellular vesicles and compositions thereof for allogeneic or autologous human administration and use.Established techniques for the generation of extracellular vesicles typically employ reagents and / or conditions that are not compatible with clinical or therapeutic use, or GMP standards. Furthermore, extracellular vesicles produced by one method would have differentfunctionalities and properties from extracellular vesicles or secretomes produced by another similar method, see Thery et al. (J Extracell Vesicles. 2018 Nov 23;7(l):1535750).Therefore, therapeutic administration, efficacy and safety of an extreacellular vesicle containing composition can be method and process dependant. For instance, regulatory approval of production of drugs and biological substances requires strict adherence to laws and regulations that are promulgated with the goal of establishing safe and effective manufacturing facilities and products. As a non-limiting example, “Good Manufacturing Practices” (GMP) and “Good Laboratory Practices” (GLP) are established by regulation and implemented by the FDA (the U.S. Food and Drug Administration), CDER (Center for Drug Evaluation and Research), and CBER (Center for Biologies Evaluation and Research), with regard to drugs and biologies. Similar GMP and / or GLP laws are implemented worldwide, for instance in the EMEA.For example, the use of serum in culturing protocols raises reliability- and biosafetyconcerns, especially where serum obtained from an animal may be contaminated with, for example, infectious agents such as viruses or prions. Fetal bovine serum (FBS) is a widely used growth supplement for cell and tissue culture media; however, FBS is not well suited for clinical or therapeutic use for these reasons.In contrast, the use of serum-free media confers many advantages, including consistency in formulations and safety. However, using only serum-free media can have disadvantageous effects on cell metabolism and growth, and there exists a need for good manufacturing practices (GMP)-ready compositions / methods for generating, purifying, isolating, and / or enriching, secretome compositions.Additionally, there is a need for improved treatments for patients such as cancer survivors who have been treated with anthracyclines, and who are at risk of developing a left ventricular (LV) dysfunction (sometimes as late as even 10 to 20 years after the end of their cancer treatment). Several factors, like cumulative dose, age, and cardiovascular risk, increase the probability of developing anthracycline-induced cardiotoxicity, but in all cases, patients require careful monitoring, sometimes a reduction in dosing regimens, or the use of classical neuro-hormonal blockade preventive therapies (the benefit of which is still unclear).Anthracycline treatment causes DNA damage, oxidative and energetic stress leading to inflammation, extracellular matrix remodeling, and defects in heart contractility (which, in the long term, lead to LV dysfunction).In order to develop, optimize and release products for human therapeutic use, it is important to establish their safety and efficacy in appropriate models. A combined approach of testing products on in vitro human cells, together with animal studies will provide a strong data set describing product efficacy and safety, and predicting efficacy, safety, and use of the product in human subjects.SUMMARY OF THE INVENTIONThe present disclosure addresses the above-described limitations in the art, by providing methods for generating, purifying, isolating, and / or enriching, secretomes using serum-free media, thereby permitting a GMP-ready, scalable, quality-controlled culture protocol for the release of clinic-ready secretomes.The present disclosure also provides methods for generating, purifying, isolating, and / or enriching, secretomes, extracellular vesicles, and fractions thereof, from cells (such as, but not limited to, progenitor cells); and provides compositions containing such generated, purified, isolated, and / or enriched, secretomes, extracellular vesicles, and fractions thereof. The present disclosure further provides methods for analyzing one or more activities, properties, and / or characteristics, of such secretomes, extracellular vesicles, and fractions thereof, as well as the therapeutic use of secretomes, extracellular vesicles, and fractions thereof.The present disclosure also provides assays for determining the effect of secretomes, extracellular vesicles, and fractions thereof, on the treatment of chemotherapy-induced cardiomyopathy. The present disclosure further provides compositions containing generated, purified, isolated, and / or enriched, secretomes, extracellular vesicles, and fractions thereof, for the treatment and / or prevention of chemotherapy-induced cardiomyopathy in a subject.Non-limiting embodiments of the disclosure include as follows:[1] A method for generating a secretome, said method comprising: (a) culturing one or more progenitor cells in a first serum-free culture medium, wherein said first serum-free culture medium comprises basal medium, human serum albumin, and one or more growth factors; (b) removing said first serum-free culture medium from said one or more progenitor cells; (c) culturing said one or more progenitor cells in a second serum-free culture medium, wherein said second serum-free culture medium comprises basal medium, but does not comprise human serum albumin or growth factors; and (d) recovering the second serum-free culture medium after the culturing ofstep (c), to thereby obtain conditioned medium comprising the secretome of the one or more progenitor cells.[2] The method of [1], wherein one of said one or more growth factors is fibroblast growth factor 2 (FGF-2).[3] The method of [1] or [2], wherein said first and second serum-free media are supplemented with a carbohydrate source.[4] The method of [3], wherein said carbohydrate source is glucose.[5] The method of any one of [1]-[4], wherein said first and second scrum-free media are supplemented with an antibiotic.[6] The method of [5], wherein said antibiotic is gentamicin.[7] The method of any one of [1]-[6], wherein said first serum-free media further comprises one or more selected from the group consisting of: glutamine; biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine, glutathione; L-carnitine; linoleic acid; progesterone; putrescine; sodium selenite; triodo-I-thyronine; an amino acid; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.[8] The method of any one of [1]-[7], wherein said basal medium is a Minimum Essential Medium (MEM).[9] The method of [8], wherein said MEM is α-MEM.

[0010] The method of any one of [1]-[9], wherein the culturing of step (a) is for 6-96 hours.

[0011] The method of

[0010] , wherein the culturing of step (a) is for 12-96 hours.

[0012] The method of

[0011] , wherein the culturing of step (a) is for 36-84 hours.

[0013] The method of

[0012] , wherein the culturing of step (a) is for about 72 hours.

[0014] The method of any one of [1]-

[0013] , wherein the culturing of step (c) is for 6-96 hours.

[0015] The method of

[0014] , wherein the culturing of step (c) is for 12-72 hours.

[0016] The method of

[0015] , wherein the culturing of step (c) is for 36-60 hours.

[0017] The method of

[0016] , wherein the culturing of step (c) is for about 48 hours.

[0018] The method of

[0014] , wherein the last 12-36 hours of the culturing of step (c) is conducted under hypoxic conditions.

[0019] The method of

[0018] , wherein said culture conditions comprise culturing in an atmosphere having 1-21% oxygen.

[0020] The method of any one of [1]-

[0019] , wherein after step (b), but before step (c), said one or more progenitor cells are washed.

[0021] The method of any one of [1]-

[0020] , wherein said one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac pregenitor cells, and cardiovascular progenitor cells.

[0022] The method of any one of [1]-

[0021] , wherein said one or more progenitor cells are obtained from induced pluripotent stem cells (iPSCs).

[0023] The method of any one of [1]-[4] and [7]-

[0022] , wherein said first and second scrum- free media do not contain an antibiotic.

[0024] The method of any one of [1]-

[0023] , wherein the culturing in one or more of steps (a) and (c) is two-dimensional cell culture.

[0025] The method of

[0024] , wherein said two-dimensional cell culture comprises culturing said one or more progenitor cells on a surface of a culture vessel.

[0026] The method of

[0025] , wherein said culture vessel surface is coated with a substance to promote cell adhesion.

[0027] The method of

[0026] , wherein said substance to promote cell adhesion is vitronectin or fibronectin.

[0028] The method of any one of [1]-

[0023] , wherein the culturing in one or more of steps (a) and (c) is three-dimensional cell culture.

[0029] The method of

[0028] , wherein the three-dimensional cell culture comprises culturing cell aggregates in suspension in a bioreactor, spinner flask, or stirred culture vessel, or comprises culturing cells in a microcarrier culture system.

[0030] The method of any one of [1]-

[0029] , wherein said method further comprises preclearing the medium recovered in step (d) by centrifugation, filtration, or a combination of centrifugation and filtration.

[0031] The method of any one of [1]-

[0030] , wherein said method further comprises freezing the medium recovered in step (d).

[0032] The method of any one of [1]-

[0031] , wherein said one or more progenitor cells cultured in step (a) have previously been frozen.

[0033] The method of any one of [1]-

[0032] , wherein said method further comprises concentrating, and / or enriching for a small extracellular vesicle-enriched fraction (sEV) from the medium recovered in step (d).

[0034] The method of

[0033] , wherein said sEV is concentrated, and / or enriched, from the recovered medium by at least one process selected from the group consisting of ultracentrifugation, filtration, ultrafiltration, tangential flow filtration, size exclusion chromatography, and affinity capture.

[0035] The method of

[0033] , wherein said enriching enriches for extracellular vesicles that have one or more of the following characteristics: (a) are CD63+, CD81+and / or CD9+; (b) are between 50-200 nm in diameter; (c) are positive for one or more of CD49e, ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1), SSEA-4 (Stage-specific embryonic antigen 4), MSCP (Mesenchymal stem cell-like protein), CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29 and CD142; and / or (d) are negative for one or more of CD19, CD4, CD209, HLA-ABC (human leukocyte antigen-ABC), CD62P, CD42a and CD69.

[0036] The method of

[0033] , wherein said sEV comprises one or more of exosomes, microparticles, extracellular vesicles and secreted peptides / proteins.

[0037] A secretome-containing composition obtained by the method of any one of [1]-

[0032] .

[0038] An sEV-containing composition obtained by the method of any one of

[0033] -

[0036] .

[0039] A method for producing a therapeutic composition suitable for administration to a patient, said method comprising producing a secretome-containing composition according to the method of any one of [1]-

[0032] .

[0040] The method of

[0039] , wherein said method further comprises purifying, concentrating, isolating, and / or enriching, said secretome-containing composition by one or more purification, concentrating, isolation, and / or enrichment, steps.

[0041] The method of

[0039] , wherein said method further comprises adding a pharmaceutically acceptable excipient or carrier to the secretome-containing composition.

[0042] A method for producing a therapeutic composition suitable for administration to a patient, said method comprising producing an sEV-containing composition according to the method of any one of

[0033] -

[0036] .

[0043] The method of

[0042] , wherein said method further comprises purifying, concentrating, isolating, and / or enriching, said sEV-containing composition by one or more purification, concentration, isolation, and / or enrichment, steps.

[0044] The method of

[0042] , wherein said method further comprises adding a pharmaceutically acceptable excipient or carrier to the sEV-containing composition.

[0045] A therapeutic composition comprising the secretome-containing composition of

[0037] , and a pharmaceutically acceptable excipient or carrier.

[0046] A therapeutic composition comprising the sEV-containing composition of

[0038] , and a pharmaceutically acceptable excipient or carrier.

[0047] A secretome-containing composition obtained by the method of [1], wherein said one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

[0048] An sEV-containing composition obtained by the method of

[0033] , wherein said one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

[0049] A therapeutic composition comprising the composition of

[0047] , and a pharmaceutically acceptable excipient or carrier.

[0050] A therapeutic composition comprising the composition of

[0048] , and a pharmaceutically acceptable excipient or carrier.

[0051] A method for treating acute myocardial infarction, heart failure, myocarditis, ischemic cardiomyopathy, cardiomyopathy, ventricular dysfunction, atrial dysfunction, or arrhythmia in a subject in need thereof comprising administering to the subject the therapeutic composition of

[0049] or

[0050] ,

[0052] A method for improving angiogenesis, comprising administering to a subject in need thereof the therapeutic composition of

[0049] or

[0050] .

[0053] A method for improving cardiac performance, comprising administering to a subject in need thereof the therapeutic composition of

[0049] or

[0050] ,

[0054] The method of

[0011] , wherein the culturing of step (a) is for 60-84 hours.

[0055] The method of

[0014] , wherein the last 12-36 hours of the culturing of step (c) is conducted under normoxic conditions.

[0056] The method of

[0055] , wherein said normoxic conditions comprise culturing in an atmosphere containing 20-21% oxygen.

[0057] The method of

[0029] , wherein the bioreactor is a vertical wheel bioreactor.

[0058] The method of

[0039] , wherein said method further comprises cryopreserving, freezing, or lyophilizing, said secretome-containing composition.

[0059] The method of

[0042] , wherein said method further comprises cryopreserving, freezing, or lyophilizing, said sEV-containing composition.

[0060] The method of [2], wherein said first scrum-frcc media comprises 0.1-10 μg / mL FGF-

[0061] The method of

[0060] , wherein said first serum-free media comprises 0.5-5 μg / mL FGF-

[0062] The method of

[0061] , wherein said first serum-free media comprises 0.5-2.5 μg / mL FGF-2.

[0063] The method of

[0062] , wherein said first serum-free media comprises about 1 μg / mL FGF-2.

[0064] The method of any of [1]-

[0036] ,

[0039] -

[0044] and

[0054] -

[0063] , wherein said method is Good Manufacturing Practices (GMP)-ready.

[0065] The secretome-containing composition of

[0037] , wherein said composition is GMP- ready.

[0066] The sEV-containing composition of

[0038] , wherein said composition is GMP-ready.

[0067] The method of

[0014] , wherein the last 12-36 hours of the culturing of step (c) is conducted under normoxic conditions.

[0068] The method of

[0067] , wherein said normoxic conditions comprises culturing in an atmosphere containing between 20-21% of oxygen.

[0069] The method of

[0030] , wherein said pre-clearing comprises at least three filtration steps.

[0070] The method of

[0034] , wherein the separation of said sEV from the recovered medium comprises tangential flow filtration.

[0071] The secretome-containing composition of

[0037] comprising trehalose and L-histidine.

[0072] The sEV-containing composition of

[0038] comprising trehalose and L-histidine.

[0073] The secretome-containing composition of

[0037] or

[0065] , wherein said composition is able to promote wound scratch healing in an in vitro wound scratch healing assay, and / or is able to promote cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

[0074] The sEV-containing composition of

[0038] or

[0066] , wherein said composition is able to promote wound scratch healing in an in vitro wound scratch healing assay, and / or is able to promote cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

[0075] The secretome-containing composition of

[0037] or

[0065] , wherein said composition is at least one of the following: a composition that has been enriched for extracellular vesicles having a diameter of between about 50-200 nm or between 50-200 nm, preferably having a diameter of between about 50-150 nm or between 50-150 nm; a composition that is substantially free or free of whole cells; and / or a composition that is substantially free of one or more culture medium components.

[0076] The sEV-containing composition of

[0038] or

[0066] , wherein said composition is at least one of the following: a composition that has been enriched for extracellular vesicles having a diameter of between about 50-200 nm or between 50-200 nm, preferably having a diameter of between about 50-150 nm or between 50-150 nm; a composition that is substantially free or free of whole cells; and / or a composition that is substantially free of one or more culture medium components.

[0077] The method of

[0051] , wherein the heart failure is acute heart failure, chronic heart failure, ischemic heart failure, non-ischemic heart failure, heart failure with ventricular dilation, heart failure without ventricular dilation, heart failure with reduced left ventricular ejection fraction, or heart failure with preserved left ventricular ejection fraction.

[0078] The method of

[0077] , wherein the heart failure is selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy induced heart failure.

[0079] The secretome-containing composition of

[0037] or

[0065] , wherein said composition is able to promote cardiomyocyte viability in an in vitro chemotherapy-induced cardiomyopathy viability assay.

[0080] The sEV-containing composition of

[0038] or

[0066] , wherein said composition is able to promote cardiomyocyte viability in an in vitro chemotherapy-induced cardiomyopathy viability assay.

[0081] The secretome-containing composition of

[0079] , wherein in said in vitro chemotherapy- induced cardiomyopathy viability assay, said chemotherapy is an anthracycline.

[0082] The secretome-containing composition of

[0081] , wherein said anthracycline is doxorubicin.

[0083] The sEV-containing composition of

[0080] , wherein in said in vitro chemotherapy- induced cardiomyopathy viability assay, said chemotherapy is an anthracycline.

[0084] The sEV-containing composition of

[0083] , wherein said anthracycline is doxorubicin.

[0085] The method according to

[0051] , wherein said chemotherapy-induced cardiomyopathy is caused by an anthracycline.

[0086] The method according to

[0085] , wherein said anthracycline is doxorubicin.

[0087] A method of maintaining physiological heart volume in a subject by administering to said subject the therapeutic composition of any one of

[0045] ,

[0046] ,

[0049] and

[0050] .

[0088] The method of any one of

[0051] -

[0053] and

[0087] , wherein said method maintains Left Ventricular End Systolic Volume (LVESV) within 15% of the pre-treatment LVESV.

[0089] The method of anyone of

[0051] -

[0053] and

[0087] , wherein said method maintains LVEDV within 2% of the pre-treatment volume.

[0090] The method of any one of

[0051] -

[0053] and

[0087] , wherein the method prevents progressive post-ischemic heart failure.

[0091] The method of any one of

[0051] -

[0053] and

[0087] , wherein the method improves endothelial cell survival, health and function in said subject.

[0092] The method of anyone of

[0051] -

[0053] and

[0087] , wherein said method reduces fibrosis in stimulated cardiac fibroblasts.

[0093] The method of

[0092] , wherein said method reduces the expression of the pro-fibrotic marker, POSTN, in TGF-β1 -stimulated cardiac fibroblasts to level prior to stimulation with TGF- β1 or below.

[0094] The method of any one of

[0051] -

[0053] and

[0087] , wherein the method does not induce an allogeneic inflammatory response in a subject.

[0095] The method of any one of

[0051] -

[0053] and

[0087] , wherein said method does not induce an allogeneic peripheral blood mononuclear cell (PBMC) activation.

[0096] The method of any one of

[0051] -

[0053] and

[0087] , wherein said method does not induce a significant increase in the percentage of IFNg or IL-2 expressing PBMCs.

[0097] The method of any one of

[0051] -

[0053] and

[0087] , wherein the method does not induce allogeneic natural killer (NK) cell degranulation.

[0098] The method of any one of

[0051] -

[0053] and

[0087] , wherein the method does not induce a significant increase in the percentage of CD 107 expressing NK cells.

[0099] A method of improving heart function in a patient experiencing heart failure by administering the therapeutic composition of any one of

[0045] ,

[0046] ,

[0049] and

[0050] .

[0100] The method of

[0099] , wherein the method improves survival of stressed cardiomyocyte cells.

[0101] The method of

[0099] , wherein the method improves one or more of the seeding, survival, viability and proliferation of stressed endothelial cells in vitro.

[0102] The method of

[0099] , wherein the method improves cell migration and / or wound healing capabilities in stressed endothelial cells.

[0103] The method of any one of

[0099] -

[0102] , wherein the method improves wound healing in said subject.

[0104] The method of any of one of

[0099] -

[0103] , wherein the method reduces signs of fibrosis in fibroblast cells of said subject.

[0105] The method of

[0104] , wherein the fibroblasts are activated with TGF-β1.

[0106] The method of any one of

[0099] -

[0105] , wherein the method does not stimulate allogeneic human PBMC activation.

[0107] The method of any of

[0099] -

[0106] , wherein the method does not induce NK degranulation of allogeneic human NK cells.

[0108] The method of any of

[0099] -

[0107] , wherein the composition is non-toxic in mice and rats at a dose of 4 x 1011particles / kg.

[0109] The method of any of

[0099] -

[0108] , wherein the composition is not tumorigenic in mice at dose of 4 x 1011particles / kg.

[0110] The method of any of

[0099] -

[0109] , wherein the composition does not contain DNA fragments ranging from 179 to 742 pb, at concentrations in the μg / mL range.

[0111] The method of any of

[0051] -

[0053] and

[0087] -

[0110] , wherein said therapeutic composition is administered as an intravenous infusion, direct cardiac injection or is administered intraarterially.

[0112] The method of

[0111] , wherein the therapeutic composition is administered at a dose containing secretome obtained from 0.1 to 10 million cells per kg weight of said subject per administration.

[0113] The method of

[0111] , wherein the therapeutic composition is administered at a dose containing sccrctomc obtained from 0.5 to 5 million cells per kg weight of said subject per administration.

[0114] The method of

[0111] , wherein the therapeutic composition is administered at a dose containing secretome obtained from 1 to 3 million cells per kg weight of said subject.

[0115] The method of

[0111] , wherein the therapeutic composition is administered at a dose containing secretome obtained from 1 to 2 million cells per kg weight of said subject.

[0116] The method of

[0111] , wherein said therapeutic composition is administered at a dose containing from 1 x 109to 60 x 109particles, as measured by Nanoparticle Tracking Analysis (NT A), per kg weight of the subject.

[0117] The method of

[0111] , wherein said therapeutic composition is administered at a dose containing from 10 x 109to 60 x 109particles, as measured by NT A, per kg weight of the subject.

[0118] The method of

[0111] , wherein said therapeutic composition is administered at a dose containing from 10 x 109to 40 x 109particles, as measured by NT A, per kg weight of the subject.

[0119] The method of

[0111] , wherein said therapeutic composition is administered at a dose containing from 20 x 109to 40 x 109particles, as measured by NT A, per kg weight of the subject.

[0120] The method of

[0111] , wherein said therapeutic composition is administered at a cumulative daily dose containing from 20 x 109to 200 x 109particles, as measured by NT A, per kg weight of the subject.

[0121] The method of

[0111] , wherein said therapeutic composition is administered at a cumulative daily dose containing from 30 x 109to 100 x 109particles, as measured by NT A, per kg weight of the subject.

[0122] The method of

[0111] , wherein said therapeutic composition is administered at a cumulative daily dose containing 60 x 109particles, as measured by NTA, per kg weight of the subject.

[0123] The method of

[0111] , wherein said therapeutic composition is administered at a cumulative daily dose containing 40 x 109particles, as measured by NTA, per kg weight of the subject.

[0124] The method of any one of

[0111] -

[0123] , wherein said composition is administered from 1 to 10 times per day.

[0125] The method of any one of

[0111] -

[0123] , wherein said composition is administered from 3 to 6 times per day.

[0126] The method of any one of

[0111] -

[0123] , wherein said composition is administered from 1 to 5 times per day.

[0127] The method of any one of

[0111] -

[0123] , wherein said composition is administered 3 times per day.

[0128] The method of any one of

[0111] -

[0123] , wherein said composition is administered 2 times per day.

[0129] The method of any one of

[0051] -

[0053] and

[0087] -

[0128] , wherein the duration of treatment is 60 days or less.

[0130] The method of

[0129] , wherein the duration of treatment is from 5 to 50 days.

[0131] The method of

[0129] , wherein the duration of treatment is from 10 to 50 days.

[0132] The method of

[0129] , wherein the duration of treatment is from 20 to 45 days.

[0133] The method of

[0129] , wherein the duration of treatment is 42 days.

[0134] The method of any one of

[0111] -

[0133] , wherein said therapeutic composition is administered every day.

[0135] The method of any one of

[0111] -

[0133] , wherein said therapeutic composition is administered every other day.

[0136] The method of any one of

[0111] -

[0133] , wherein said therapeutic composition is administered at a frequency of from every day to every 30 days.

[0137] The method of any one of

[0111] -

[0133] , wherein said therapeutic composition is administered at a frequency of from every 7 days to every 21 days.

[0138] The method of any one of

[0111] -

[0133] , wherein said therapeutic composition is administered every 21 days.

[0139] The method of any one of

[0111] -

[0138] , wherein said therapeutic composition is formulated in a solution comprising one or more pharmaceutically acceptable excipient.

[0140] A secretome-containing composition obtained by the method of any one of

[0001] -

[0032] , wherein said secretome-containing composition comprises extracellular vesicles secreted from said progenitor cells.

[0141] The secretome-containing composition of

[0140] , wherein said extracellular vesicles comprise one or more miR selected from hsa-miR-302a-5p, hsa-miR-16-5p, hsa-miR-93-5p, hsa- miR-126-3p, hsa-miR-148a-3p, hsa-miR-21-5p, hsa-miR-20a-5p, hsa-miR-143 -3p, hsa-miR-335- 5p, hsa-miR-218-5p, hsa-miR-101-3p, hsa-miR-302d-3p, hsa-miR-25-3p, hsa-miR-126-5p, hsa- miR-423-5p, hsa-miR-532-5p, hsa-miR-1246, hsa-miR-302a-3p, hsa-miR-20b-5p, hsa-miR-148b- 3p, hsa-miR-34a-5p, hsa-miR-l-3p, hsa-miR-191 -5p, hsa-miR-26b-5p, hsa-miR-151a-3p, hsa- miR-103a-3p / 107, hsa-miR-660-5p, hsa-miR-320a-3p / 320b / 320c / 320d / 320e, hsa-miR-130a-3p, hsa-miR-19b-3p, hsa-miR-27a-3p / 27b-3p, hsa-miR-186-5p, hsa-miR-26a-5p, hsa-miR-125b-5p, hsa-miR-7-5p, hsa-miR-24-3, hsa-miR-483-5p, hsa-miR-99b-5p, hsa-miR-205-5p, and hsa-miR- 302b-3p.

[0142] The secretome-containing composition of

[0140] , wherein said extracellular vesicles comprise one or more miR selected from hsa-miR-l-5p, hsa-miR-11401, hsa-miR-1263-3p, hsa- miR-3085-3p, hsa-miR-3161-5p, hsa-miR-3678-3p, hsa-miR-3942-5p, hsa-miR-4652-5p, hsa- miR-4758-5p, hsa-miR-4760-5p, hsa-miR-4779-3p, hsa-miR-508-5p, hsa-miR-548ad-3p, hsa- miR-5580-5p, hsa-miR-559-5p, hsa-miR-6791-5p, hsa-miR-6889-5p, and hsa-miR-96-3p.

[0143] The secretome-containing composition of

[0140] , wherein said extracellular vesicles comprise at least five miR selected from hsa-miR-l-5p, hsa-miR-11401, hsa-miR-1263-3p, hsa- miR-3085-3p, hsa-miR-3161-5p, hsa-miR-3678-3p, hsa-miR-3942-5p, hsa-miR-4652-5p, hsa- miR-4758-5p, hsa-miR-4760-5p, hsa-miR-4779-3p, hsa-miR-508-5p, hsa-miR-548ad-3p, hsa- miR-5580-5p, hsa-miR-559-5p, hsa-miR-6791-5p, hsa-miR-6889-5p, and hsa-miR-96-3p.

[0144] The secretome-containing composition of any one of

[0141] -

[0143] , wherein an expression level of the one or more miR ranges from -5 to +5 units.

[0145] A secretome-containing composition comprising a secretome from progenitor cells, said secretome comprising extracellular vesicles secreted from said progenitor cells.

[0146] The secretome-containing composition of

[0145] , wherein said progenitor cells are cardiovascular progenitor cells.

[0147] The secretome-containing composition of

[0145] or

[0146] , wherein said extracellular vesicles comprise one or more miR selected from hsa-miR-302a-5p, hsa-miR-16-5p, hsa-miR-93- 5p, hsa-miR-126-3p, hsa-miR-148a-3p, hsa-miR-21-5p, hsa-miR-20a-5p, hsa-miR-143-3p, hsa- miR-335-5p, hsa-miR-218-5p, hsa-miR-101-3p, hsa-miR-302d-3p, hsa-miR-25-3p, hsa-miR-126- 5p, hsa-miR-423-5p, hsa-miR-532-5p, hsa-miR-1246, hsa-miR-302a-3p, hsa-miR-20b-5p, hsa- miR-148b-3p, hsa-miR-34a-5p, hsa-miR-1 -3p, hsa-miR-191-5p, hsa-miR-26b-5p, hsa-miR-151a- 3p, hsa-miR-103a-3p / 107, hsa-miR-660-5p, hsa-miR-320a-3p / 320b / 320c / 320d / 320e, hsa-miR- 130a-3p, hsa-miR-19b-3p, hsa-miR-27a-3p / 27b-3p, hsa-miR-186-5p, hsa-miR-26a-5p, hsa-miR- 125b-5p, hsa-miR-7-5p, hsa-miR-24-3, hsa-miR-483-5p, hsa-miR-99b-5p, hsa-miR-205-5p, and hsa-miR-302b-3p .

[0148] The secretome-containing composition of

[0145] or

[0146] , wherein said extracellular vesicles comprise one or more miR selected from hsa-miR-1 -5p, hsa-miR-11401, hsa-miR-1263- 3p, hsa-miR-3085-3p, hsa-miR-3161-5p, hsa-miR-3678-3p, hsa-miR-3942-5p, hsa-miR-4652-5p, hsa-miR-4758-5p, hsa-miR-4760-5p, hsa-miR-4779-3p, hsa-miR-508-5p, hsa-miR-548ad-3p, hsa-miR-5580-5p, hsa-miR-559-5p, hsa-miR-6791-5p, hsa-miR-6889-5p, and hsa-miR-96-3p.

[0149] The secretome-containing composition of

[0145] or

[0146] , wherein said extracellular vesicles comprise at least five miR selected from hsa-miR-1 -5p, hsa-miR-11401, hsa-miR-1263- 3p, hsa-miR-3085-3p, hsa-miR-3161-5p, hsa-miR-3678-3p, hsa-miR-3942-5p, hsa-miR-4652-5p, hsa-miR-4758-5p, hsa-miR-4760-5p, hsa-miR-4779-3p, hsa-miR-508-5p, hsa-miR-548ad-3p, hsa-miR-5580-5p, hsa-miR-559-5p, hsa-miR-6791-5p, hsa-miR-6889-5p, and hsa-miR-96-3p.

[0150] The secretome-containing composition of any one of

[0147] -

[0149] , wherein an expression level of the one or more miR ranges from -5 to +5 units.

[0151] A method of increasing a maximal oxygen consumption at exercise in a subject by administering compositions disclosed herein.

[0152] The method of

[0051] ,

[0052] ,

[0053] ,

[0087] ,

[0099] or

[0151] , wherein the maximal oxygen consumption at exercise in a subject is increased by 0.1% to 50%, 1% to 20%, 5% to 10%, 0.1 to 6.0 mL / kg / min, 0.5 to 5.0 mL / kg / min, or 1.0 to 3.0 mL / kg / min.

[0153] The method of

[0051] ,

[0052] ,

[0053] ,

[0087] ,

[0099] ,

[0151] or

[0152] , wherein the subject has a mutation in the tropomyosin gene.

[0154] The method of

[0051] ,

[0052] ,

[0053] ,

[0087] ,

[0099] ,

[0151] or

[0152] , wherein the subject has a mutation in the MYH7 gene.

[0155] The method of

[0051] ,

[0052] ,

[0053] ,

[0087] ,

[0099] ,

[0151] or

[0152] , wherein the subject has a mutation in the LMNA II gene.INCORPORATION BY REFERENCEAll patents, publications, and patent applications cited in the present specification are herein incorporated by reference as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.BRIEF DESCRIPTION OF THE DRAWINGSThe patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.FIG. 1 depicts an iPSC to CPC process flow diagram, illustrating the generation of cardiovascular progenitor cells from hiPSCs (steps 1-4). After CPC generation, cells were maintained as fresh aggregates (5 a) or dissociated to single cells (step 5b) for the vesiculation process. Single cells were plated fresh or cryo-preserved and plated post-thaw (steps 6-7) for the vesiculation process.FIG. 2 and FIG. 3 depict flowcharts showing the material generated in Example 1. As shown in FIG. 2 and FIG. 3, two batches of CPCs (CPC1, CPC2) were generated and each were divided into three vesiculation conditions: aggregate vesiculation, fresh CPC plated vesiculation, and thawed CPC plated vesiculation. The conditioned media from each condition were collected, pre-cleared, and frozen (MC 1-6). The cells at the end of four days of the vesiculation process (day +4) were also collected and analyzed (C+4 # 1-6). Conditioned media were subjected to ultracentrifugation (UC) to isolate the small vesicular fraction (sEV 1-6). For MC5, three separate rounds of UC were performed on separate aliquots of MC5. In parallel, vessels containing media but no cells were incubated in the same conditions as the cell-containing vessels as described above.The media generated by this process, referred to as virgin media, were collected (virgin media 1- 3). Subsequently, mock EV (also called MV) controls were generated from the virgin media via the same UC protocol as described above (MV 1.1-3).FIG. 4 and FIG. 5 depict heatmaps of the gene expression of 48 relevant genes to CPC differentiation and potential off targets. Data were generated using a custom Fluidigm qPCR panel. (A) Heatmap generated using the “SINGULAR Analysis Toolset” package in R3.1.1 by calculating the global z-score. (B) Heatmap generated by calculating the gene Z-Score followed by hierarchical clustering in IMP software version 17 (ward method, unstandardized). The Ct values are presented in TABLE 1. Data from CPCs at the end of the differentiation process (CPC), as well as four days into the vesiculation process (C+4), are shown in addition to iPSC and cardiomyocyte (CM) controls. Under these conditions, CPC are clustered and separate from C+4 cells, which are more mature than CPCs but less mature than CM. Fourth vesiculation day aggregates (Agg+4) are distinct from fourth day hyperflask plated cells (HF+4). Both conditions show increased cTNT (cardiac Troponin T) and alpha-MHC (alpha-myosin heavy chain) expression compared to CPC. This supports the idea that CPC in the vesiculation process remain on the cardiac differentiation lineage, but do not attain the CM differentiation state, as shown by the persistence of CPC marker expression such as PDGFRa, ISL-1 and KDR.FIG. 6 depicts a process flow diagram for the generation of conditioned media and virgin media controls.FIG. 7 depicts a process flow diagram for the isolation of sEV or mock (virgin media) control samples.FIG. 8 depicts representative size distribution curves from two sEVs and two control MV samples. Suspension culture yielded higher concentrations of particles than plated culture, and both were much higher than controls. Mode particle sizes for sEV 1 and sEV 2 (74 nm, 99 nm respectively) are consistent with exosomes or small microparticles.FIG. 9 depicts ELISA results for the detection of CD-63. Bars numbered one through nine from left to right. sEVs (bars one, four, five, six, and seven) and MV controls (bars two, three, eight, and nine) were analyzed by FUJIFILM Wako Elisa kit for the detection of CD-63, a protein found on the surface of EV, especially exosomes. The results show that for a given protein input, MVs contain no CD-63 signal, whereas sEVs from both aggregate and plated cultures do. Aggregate sEV (bar one) produced more CD-63 / protein signal than sEV from plated vesiculationprotocols (bars four through seven). Replicate preparations of sEV from the same MC (5.1, 5.2 and 5.3, bars five, six, and seven) yielded similar CD63 signals. Furthermore, sEV isolated from different MCs generated from separate lots also yielded similar CD-63 / μg protein (sEV 2 (bar four) vs sEV 5.1 / .2 / .3). Protein signal is given as (absorbance 450 - absorbance 620) minus the result for the blank (“abs 450-620, blank adj).FIG. 10 depicts relative scratch wound closure in a HUVEC scratch wound healing assay. Bars numbered one through seven from left to right. sEVs from suspension and plated vesiculation processes (bars four and six) as well as their corresponding mock EV controls (MV) (bars five and seven) were tested in a HUVEC scratch wound healing assay. Controls were complete HUVEC media (positive control, “Positive”, bar one), poor HUVEC media (no supplements, negative control, “Negative”, bar two), and poor media + the sEV isolated from fetal bovine serum by UC (“FBS-EV”, additional positive control, bar three). sEV from suspension (bar four) and plated (bar six) vesiculation processes showed improved wound healing compared to Negative and MV controls.FIG. 11 depicts the results of an H9c2 viability assay. Bars are labeled one through seven from left to right. The results of the H9c2 cell viability assay show that the sEVs from suspension (bar three) and plated (bars five and seven) cultures improve H9c2 survival in a serum deprivation assay. MVs (bars four and six) showed minimal to no positive effect in this assay. sEV generated from the suspension vesiculation method showed an improvement in fold change over negative control over the positive control, suggesting increased cell proliferation in addition to sustained survival.FIG. 12 depicts a time course of cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay. Condition lines labeled A through F from top to bottom, according to last data point. sEV from plated (line C) and aggregate (line B) cultures improve CM survival in this staurosporine assay. Aggregate cultures are suspension cultures in this experiment. MVs (lines D and F) showed little to no effect on CM survival. Arrows link each sEV with its corresponding MV control. The 18-hour data points from FIG. 12 are given in TABLE 2.FIGS. 13 and 14 depict flowcharts illustrating the stages of production (vesiculation, conditioned media clarification, and TFF for Test Example 20, FIG. 13; followed by final formulation, FIG. 14) in a first GMP-compatible process, described in Example 5 and Example 6. The final formulation in this example was produced with and without trehalose addition prior tosterilizing filtration. The different stages at which smples were taken for in-process testing and quality control testing was undertaken are indicated with a “*” (e.g., *1, *2, *3, etc.).FIG. 15 depicts the results of flow cytometry experiments to analyze the cell marker expression profile of CPCs at different times during the vesiculation process (D+0, D+3 and D+5). iPSCs and cardiomyocytes (CM) were used as control cells and were analyzed separately. The values shown are average values.FIG. 16 depicts the results of transcriptome analysis of CPCs at different times during the vesiculation process (D+0, D+3 and D+5). RNA was extracted from CPCs at D+0, and from cells at D+3 and D+5 of the vesiculation process. RNA was also extracted from iPSCs (pluripotent cell controls), and from iPSC-derived cardiomyocytes (differentiated cardiomyocyte controls; CM). Total RNA was sequenced on the Illumina NovaSeq 6000 platform, and differential gene expression was determined on normalized data. The FIG.16 and FIG. 17 heatmaps were generated based on hierarchical clustering analysis using the UPGMA clustering method, with correlation distance metric in TIBCO Spotfire software v 11.2.0. The FIG. 16 heatmap has a blue to red color scale where dark blue represents low expression and dark red represents high expression. The FIG. 17 heatmap is in grey scale where white represents low expression and dark grey / black represents high expression. The data (log2FPKM) used to generate both heatmaps are given in TABLE 3.FIG. 18 depicts the morphology of CPCs during the vesiculation process, as observed under light microscopy. Cell morphology was analyzed in cells within both T75 and selected CS10 flasks. The left image is a representative image showing the typical D+3 morphology observed in all vessels analyzed at D+3. The right image is a representative image showing the typical D+5 morphology observed in all vessels analyzed at D+5. T75 flasks were used for image capture for clarity.FIGS. 19 and 20 depict the results of an analysis of particle concentration and size distribution of EVs. FIG. 19 depicts the particle concentration and size distribution of EVs in clarified conditioned media before tangential flow filtration (TFF) (*5(Test 20)), and in final formulations without trehalose (*7, samples a (Test 20)) and with trehalose (*7, sample b (Test 20)), using nanoparticle tracking analysis. FIG. 20 depicts the particle concentration and size distribution of EVs in clarified conditioned media before tangential flow filtration (TFF) (*5(Test 20)), and in stored retentate samples without trehalose or histidine (*6, sample a (Test 20)), withtrehalose (*6, sample b (Test 20)) or with histidine (*6, sample c (Test 20)) which were not fdter sterilized. As FIGS. 19 and 20 show, TFF increased the particle concentration by about 32-fold.FIGS. 21-24 depict the results of MACSPlex analysis. FIGS. 21 and 22 depict the results of analysis of small EV-enriched secretome final formulations with and without trehalose, for expression of extracellular vesicle tetraspanins often expressed on the surface of extracellular vesicles (CD9, CD81 and CD63) (FIG. 21); and for various additional markers, which exhibited little or no expression (FIG. 22). FIGS. 23 and 24 depict the results of analysis of stored retentate samples (with and without trehalose or histidine) which were not filter sterilized [sec FIG. 14; *6, sample a (Test 20); *6, sample b (Test 20); *6, samples c (Test 20)], for expression of extracellular vesicle tetraspanins often expressed on the surface of extracellular vesicles (CD9, CD81 and CD63) (FIG. 23); and for various additional markers, which exhibited little or no expression (FIG. 24).FIGS. 25 and 26 depict the results of analysis of samples *7, sample a (Test 20); *7, sample b (Test 20); (*6, sample 1 (Test 20); *6, sample b (Test 20); *6, sample c (Test 20) for the presence of cardiac-related markers. FIG. 25 depicts the results for small EV-enriched secretome final formulations with and without trehalose, for expression of cardiac-related markers. FIG. 26 depicts the results for stored retentate samples (with and without trehalose or histidine) which were not filter sterilized, for expression of cardiac-related markers. For all samples depicted in FIG.25 and FIG. 26, the interrogated markers were found to be present.FIG. 27 depicts relative scratch wound healing in a HUVEC scratch wound healing assay. Bars labeled one through seven from left to right. Small EV-enriched secretome final formulations with (bars six and seven) and without (bars four and five) trehalose, were tested in a HUVEC scratch wound healing assay. The positive control (“+ve”, bar 1) consisted of culturing the scratched well in complete HUVEC cell medium (“Comp”) plus PBS “treatment,” and the negative control (“-ve”, bar 2) consisted of culturing the scratched wells in basal medium (“Poor”) plus PBS “treatment.” FBS-derived EV served as an EV control (“EV Ctl”, bar three). A 1x treatment equals the secretome derived from 150,000 cells. Values are baseline (negative control) subtracted and normalized to the positive control.FIG. 28 depicts cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay. Bars labeled one to seven from left to right. Small EV-enriched secretome final formulations with (bars six and seven) and without (bars four and five) trehalose, were tested in a cardiomyocyte survival assay. A 1x treatment equals the secretome derived from 150,000 cells. PBS controlswith (bar two) and without (bar one) staurosporine served as negative (“-ve”) and positive (“+ve”) controls, respectively. Mesenchymal Stem Cell (MSC)-derived EV served as an EV control (“EV Ctl”, bar three). Plated cells were either stressed with staurosporine for 4 hours prior to treatment (“+”), or were not stressed with staurosporine (“-“),FIGS. 29 and 30 depict flowcharts illustrating the stages of production (vesiculation, conditioned media clarification, and TFF, FIG. 29; and final formulation, FIG. 30) in a second GMP-compatible process, described in Example 12 and Example 13, i.e., for Test Example 22. The final formulation in this example was produced with and without trehalose addition prior to sterilizing filtration. The different samples which underwent in-process and quality control testing are indicated with a “*” (e.g., *6, *7, etc.).FIG. 31 depicts the results of flow cytometry experiments to analyze the cell marker expression profile of CPCs at different times during the vesiculation process (D+0, D+3 and D+5). iPSCs and cardiomyocytes (CM) were used as control cells and were analyzed separately. The values shown are average values.FIG. 32 depicts the morphology of CPCs during the vesiculation process, as observed under light microscopy. Cell morphology was analyzed in cells within both T75 and selected CS10 flasks. The left image is a representative image showing the typical D+3 morphology observed in all vessels analyzed at D+3. The right image is a representative image showing the typical D+5 morphology observed in all vessels analyzed at D+5. T75 flasks were used for image capture for clarity.FIGS. 33 and 34 depict the results of an analysis of particle concentration and size distribution of EVs. FIG. 33 depicts the particle concentration and size distribution of EVs in conditioned media, before clarification, conditioned media after clarification, in the final formulation (i.e., after TFF) and in the final formulation with trehalose using nanoparticle tracking analysis. Sample naming is depicted in FIG. 29 and FIG 30. FIG. 34 depicts the concentration and size distribution of particles detected by NT A in *6, sample a (Test 22); *7, sample c (Test 22); and *7, sample d (Test 22).FIGS. 35-36 depict the MACSPlex results of analysis of small EV-enriched secretome final formulations with and without trehalose, for expression of extracellular vesicle tetraspanins often expressed on the surface of extracellular vesicles (CD9, CD81 and CD63) (FIG. 35); and for various other markers, which exhibited little or no expression (FIG. 36).FIG. 37 depicts the MACSPlex results for small EV-enriched secretome final formulations with and without trehalose, for expression of cardiac-related markers. For all samples depicted in FIG. 37, the markers depicted in FIG. 37 were found to be expressed.FIGS. 38 and 39 depict relative scratch wound healing in a HUVEC scratch wound healing assay. Bars labeled 1 through 15 from left to right in FIG. 38 and 16 though 30 from left to right in FIG. 39. The results for samples *7, sample a (Test 22) (bars 4 through 9) and *7, sample b (Test 22) (bars 10 through 15) (depicted in FIG. 30) are shown in FIG. 38. The results for samples *7, sample c (Test 22) (bars 19 through 24) and *7, sample d (Test 22) (bars 25 through 30) (depicted in FIG. 30) are shown in FIG. 39. The positive control (“+ve”, bars 1 and 16) consisted of culturing the scratched well in complete HUVEC cell medium (“Comp”) plus PBS “treatment”, and the negative control (“-ve”, bars 2 and 17) consisted of culturing the scratched wells in basal medium (Poor) plus PBS “treatment”. FBS-derived EV served as an EV control (EV Ctl, bars 3 and 18). A 1x treatment equals the secretome derived from 150,000 cells. Values are baseline subtracted (negative control) and normalized to the positive control. Samples *7, sample a (Test 22); *7, sample b (Test 22); *7, sample c (Test 22)”; and “*7 sample d (Test 22), whose preparation is described in detail in Example 12 and Example 13 were tested in a scratch wound healing assay as described in Example 17. These four samples are derived from the same TFF retentate but differ in their method of final formulation. These four variations are to use fresh retentate and filter sterilize with Sterivex-GP, 0.22 μm filter (resulting in sample a); to use fresh retentate, supplement with trehalose, and filter sterilize with a Sterivex-GP, 0.22 μm filter (resulting in sample b); freezing a retentate, thawing it, and then filter sterilizing with a Sterivex-GP, 0.22 μm filter (resulting in sample c); or freezing a retentate, thawing it, and then filter sterilizing with a Sartopore 2; 0.45+0.2μm filter (resulting in sample d).In FIG. 38, the +ve control result is at 100% (first bar on the left). The -ve control is at 0% (second bar from the left). The EV Ctl is 29.8% (third bar from the left). The Final Formulations *7, sample a (Test 22) and *7, sample b (Test 22) gave similar results. Both materials improved scratch wound healing, with indications of a dose-response from the doses ranging from 0.25x to 2.6x. The lowest dose tested, which was 0.25x gave at least a 17% increase in wound healing capacity over the -ve control for both samples. At a dose of 2.6x, both samples improved scratch wound healing by greater than 25% over the negative control. The *7, sample a (Test 22) improved scratch wound healing by 35.6% at the 2.6x dose. Taken together, the data here indicate that bothSterivex-GP, 0.22 μm filtered and Sartopore 2; 0.45+0.2 μm filtered *7 Final formulations are equally potent in the scratch wound healing assay. This indicates that both filtration devices are equally suitable filtration devices for maintaining scratch wound healing potency of the CPC-EV- enriched secretomes prepared by the GMP-Compatible process for producing small extracellular vesicle-enriched fraction (sEV) formulations described as in Example 12 and 13. In terms of potency in a scratch wound healing assay, all four variations of final formulation method depicted in FIG. 30 and described in Example 13 are equally suitable.In FIG. 39, the +vc control result is at 100% (first bar on the left). The -ve control is at 0% (second bar from the left). The EV Ctl is a 25.2% (third bar from the left). The Final Formulations *7, sample c (Test 22) and *7, sample d (Test 22) gave similar results. Both materials improved scratch wound healing, with indications of a dose-response from the doses ranging from 0.25x to 2.6x. The lowest dose tested, which was 0.25x gave at least a 17% increase in wound healing capacity over the -ve control for both samples. At a dose of 2.6x, both samples improved scratch wound healing by greater than 30% over the negative control. Taken together, the data here indicate that both Sterivex-GP, 0.22 μm filtered and Sartopore 2; 0.45+0.2 μm filtered *7 Final formulations are equally potent in the scratch wound healing assay. This indicates that both filtration devices are equally suitable filtration devices for maintaining scratch wound healing potency of the CPC-EV-enriched secretomes prepared by the GMP-Compatible process for producing small extracellular vesicle-enriched fraction (sEV) formulations described as in Example 12 and 13. In terms of potency in a scratch wound healing assay, all four variations of final formulation method depicted in FIG. 30 and described in Example 13 are equally suitable.FIGS. 40 and 41 depict cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay. The results for samples *7, sample a (Test 22) and *7, sample b (Test 22) (depicted in FIG. 30) are shown in FIG. 40, bars referred to as bars one through nine from left to right. The results for samples *7, sample c (Test 22) and *7, sample d (Test 22) (depicted in FIG. 30) are shown in FIG. 41, bars referred to as bars 1 through 15 from left to right, 1x equals the secretome derived from 150,000 cells. PBS controls with and without staurosporine served as negative control (“- ve”, bar two in both figures) and positive control (“+ve”, bar one in both figures), respectively. Mesenchymal Stem Cell (MSC)-derived EV served as an EV control (“EV Ctl”, bar three in both figures). Plated cells were either stressed with staurosporine for 4 hours prior to treatment (“+”, bars two through nine in FIG. 40 and bars two through 15 in FIG. 41), or were not stressed withstaurosporine (“-”, bar one in both figures). *7, sample a (Test 22); *7, sample b (Test 22); *7, sample c (Test 22); *7, sample d (Test 22) (depicted in FIG. 30) whose preparation is described in detail in Example 12 and Example 13 were tested in a cardiomyocyte survival assay as described in Example 17. These four samples are derived from the same TFF retentate but differ in their method of final formulation. These four variations are to use fresh retentate and filter sterilize withSterivex-GP, 0.22 μm filter (resulting in *7, sample a (Test 22)); to use fresh retentate, supplement with trehalose, and filter sterilize with a Sterivex-GP, 0.22 μm filter (resulting in *7, sample b (Test 22)); to freeze retentate, thaw it, and then filter sterilize with a Sterivex-GP, 0.22 μm filter (resulting in *7, sample c (Test 22)); or freeze retentate, thaw it, and then filter sterilize with a Sartopore 2; 0.45+0. μm filter (resulting in *7, sample d (Test 22)).In FIG. 40, at the 24-hour time point illustrated in the figure, the greatest effect seen for *7, sample a (Test 22) was at the 0.75x dose (bar six), which corresponded to an increase (improvement) in cell survival of 10.64% more than the -ve control. At the 24-hour time point illustrated in the figure, the greatest effect seen for *7, sample b (Test 22) was at the 0.5x dose (bar eight), which corresponded to an increase (improvement) in cell survival of 11.85% improvement over the -ve control.In FIG. 41, at the 24-hour time point illustrated in the figure, the greatest effect seen for *7, sample c (Test 22) was at the 0.75x dose (bar six), which corresponded to an increase (improvement) in cell survival of 14.82% more than the -ve control. At the 24-hour time point illustrated in the figure, the greatest effect seen for *7, sample d (Test 22) was at the 0.5x dose (bar 11), which corresponded to an increase (improvement) in cell survival of 11.90% improvement over the -ve control.FIG. 42 depicts echocardiography results of mice with induced chronic heart failure following administration of CPC EVs (“sEV5.3”), or PBS (as a control). The data depicts the absolute changes in Left Ventricular End Systolic Volume (LVESV); Left Ventricular End Diastolic Volume (LVEDV); and ejection fraction (EF). The bottom three graphs show the absolute change in each animal as as individual point with an overlayed quantile plot for the group. For illustrative purposes, a dotted horizontal line is added to each graph to indicate the approximate location of the threshold used to define severely progressive heart failure for each of the three parameters. For illustrative purposes, a dotted-line-box has been added to the figure to identify the animals considered to have severely progressive heart failure in each graph. The actual number ofanimals with and without severely progressive heart failure is noted in large font on each graph. The graph on the left shows a threshold of 9.1 μL. Animals at or above this threshold have severely progressive heart failure. The middle graph shows a threshold of 4 μL. Animals at or above this threshold have severely progressive heart failure. The graph on the right shows a threshold of - 5.5%. Animals at or below this threshold have severely progressive heart failure, with severely decreasing EF. In all three graphs, less of the sEV treated animals have severely progressive heart failure than in the PBS group. For the Absolute Change in LVESV, the sEV5.3 group had significantly less animals with severely progressive heart failure than PBS controls (5 of 11 animals versus 10 of 11 for PBS controls, p<0.05). For the Absolute Change in LVEDV, the sEV5.3 group had significantly less animals with severely progressive heart failure than PBS controls (5 of 11 animals versus 10 of 11 for PBS controls, p<0.05). For the Absolute Change in EV, the sEV5.3 group had less animals with severely progressive heart failure than PBS controls, approaching significance (1 of 11 animals versus 5 of 11 for PBS controls, p<0.05). Taken together, these results indicate that cardiac therapy candidate- 1 -extracellular vesicle enriched secretome (CTC1-EV) improves heart failure outcomes in animals with chronic heart failure by limiting the progression of that heart failure.FIG. 43 depicts the results of Lunatic analysis for cellular RNA extracted from *3 (Test 25) as depicted in FIG. 105. The RNA extracted from *3 (Test 25) is labeled as sample “546” in the figure. FIG. 43 also depicts the results of Lunatic analysis for cellular RNA extracted from *3 (Test 26) as depicted in FIG. 111. The RNA extracted from *3 (Test 26) is labeled as sample “547” in the figure. The preparation of samples *3 (Test 25) and *3 (Test 26) is described in detail in Example 19.FIG. 44 depicts the results for quality control (QC) testing of cellular RNA that was extracted from *3 (Test 25). This RNA is described as “546RNA” in the figure. This analysis was completed to assess the quality of the extracted RNA.FIG. 45 depicts the results for quality control (QC) testing of cellular RNA that was extracted from*3 (Test 26). This RNA is described as “547RNA” in the figure. This analysis was completed to assess the quality of the extracted RNA.FIG. 46 depicts the results of Lunatic analysis for CTC1-EV RNA extracted from *9 (Test 27). This RNA is described as “45.evma” in the figure. This analysis was completed to assess the quality of the extracted RNA.FIG. 47 depicts the results for quality control (QC) testing of CTC1-EV RNA extracted from *9 (Test 27). This RNA is described as “45.evma” in the figure. This analysis was completed to assess the quality of the extracted RNA.FIG. 48 depicts the results for quality control (QC) testing of the cDNA libraries produced from three different RNA samples. The “Library from 546RNA” is the cDNA library generated from the RNA extracted from *3 (Test 25). The “Library from 547RNA” is the cDNA library generated from the RNA extracted from *3 (Test 26). The “Library from 45.evma” is the cDNA library from the RNA extracted from *9 (Test 27). These assessments were performed to assess the quality of the cDNA libraries.FIG. 49 depicts the results of the analysis of the sequencing read lengths for the small RNA sequencing analysis of CTC1-EV, which is *9 (Test 27) in this experiment.FIG. 50 depicts the prevalence (read distribution) of different RNA biotypes in CTC1-EV, which is *9 (Test 27) in this experiment. The RNA biotypes illustrated here were determined by sequence mapping. Results for this sample are identified as “45RNA” in the figure.FIG. 51 depicts the results of the analysis of read distributions for isomirs of the top 20 miRs identified in CTC1-EV, which is *9 (Test 27) in this experiment. Results for this sample are identified as “45RNA” in the figure.FIG. 52 depicts the top 40 most abundant miRNA identified in CTC1-EV, which is *9 (Test 27) in this experiment. The data are displayed as a honeycomb representation. The results for this sample are labeled as “45RNA” in this figure. The data which was used to generate FIG. 52 are tabulated in TABLE 9.FIG. 53 shows a wordcloud indicating the top localization terms associated with the RNA sequences identified in CTC1-EV, which is *9 (Test 27) in this experiment.FIG. 54 shows a scatterplot identifying an miRNAs signature in CTC1-EV as compared to extracellular vesicles from other cell types included in this study (astrocyte, cardiac fibroblast, cardiomyocyte, neurons (GABAergic, Glutamatergic, Dopaminergic, Motor Neurons, and induced Neurons by forward reprogramming), endothelial, hematopoietic progenitor cells, hepatocyte, induced pluripotent stem cell, microglia, macrophage, mesenchymal stem cells, pericytes, and retinal pigment epithelial). The CTC1 EV miR signature was extracted by calculating the genewise 10thpercential of log2FPKM values of CTC1-EV sample replicates and 90thpercentile of all the other samples in the study.FIG. 55 and FIG. 56 depict cryo-electron micrographs of extracellular vesicles identified in CTC1-EV, which is *9 (Test 27) in this experiment. Scale bar = 100nm.FIG. 57 depicts a cryo-electron micrograph of a large bilipid membrane vesicle (identified in CTC1-EV, which is *9 (Test 27) in this experiment) of approximately 200 nm in diameter, which contains therein a second bilipid membrane vesicle of a similar diameter as well as a third, smaller (approximately 50 nm in diameter) bilipid membrane. Scale bar = 100nm.FIG. 58 depicts a 96-well platemap for the analysis of the effects of CTC 1 -EV in a HUVEC plating assay as described in Example 23. CTC1-EV in this experiment is *5b.uc (Test 26). This sample is labeled “EV 481" in the figure. A mock-EV control is also included (labeled “EV 457” in the figure).FIG. 59 depicts the effects of CTC 1 -EV in the HUVEC plating assay, as measured by Tecan for Life Science® plate reader. Bars referred to as bars one through seven from left to right. CTC1-EV is *5b.uc (Test 26) in this experiment (results depicted in bars four and five) was analyzed in a HUVEC plating assay. In this assay, the number of HUVEC cells in each well are determined by measuring the amount of intracellular ATP in the well, which is a surrogate for the number of cells. The amount of ATP is determined using the Cell Titer Gio kit as described in Example 23. The readout is luminescence. The higher the luminescence, the more ATP was present in the well, which means more cells were present in the cell. The higher luminescence therefore means the better the HUVEC plating. In this assay, the positive control (“+ Control", bar one) is HUVEC cells plated in their complete media as described in Example 23. The negative control (“- Control", bar two) is the HUVEC cells plated in poor media as described in Example 23. For the remaining conditions, the HUVEC cells are plated in poor media supplemented with FBS-EV (bar three), *5b.uc (Test 26) (bars four and five), or matched mock-EV controls (“mock-EV”, bars six and seven) as described in Example 23. The results are double normalized such that the negative control is set to 0% and the positive control is set to 100%. The results for the positive control are in the first bar on the left (100%). The results for the negative control are in the second bar from the left (0%). The FBS-EV condition gave a 60.37% result. The *5b.uc (Test 26) resulted in 29.11% luminescence of the positive control when dosed at first dose (“1x"). The *5b.uc (Test 26) resulted in 49.37% luminescence of the positive control when dosed at a three times higher dose than the first dose (“3x”). The matched mock-EV controls were also dosed at 1x and 3x doses, resulting in 9.66% and 17.90% luminescence of the positive control. The greater the %luminescence in this assay, the greater the improvement the material tested has on HUVEC cell plating. Both the 1x and 3x doses of the CTC1-EV tested here improve HUVEC plating in this assay as compared to the negative control, and as compared to their matched mock-EV controls. The improvement in HUVEC seeding in this assay is more than twice the improvement seen from the matched mock-EV controls.FIG. 60 depicts the effects of CTC1-EV (which is sample *5b.uc (Test 26) in this experiment) in a HUVEC plating assay, as measured by visual inspection (the nuclei of living cells arc labeled in green, which resembles a bright light grey in black and white rendering). This sample is labeled “CTC1-EV *5b.uc (Test 26)” in the figure. The mock-EV control is labeled “mock-EV” in this figure.FIG. 61 depicts the effects of CTC1-EV, which is sample *5b.uc (Test 26) in this experiment, in the HUVEC plating assay, as determined by CyQuant nucleic acid stain (bars referred to as one through seven from left to right). Sample *5b.uc was analyzed in a HUVEC plating assay as described in Example 23. In this assay, the number of HUVEC cells in each well is determined by measuring the amount of fluorescence in each well. The fluorescence comes from the CyQuant Green dye, which is fluorescent inside cells. The higher the fluorescence signal at the end of the assay, the more cells are present in the well. The greater the number of cells present in the well, the better the tested material is at improving HUVEC cell plating. In this assay, the positive control (“+ Control”, bar one) is HUVEC cells plated in their complete media as described in Example 23. The negative control (“- Control”, bar two) is the HUVEC cells plated in poor media as described in Example 23. For the remaining conditions, the HUVEC cells are plated in poor media supplemented with FBS-EV (bar three), *5b.uc (bars four and five), or matched mock- EV controls (“mock-EV”, bars six and seven) as described in Example 23. The results are double normalized such that the negative control is set to 0% and the positive control is set to 100%. The results for the positive control are in the first bar on the left (100%). The results for the negative control are in the second bar from the left (0%). The FBS-EV condition gave a 36.34% result. The *5b.uc (Test 26) resulted in 15.43% of the positive control when dosed at a first dose (“1x”). The *5b.uc (Test 26) resulted in 36.75% of the positive control when dosed at a three times higher dose than the first dose (“3x”). The matched mock-EV controls were also dosed at 1x and 3x doses, resulting in -1.07% and 8.42% of the positive control. Both the 1x and 3x doses of the CTC1-EV tested here [*5b.uc (Test 26)] improve HUVEC plating in this assay as compared to the negativecontrol, and as compared to their matched mock-EV controls. The improvement in HUVEC seeding in this assay by the CTC1-EV tested here [*5b.uc (Test 26)] is more than four times any improvement seen from the matched mock-EV controls.FIG. 62 depicts the effects of CTC1-EV, which is sample *5b.uc (Test 26) in this experiment, in the HUVEC plating assay, as determined by CyQuant nucleic acid stain (bars referred to as one through seven from left to right) . Sample *5b.uc (Test 26) (bars four and five) was analysed in a HUVEC plating assay as described in Example 23. In this assay, the number of HUVEC cells in each well arc determined by analysing microscope images where the cells arc easily identified by CyQuant green straining. The greater the number of cells present in the well, the better the tested material is at improving HUVEC cell plating. In this assay, the positive control (“+ Control”, bar one) is HUVEC cells plated in their complete media as described in Example 23. The negative control (“- Control”, bar two) is the HUVEC cells plated in poor media as described in Example 23. For the remaining conditions, the HUVEC cells are plated in poor media supplemented with FBS-EV (bar three), Sample *5b.uc (Test 26) (bars four and five), or matched mock-EV controls (“mock-EV”, bars six and seven) as described in Example 23. The results are double normalized such that the negative control is set to 0% and the positive control is set to 100%. The results for the positive control are in the first bar on the left (100%). The results for the negative control are in the second bar from the left (0%). The FBS-EV condition gave a 54.47% result. The *5b.uc (Test 26) resulted in 20.42% of the positive control when dosed at a first dose (“1x”). The *5b.uc (Test 26) resulted in 48.09% of the positive control when dosed at a three times higher dose than the first dose (“3x”). The matched mock-EV controls were also dosed at 1x and 3x doses, resulting in -2.13% and 11.71% of the positive control. Both the 1x and 3x doses of the CTC1-EV tested here [*5b.uc (Test 26)] improve HUVEC plating in this assay as compared to the negative control, and as compared to their matched mock-EV controls. The improvement in HUVEC seeding in this assay by the CTC1-EV tested here [*5b.uc (Test 26)] is more than four times any improvement seen from the matched mock-EV controls.FIG. 63 depicts the results of an analysis of CTC1-EV in a HUVEC stress assay, in which HUVECs were stressed with staurosporine as described in Example 24. Bars are referred to as bars one through six from left to right. Three different EV types were tested in a HUVEC Stress Assay. In this assay, HUVEC cells in culture are not stressed (“Complete”; positive control; bar one), stressed by culturing in serum-free media (“Poor”; bar two) or stressed by culturing in serum-freemedia containing staurosporine (“Poor + Staurosporine” conditions; bars three through six. For the “Poor + Staurosporine” conditions, either vehicle control was added to the culture media (“dPBS”, bar three) or a dose of 5 x 109particles was added from one of three different EV- enriched secretome preparations. These EV-enriched secretome preparations were isolated from MSC conditioned media (“MSC-EV”, bar four), from iCell CPC conditioned media (“iCell-CPC- EV”, bar five) or from CTC 1 conditioned media (“CTC 1 -EV”, bar six). In this example, the CTC 1 - EV (bar six) is *9 (Test 27). The number of HUVEC cells remaining in culture at the end of the assay period was determined. The result for each condition was normalized to the “dPBS” (bar three) vehicle control condition. The CTC1-EV sample tested here [*9 (Test 27)] improved HUVEC cell survival by 40% in this experiment.FIG. 64 depicts the results of an analysis of EV-CPC in an in-vitro chemotherapy-induced cardiomyopathy assay, as determined by measuring intracellular ATP concentration (A) at day 6, (B) at day 8, and (C) at day 10, in doxorubicin-stressed cardiomyocytes (and non-stressed control cardiomyocytes) as described in Example 25. The results were normalized to the control (“DOX+Placebo”) at the day of the measurements. The results are from five separate experiments, with each sample within each experiment being performed in triplicate. The bars show the mean+7-SEM. *p<0.05 (Kruskal-Wallis with Dunn’s multiple comparisons test). CM: complete maintenance cardiomyocyte medium; DOX: doxorubicin, EV-CPC: extracellular vesicles derived from cardiac progenitor cells; VM-CPC: CPC-virgin medium; ATP: Adenosine Triphosphate.As can be seen from FIG. 65, CTC 1 -EV sample (which is *7, sample a (Test 20) in this experiment; labeled “CTC 1 -EV (prod 20)” in the figure), improved (increased) the amount of intracellular ATP per cell in the doxorubicin stressed cardiomyocytes by 40% over the stressed control. This result shows that CTC1-EV [*7, sample a (Test 20)] was able to promote cardiomyocyte metabolic health in surviving cells. The results of the positive control are shown in bar 1 (as numbered left to right, 1 through 3). The results of the negative control, which is doxorubicin stressed cells, are shown in bar 2. The results of CTC 1 -EV treatment of doxorubicin stressed cells are shown in bar 3.FIG. 66 and FIG. 67 depict the results of an anti-fibrosis assay in which HCF cells were stimulated with TGF-β1, and the effects of MSC-EV and CTC1-EV [*9 (Test 27) in this experiment] on various fibrosis-associated markers were then analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) as described in Example 26 (bars referred toas one through ten in both figures from left to right). The results for the MMP2 expression analysis are shown in FIG. 66. The results for the Periostin (“Postn”) expression analysis are shown in FIG. 67. In this example, the CTC1-EV is *9 (Test 27) (bars five, six, nine, and ten in each figure).FIG. 68 depicts the experimental schedule for the experiment described in Example 27. A timeline is presented illustrating the five days on which rats received IP injections of doxorubicin (where applicable), the three days on which rats were evaluated by echocardiography, and the three days on which rats received TV injection of placebo (NaCl) or CTC1-EV where applicable, where CTC1-EV is *7, sample a (Test 20) in this experiment. In this figure, *7, sample a (Test 20) is labeled as “GMP-EV”.FIG. 69A and FIG. 69B depict the effects of CTC1-EV (which is *7, sample a (Test 20) in this experiment), on cardiac function in a rat chemotherapy (doxorubicin)-induced cardiomyopathy (CCM) model as described in Example 27. In this figure, *7, sample a (Test 20) is labeled as “GMP-EV”. FIG. 69A depicts the % change in LV-ESV since DIO. FIG 69B depicts the % change in LV-EDV since day 10. Results were measured by echocardiography and expressed as a percent change (Median+ / -IQR) from day 10 (post-DOX administration). There were 6 animals that did not receive doxorubicin (Sham: n=6). There were 11 animals that received the doxorubicin stress and then NaCl injections (“DOX+Placebo”; n=11) and there were 12 animals that received the doxorubicin stress and then *7, sample a (Test 20) injections (“DOX+GMP-EV”; n=12). In this figure, the symbol ‘*’ on the figure indicates p<0.05, Kruskal Wallis with Dunn’s correction test. LV-ESV / LV-EDV: left ventricular systolic / diastolic function.Cardiac function is related to heart volumes. Two types of heart volumes are examined here: the left ventricular end systolic volume (LVESV, or LV-ESV) and the left ventricular end diastolic volume (LVEDV, or LV-EDV). During heart failure, these two volumes increase. The more they increase, the worse the heart failure has progressed. The two volumes are measured by echocardiography (echo). The two volumes for each animal are measured once before doxorubicin injection (or before sham injections for “Sham” animals) (baseline echo, echo #1), then on the tenth day after the first doxorubicin administration / sham injection (which is before CTC1-EV treatment or placebo administration; echo #2), and finally at the end of the study period on or around 28 or 29 days after the first doxorubicin injection / sham injection (echo #3). The CTC1- EV in this experiment was *7, sample a (Test 27). The group of rats receiving this material is referred to as “Dox+GMP-EV” in FIG. 69 A and FIG. 69B. The Placebo group received isotonicbuffer, NaCl 0.9%, “Placebo”; this group of animals is referred to as “DOX+Placebo” in FIG. 69A and FIG. 69B). CTC1-EV or Placebo were administered 11, 14 and 16 days after the first doxorubicin injection as depicted in FIG. 68. The more the heart volumes increase between the echo #2 and echo #3, the more the heart failure has progressed in that animal during that time period. The “Sham” animals are not in heart failure; they show no markers of failing hearts. The Sham animals were not administered any doxorubicin or CTC1-EV.In the experiment depicted in FIG. 69 A and FIG. 69B, the results are expressed as a percent change (Mcdian+ / -IQR) from day 10 post-DOX administration (echo #2) to the end of the study period (echo #3). There were 6 Sham animals, 11 Placebo injected animals, and 12 animals injected with CTC1-EV. (Sham: n=6; DOX+Placebo: n=11, Dox+GMP-EV: n=12).For the LV-ESV results (depicted in FIG. 69A), the Sham group, on average, had a -3.0% change in volume; the DOX+Placebo group, on average, increased LVESV by 28.1%; the Dox+GMP-EV increased LVESV by 12.9%, which means that their heart failure progressed less than 1 / 2 as much as the placebo group as determined by LV-ESV change, which is a 2.2-times improvement in outcome.For the LV-EDV results (depicted in FIG. 69B), the Sham group, on average, had a -0.1% change in volume; the DOX+Placebo group, on average, increased LVEDV by 19.2%; the Dox+GMP-EV increased LVEDV by 0.7%, which means that their heart failure progressed less than 4-tenths (0.7 / 19.2) as much as the placebo group as determined by LV-ESV change, which is a 27-times improvement in outcome.While LV-ESV volumes were significantly increased in placebo-injected hearts compared with Sham (p=0.033), they were preserved by GMP-EV injections (effect size Hedges' g index of 0.4). Likewise, the percentages of responder rats which did not increase their LV-EDV volumes by more than 5% from their post-DOX pre-treatment values were 58% (7 out of 12) vs. 28% (3 out of 11) in the Dox+GMP-EV and DOX+Placebo rat hearts, respectively (effect size Hedges' g index of 0.5, OR=3.7).FIG. 70A, FIG. 70B, FIG. 70C, FIG. 70D, FIG. 71 depict the results of experiments validating the rat model of doxorubicin-induced cardiomyopathy as described in Example 27. FIG. 70A depicts LVEF as a percent change (Mean+ / -SEM) from day 10 (post-DOX administration). FIG. 70B depicts the end-study ratio of diastolic blood pressure to LV-EDV taken as a surrogate marker for ventricular compliance. FIG. 70C depicts mean blood pressure. FIG. 70D depicts theQT interval corrected for heart rate. **p<0.005; (Mann Whitney test). LVEF: left ventricular ejection fraction (%); QTc: length of QT segment corrected to heart rate (HR) (in seconds, “sec”); DBF: diastolic blood pressure; LV-EDV: left ventricular end diastolic volume.FIG. 71 depicts the results of experiments further validating the rat model of doxorubicin- induced cardiomyopathy as described in Example 27. The figure depicts the end-of-study ratio of systolic blood pressure to LV-ESV. This ratio is taken as a surrogate marker for ventricular contractility. This ratio is termed the “End Systolic Elastance”. LV-EDV: left 'ventricular end- diastolic volume, SEP: systolic blood pressure. That data show that the average SBP / LV-ESV decreased in doxorubicin (DOX) treated animals (“DOX+Placebo” group) by 0.46 mmHg / uL (which is a decrease of 34%) from the animals that did not receive DOX (“Sham” group).FIG. 72 depicts the experimental design of two of the experiments used to establish a novel chemotherapy-induced cardiomyopathy model in rats. The first experiment, in which 6 male rats were injected with doxorubicin resulted in an unacceptable mortality rate over the 30 or 32 day long procedure. 70% of these male rats died prior to completing the study period. A second experiment is depicted in which male and female rats were included. The female rats had a survival rate much greater than the males (91% vs 40%, respectively). d= day; DOXO = doxorubicin injection; echo = echocardiographic measurements.FIG. 73 and FIG. 74 depict the results of experiments analyzing the post-thaw viability of CTC1 cells under different conditions as described in Example 28 (referred to as bars one through six from left to right in each figure respectively).To improve CTC1 post-thaw survival, plating and expansion, three modifications to the process were tested. Details of this experiment are given in Example 28. The starting process thawed cells in the same media that was used for plating and expansion (referred to as Complete Media A, “CM A”, bars one and two in both FIG. 73 and FIG. 74). The starting process used a gentle centrifugation step to pellet cells after thaw, enabling the removal of the cryopreservation media (use of the centrifugation step is referred to as the centrifuged condition, “Cent”, bars one, three, and five in both FIG. 73 and FIG. 74). The starting process used a thaw media containing 2 mg / mL human serum albumin. The starting process used a thaw media that did not contain a ROC inhibitor. The starting process is referred to as “CM A Cent” in FIG. 73 and FIG. 74. The number of viable cells which were placed into each vial at the cryopreservation stage was known. The number of viable cells recovered after the thaw process was noted. The percentage ofrecovered cells at thaw (“% Recovered”) was calculated by taking the number of viable cells per vial after the thaw process divided by the number of viable cells placed into each vial prior to cryopreservation, times 100%. The greater the percentage recovered at thaw, the more successful the thaw process was deemed. The % Recovered was calculated for six conditions illustrated in FIG. 73 and six conditions illustrated in FIG. 74, which varied by the thaw media compositions used and whether or not a centrifugation step was included in the process. The recipes for the various thaw media used are detailed in Example 28.In FIG. 73, the results of the starting process (CM A with a Centrifugation step; “CM A Cent”) are given in the bar one. The results of the modified process using the starting thaw media but omitting the centrifugation step (“CM A No Cent”) are shown in bar two. The results of the modified process using the thaw media containing higher albumin concentration (20 mg / mL HSA) with and without a centrifugation step (“CM B Cent”, “CM B No Cent”, respectively) are shown in bar three and bar four respectively. The results of the modified process using the thaw media containing higher albumin concentration (20 mg / mL) and 1 μM H1152 ROC inhibitor with and without a centrifugation step (“CM C Cent”, “CM C No Cent”, respectively) are shown in bar five and bar six, respectively.FIG. 74 depicts the results of post-thaw cell viability assays conducted under different conditions.In FIG. 74, the results of the starting process (CM A with a Centrifugation step; “CM A Cent”) are given in bar onee. The results of the modified process using the starting thaw media but omitting the centrifugation step (“CM A No Cent”) are shown in bar two. The results of the modified process using the thaw media containing 1μM H1152 with and without a centrifugation step (“CM B Cent”, “CM B No Cent”, respectively) are shown in bar three and bar four, respectively. Note that this is a different set of conditions from the “CM B” conditions illustrated in FIG. 73. The results of the modified process using the thaw media containing higher albumin concentration (20 mg / mL) and 1 μM H1 152 ROC inhibitor with and without a centrifugation step (“CM C Cent”, “CM C No Cent”, respectively) are shown inbars five and six, respectively from the left.Taken together, the results FIGS. 73 and 74 show that when the centrifugation step used to remove the cryopreservation media components is omitted, the % Recovery at Thaw increasesby an average of 6.45 percentage points over the matched centrifuged conditions. (6.45 is the average of -0.2, +11.1, +6.6, +6.5, +7.4, and +7.3)FIG. 75 depicts an experimental design for analyzing post-thaw platability of CTC1 cells as described in Example 28.FIG. 76 depicts the results of experiments analyzing cell densities (cells / cm2) after thawing and plating CTC1 cells under different conditions as described in Example 28.FIG. 77 depicts the results of experiments optimizing the CTC1 cell culture vessel for plating as described in Example 29.FIG. 78 depicts an experimental design for analyzing the effect of insulin concentration on CTC1 cell yield throughout vesiculation as described in Example 30.FIG. 79 depicts the results of experiments analyzing the effect of insulin concentration on CTC1 cell yield throughout vesiculation as described in Example 30.FIG. 80 depicts an experimental design for analyzing the effect of FGF concentration on CTC1 cells as described in Example 31.FIG. 81 depicts the results of experiments analyzing CTC1 cell counts after incubation at different FGF concentrations as described in Example 31.FIG. 82 depicts the results of scratch wound healing experiments using EV / secretome from CTC1 cells incubated with different FGF concentrations as described in Example 31 (lines referred to as A through H from highest to lowest at the 18-hour timepoint). Conditions included in this assay include positive control (line A); High FGF, MC (labeled as “our standard protocol”, line B); Mid FGF, MC (line C); Low FGF, MV (line D); Low FGF, MC (line E), Mid FGF, MV (line F); High FGF, MV (line G); and negative control (line H). The results at the 18-hour time point are also summarized in TABLE 22.FIG. 83 depicts the results of cardiomyocyte survival assay experiments usingEV / secretome from CTC1 cells incubated with different FGF concentrations as described inExample 31. The results at the 19 hour time point are also summarized in TABLE 23.FIG. 84 and FIG. 85 depict the results of a time course monitoring scratch wound healing, for fresh media samples. The y-axis is the % wound confluence, and the x-axis is hours since the start of the assay. Lines referred to as A through P from top to bottom at the 18-hour time point, UF retentates were produced from freshly collected and freshly clarified CTC1 conditioned media as described in Example 32 (lines D through H). UF retentates from freshly collected and clarifiedCPC virgin media controls were prepared as described in Example 32 (“Mock-EV controls”, lines I, K through P). Fresh conditioned media was also subjected to ultracentrifugation as described in Example 32 (“Fresh UC generated EV”, line B). An EV-enriched secretome was also prepared from FBS by ultracentrifugation (“FBS Control”, line C). These compositions were tested for the ability to stimulate HUVEC scratch wound healing in a HUVEC Scratch Wound Healing Assay, as described in Example 32. The positive control is the condition in which HUVEC are maintained in complete media (“Complete Media Control”, line A). The positive control attained 33.2% wound confluence at the 18-hour time point. The negative control is the condition in which HUVEC are cultured in serum-free, or poor, media (“Poor Media Control", line J). The negative control attained for this HUVEC scratch wound healing assay, retentate samples were dosed into the assay at a 3x dose (where 1x doses were the secretome / EV / secretome fraction produced by 150,000 mother cells). Mock-EV controls were volume matched to the corresponding condition. The Fresh UC generated EV sample was dosed at 1x. The figure depicts the results of a time course monitoring scratch wound healing, for fresh media samples. At the 18-hour time point, the Fresh UC generated EV sample (line B) had the strongest effect out of all test conditions. Fresh UF retentate fractions containing components with molecular weight in the 5 kDa- 10 kDa (line H), 10 kDa -30 kDa (line G), 30 kDa -50 kDa, (line F) 50 kDa-100 kDa (line D) and 100 kDa-0.2 μm (line E) ranges all had a greater effect on HUVEC scratch wound healing than the Mock-EV and Poor media controls. This indicates that the retentates have a positive effect on endothelial cell migration into the scratch wound. The strongest effects are caused by components of the conditioned media having molecular weights between 30 kDa and 0.2 μm. At the 15-hour timepoint, the 30 kDa-50 kDa retentate (line F) has a 1.4-fold greater % wound confluence than the negative control (line J). At the 15-hour timepoint, the 50 kDa-100 kDa retentate (line D) has a 1.7-fold greater % wound confluence than the negative control (line J). At the 15-hour timepoint, the 100 kDa-2 μm retentate (line D) has a 2.3 -fold greater % wound confluence than the negative control (line J). The 18-hour time point results depicted in FIG. 84 are given in TABLE 24. The 18-hour time point results depicted in FIG 85 are given in TABLE 25.FIG. 86 is an alternative depiction of the data presented in FIG 84. In addition, it depicts the results of a CTC1-EV secretome composition prepared by ultracentrifugation of previously frozen CTC1 conditioned medium (labeled “EV 181 Frozen UC" in the figure) and its mock-EVcontrol (labeled “EV 189 Frozen UC MV” in the figure). The 18-hour time point results depicted in FIG. 86 are given in TABLE 26.FIG. 87 depicts the results of a time course monitoring scratch wound healing, for the CTC1-EV compositions isolated from fresh or frozen / thawed media samples as described in Example 32. The y-axis is the % wound confluence, and the x-axis is hours since the start of the assay. The 18 hour timepoint results depicted in FIG. 87 are given in TABLE 27.FIGS. 88-90 depict histograms of double normalized data from cardiomyocyte survival assays as described in Example 32. Bars referred to as one through seven from left to right in FIG. 88. Bars referred to as one through fourteen from left to right in FIG. 89. Bars referred to as one through fourteen from left to right in FIG. 90.FIG. 91 depicts the results of the flow cytometry analysis described in Example 35. Samples preparation is described in detail in Example 19. The data are expressed as a Mean Fluorescence Intensity (MFI) of technical and biological replicates. The MFI for the iPSC is shown by the white bars (the “iPSC” series, which is the first series of bars, starting from the left). The average MFI for “CPC D+0” (the second series of bars, starting from the left) was calculated by averaging the results from *1 (Test 25) and from *1 (Test 26). The average MFI for “CPC D+3” (the third series of bars, starting from the left), was calculated by averaging the results from *2 (Test 25) and from *2 (Test 26). The average MFI for “CPC D+5” (the fourth series of bars, starting from the left) was calculated by averaging sample *3 (Test 25) and from *3 (Test 26). The average MFI for CM samples is shown by the black bars (the “CM” series, the fifth series of bars, starting from the left).FIG. 92 depicts the results of the transcriptomic analysis for cells at day + 3 (“D+3”) and day +5 (“D+5”) as described in Example 35. Sample preparation is described in Example 19. The transcriptome analysis shown in the figure shows that the cells collected from Test Example 25 on day+3, from Test Example 25 on day +5, Test example 26 on day +3, and from Test Example 26 on day +5 have mRNA contents consistent with cardiovascular progenitor cells (CPC). Their mRNA profiles of the CPC are similar between the four cell samples. The profiles of the mRNA from the CPC are distinct from both the iPSC and CM controls. The heatmap was generated based on hierarchical clustering analysis using the UPGMA clustering method, with correlation distance metric in TIBCO Spotfire software vl 1.2.0. The data (log2FPKM) used to generate the heatmap depicted in FIG. 92 are presented in TABLE 36.FIG. 93 depicts the results of cell morphology analysis for cells at day + 3 (labeled as "CPC D+3” in figure) and day +5 (labeled as “CPC D+5” in figure) as described in Example 35. Sample preparation is described in Example 19. The microscope images shown in the figure show that the cell morphology is similar between *2 (Test 25) and *2 (Test 26) on day +3. The microscope images shown in the figure show that the cell morphology is similar between Test *3 (Test 25) and *3(Test 26) on day +5.FIGS. 94 and 95 depict particle concentration, mean and mode for samples *5 (Test 25), *6 (Test 25), *7 (Test 25) and *5 (Test 26), *6 (Test 26), *7 (Test 26), and samples *8 (Test 27) and *9 (Test 27) as described in Example 35. Samples preparation is described in Example 19. The particle concentration increased 67-fold between sample *5 (Test 25) to sample *6 (Test 25). The particle concentration increased 58-fold between sample *5 (Test 25) to sample *7 (Test 25). The particles concentration increased by 56-fold between sample *5 (Test 26) to sample *7 (Test 26) , a very similar factor to the fold change in Test Example 25. There is no significant change in particle concentration between sample *8 (Test 27) to sample *9 (Test 27). The final concentration of particles in the *9 (Test 27) is 2.8 x 1011particles / mL by Nanosight measurement. Throughout Test Examples 25, 26 and 27, the average mean particle size remained relatively constant, ranging from 119.7 to 169.8 nm. Throughout Test Examples 25, 26, and 27, the average mode particle size also remained relatively constant, ranging from 82.4 to 121.5 nm.FIGS. 96-98 depict CTC1-EV surface marker expression evaluated as described in Example 10 and Example 35. All results are normalized to 13 μL of sample. In thes figures, the average of the results for *4 (Test 25) and *4 (Test 26) are noted as “*4 (Tests 25-26)”. In these figures, the average of the results for *5 (Test 25) and *5 (Test 26) are noted as “*5 (Test 25-26)”. In these figures, several technical replicates were evaluated for *8 (Test 27) and the average is shown. In these figures the single result obtained for *9 (Test 27) is shown.FIG. 96 shows that the MFI for the tetraspanin markers CD9, CD81 and CD63 are greatly increased from the media sample *4 (Test 25-26) to the Final Formulation *9 (Test 27) by greater than 12x, 10x and 7x for CD9, CD63 and CD81, respectively.The associated MFI of the three canonical tetraspanin EV markers (CD9, CD63, and CD81) for *9 (Test 27) are within the top six highest MFI response of all of the proteins investigated in this assay. Using 13 μL of sample, CD9 had the 6th highest MFI (27.0), CD63 had the third highest MFI (132.1), and CD81 had the second highest MFI (139.6) of the proteins investigated inthis assay. Taken together, the strong presence of the three tetraspanin markers indicate the presence of extracellular vesicles. Using 13 μL of sample, other markers with high MFI are CD326 (102.3), CD133 / 1 (333.4), and CD29 (47.7). Using 13 μL of sample, markers with MFI greater than 1.5 (= expressed markers on *9(Test 27)) are shown in FIG. 96 and FIG. 97. Markers with MFI less than 1.5 as measured in the Final Formulation (*9 (Test 27)) (= little or no expression markers on *9 (Test 27)) are in FIG. 98.FIG 99 depicts the results of fragments size obtained for *9 (Test 27). The peaks have a size of 179, 368, 537 and 742 base pairs.FIG. 100 depicts the results of the HUVEC scratch wound healing assay described in Example 37.1 (lines referred to as A through I from top to bottom based on their position at the 24-hour timepoint). The Complete Media positive control (labeled “Complete (dotted line)” in figure, line A), attained 89.33 % wound confluence after 24 hours. The Poor Media negative control (labeled “Poor (dashed line)” in the figure, line I), attained 13.22% wound confluence after 24 hours. The Control EV sample, which is the pelleted material obtained after ultracentfiguation of FBS (labeled “Control EV (dash-dot line)” in the figure, line D), obtained 41.29% wound confluence after 24 hours. *9 (Test 27) was tested in this assay at 0.25x (line G), 0.5x (line H), 0.75x (line F), 1x (line E), 2x (line C), and 2.8x (line B), where 1x is 5.79 μL of material and is the secretome collected from 150,000 cells. The results are shown as colored circles, connected by solid lines. The 24 hour timepoint results depicted in FIG. 100 are given in TABLE 51.FIG. 101 depicts the results of the HUVEC scratch wound healing assay at the 18-hour timepoint described in Example 37.1 (bars referred to as one through nine from left to right). The assay included a Complete Media positive control (labeled “+ve” in figure, bar one), a Poor Media negative control (labeled “-ve” in figure, bar two). The Control EV sample (the pelleted material obtained after FBS ultracentrifugation, labeled “EV Ctl” in figure, bar three), obtained 31% normalized wound confluence after 18 hours. *9 (Test 27), bars four through nine) was tested in this assay at 0.25x (bar four), 0.5x (bar five), 0.75x (bar six), 1x (bar seven), 2x (bar eight), and 2.8x (bar nine), where 1x is 5.79 μL of material and is the secretome prepared from 150,000 cells.FIG. 102 depicts the results of the in vitro analysis of the potency of *9 (Test 27) whose preparation is described in detail in Example 19 in a cardiomyocyte survival assay described in Example 37.2 (lines labeled A through F from top to bottom based on position at 24-hour elapsedtimepoint). Percent of positive NucLight Red cells normalized to To is shown at the indicated timepoint. The 24 hour data points as depicted in FIG. 102 are given in TABLE 52.FIG. 103 depicts the results of the in vitro analysis of the potency of *9 (Test 27) in a cardiomyocyte survival assay described in Example 37.2 at the 24-hour timepoint (bars referred to as one through six from left to right). The results were baseline (negative control) subtracted and normalized to the positive control.FIG. 104 depicts the results of the in vitro analysis of the potency of *9 (Test 27) in a Scratch Wound Healing Assay described in Example 38 (bars referred to as one through fourteen from left to right). “Complete medium” positive control (bar one) and the “FBS-EV” control (bar three) were used as positive controls. The negative control was the “Poor medium” control (bar two). The Complete medium (bar one) control consists of HUVEC cultured in Complete Medium [Endothelial Cell Basal Media (PromoCell; ref: C-22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, ref: C-39210)]. The Poor medium control (bar two) consists of HUVEC cultured in Poor medium alone [Endothelial Cell Basal Media (PromoCell; ref: C-22210]. The “FBS-EV” control (bar three) consists of HUVEC cultured in Poor medium supplemented with 5 x 109particles of FBS-EV (FBS-EV is the EV-enriched secretome produced by ultracentrifugation of fetal bovine serum). Four separate vials of *9 (Test 27) were tested (bars 4 through 14) at doses ranging from 1 x 109to 5 x 109particles per well, as measured by NT A. The particle dose per well is noted under the corresponding data bar.FIG. 105 illustrates the process used to generate Test Example 25 as described in Example 19.FIG. 106 depicts the results of the HUVEC Scratch Wound Healing Assay described in Example 41 (bars referred to as one through twelve from left to right). The positive control (labeled “+ve” in the figure, bar one) which is a HUVEC scratch wound healing assay performed in the presence of complete assay media (labeled “Complete” in figure) and treated with vehicle control, which is 0.1 μm filtered PBS (labeled “PBS” in figure). The negative control (labeled “-ve” in the figure, bar two) which is a HUVEC scratch wound healing assay performed in Poor Assay Media (labeled “Poor” in figure) and treated with vehicle control, which is 0.1 μm filtered PBS (labeled “PBS” in figure). The EV control (labeled “EV Ctl” in figure, bar three) is the pellet collected after FBS ultracentrifugation. The results of the HUVEC scratch wound healing assay for 1x (bar four), 2x (bar five), and 3x (bar six) doses of *5a.uc (Test 25), whose preparation is described in detailin Example 19 are shown. The results of the HUVEC scratch wound healing assay for 1x (bar seven), 2x (bar eight), and 3x (bar nine) doses of Test Example 26 CTC1-EV (sample *5b.uc), whose preparation is described in detail in Example 19 are shown.The results of the HUVEC scratch wound healing assay for 1x (bar ten), 2x (bar eleven), and 3x (bar twelve) doses of a mock- EV control (labeled “MV Control” in figure) are shown. The results of this assay are double normalized such that the “-ve” control (bar two) is at 0% wound confluence, and the “+ve “control (bar one) is at 100% wound confluence at the 18 hour timepoint.FIG. 107 and 108 depicts the results of the stability testing (described in Example 43) for *9 (Test 27) byNTA.FIG. 109 depicts the results of the HUVEC Survival Assay at various timepoints as described in Example 44. Sample is *9 (Test 27), (labeled “Poor media with staurospoin (0.01 μM) + EV [Test 27 (*9)]” in figure, results shown in third bar of each cluster of three bars). Data are shown as fold change over the negative control (labeled “Poor media with staurosporine (0.01 μM)” in figure, second bar in each cluster of three bars). Positive control results also shown as foldchange over the negative control (labeled “Poor media without staurosporin” in figure, results shown in first bar of each cluster of three bars).FIG. 110 depicts the results of the H9c2 cell viability assay described in Example 45. The results of the technical replicates were averaged and normalized to the Virgin Media 100 kDa test condition.FIG. 111 illustrates the process used to generate Test Example 26 as described in Example 19.FIG. 112 illustrates the process used to generate Test Example 27 as described in Example 19.FIG. 113 illustrates the correlation found between particle number (in 106particles) as measured by NT A, and CD9 MFI as measured by MACSPlex Exosome kit®.FIG. 114 illustrates the linearity and minimum linear range of CD9 MFI (which is the Mean Fluorescence Intensity determined by flow cytometry using the MASCPlex Exosome kit human (Miltenyi Ref: 130-108-813) probing for CD9) versus the input volume of *9 (Test 27) for inputs ranging from 1 and 120 μL.FIG. 115 shows a representative cluster from *9 (Test 27) visualized by ONi that is CD81 / CD63 / CD9 TP. The CD9 signal, CD81 signal and CD63 signal are shown individually andas an overlay, confirming the presence of each of the three markers in a single cluster. FIG. 116 indicates the relative abundance of each cluster sub-type in the *9 (Test 27) as detected by ONi super-resolution microscopy (shown as a % under each bar). The absolute counts are graphed in the figure and given above each bar.FIG. 117 depicts the results of a GO (Gene Ontology) enrichment analysis in terms of biological Process. Analysed using String Prot.FIG. 118 depicts some of the molecular components identified in CTC1-EV. Image created with BioRcndcr.com.FIG. 119 depicts some of the protein components identified in CTC1-EV and the biological processes in which those components are implicated. The varied processes depicted in the figure highlight the potential for CTC1-EV to have multiple beneficial effects on multiple cell types, culminating in improved physiological outcomes.FIG. 120 summarizes some of the important biological effects of CTC1-EV treatment as described in this specification. Importantly, mouse, rat, human cell, and human patient data agree that CTC1-EV can impart molecular, cellular and physiological effects on target cells and tissues which are beneficial to stressed human cells and mammals with impaired ventricular function, including humans with heart failure.FIG. 121 Summary illustration of the clinical trial design.DETAILED DESCRIPTION OF THE INVENTIONIt is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the present specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes one or more cells.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other methods and materials similar, or equivalent, to those described herein can be useful in the present invention, preferred materials and methods are described herein.As used herein, “subject,” “individual,” or “patient” are used interchangeably herein and refer to any member of the phylum Chordata, including, without limitation, humans and otherprimates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like. The term does not denote a particular age or gender. Thus, the term includes adult, young, and newborn individuals as well as males and females. In some embodiments, cells (for example, stem cells, including pluripotent stem cells, progenitor cells, or tissue-specific cells) arc derived from a subject. In some embodiments, the subject is a non-human subject.As used herein, “differentiation” refers to processes by which unspecialized cells (such as pluripotent stem cells, or other stem cells), or multipotent or oligopotent cells, for example, acquire specialized structural and / or functional features characteristic of more mature, or fully mature, cells. “Transdifferentiation” is a process of transforming one differentiated cell type into another differentiated cell type or to a certain fate.As used herein, “embryoid bodies” refers to three-dimensional aggregates of pluripotent stem cells. These cells can undergo differentiation into cells of the three germ layers, the endoderm, mesoderm and ectoderm. The three-dimensional structure, including the establishment of complex cell adhesions and paracrine signaling within the embryoid body microenvironment, enables differentiation and morphogenesis.As used herein, “stem cell” refers to a cell that has the capacity for self-renewal, i.e., the ability to go through numerous cycles of cell division while maintaining their non-terminally- differentiated state. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells may be, for example, embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.As used herein, “pluripotent stem cell” (PSC) refers to a cell that has the ability to reproduce itself indefinitely, and to differentiate into any other cell type of an adult organism. Generally, pluripotent stem cells are stem cells that are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; are capable of differentiating into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal, cell types); and express one or more markers characteristic of PSCs. Examples of such markers expressed byPSCs, such as embryonic stem cells (ESCs) and iPSCs, include Oct 4, alkaline phosphatase, SSEA- 3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and REX1.As used herein, “induced pluripotent stem cell” (iPSC) refers to a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell, typically a somatic cell. In some embodiments, the somatic cell is a human somatic cell. Examples of somatic cells include, but are not limited to, dermal fibroblasts, bone marrow-derived mesenchymal cells, cardiac muscle cells, keratinocytes, liver cells, stomach cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Additional examples of somatic cells include cells of the immune system, including, but not limited to, B-cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes, and hematopoietic stem cells. iPSCs may be generated by reprogramming a somatic cell, by expressing or inducing expression of one or a combination of factors (herein referred to as reprogramming factors) in the somatic cell. iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In some instances, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, OCT4 (OCT3 / 4), SOX2, c-MYC, and KLF4, NANOG, and LIN28. In some instances, somatic cells may be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors, to reprogram a somatic cell to a pluripotent stem cell. The cells may be reprogrammed by introducing reprogramming factors using vectors, including, for example, lentivirus, retrovirus, adenovirus, and Sendai virus vectors. Alternatively, non-viral techniques for introducing reprogramming factors include, for example, mRNA transfection, miRNA infection / transfection, PiggyBac, minicircle vectors, and episomal plasmids. iPSCs may also be generated by, for example, using CRISPR-Cas9-based techniques, to introduce reprogramming factors, or to activate endogenous programming genes.As used herein, “embryonic stem cells” are embryonic cells derived from embryo tissue, preferably the inner cell mass of blastocysts or morulae, optionally that have been serially passaged as cell lines. The term includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo. The term also includes cells produced by somatic cell nuclear transfer. ESCs can be produced or derived from a zygote, blastomere, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nucleartransfer, or parthenogenesis, for example. Human ESCs include, without limitation, MA01, MA09, ACT-4, No. 3, Hl, H7, H9, H14 and ACT30 embryonic stem cells. Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of blastocyst stage embryos, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo. These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone cannot develop into a fetal or adult animal when transplanted in utcro because they lack the potential to contribute to all extraembryonic tissue (e.g., placenta in vivo or trophoblast in vitro).As used herein, the term “progenitor cell” refers to a descendant of a stem cell which is capable of further differentiation into one or more kinds of specialized cells, but which cannot divide and reproduce indefinitely. That is, unlike stem cells (which possess an unlimited capacity for self-renewal), progenitor cells possess only a limited capacity for self-renewal. Progenitor cells may be multipotent, oligopotent, or unipotent, and are typically classified according to the types of specialized cells they can differentiate into. For instance, a “cardiomyocyte progenitor cell” is a progenitor cell derived from a stem cell that has the capacity to differentiate into a cardiomyocyte. Similarly, “cardiac progenitor cells" may differentiate into multiple specialized cells constituting cardiac tissue, including, for example, cardiomyocytes, smooth muscle cells, and endothelial cells. Additionally, a “cardiovascular progenitor cell" has the capacity to differentiate into, for example, cells of cardiac and vascular lineages.As used herein, “expand” or “proliferate" may refer to a process by which the number of cells in a cell culture is increased due to cell division.“Multipotent” implies that a cell is capable, through its progeny, of giving rise to several different cell types found in an adult animal.“Pluripotent” implies that a cell is capable, through its progeny, of giving rise to all the cell types that comprise the adult animal, including the germ cells. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.The term “autologous cells" as used herein refers to donor cells that are genetically identical with the recipient.As used herein, the term “allogeneic cells” refers to cells derived from a different, genetically non-identical, individual of the same species.The term “totipotent” as used herein can refer to a cell that gives rise to a live bom animal. The term “totipotent” can also refer to a cell that gives rise to all of the cells in a particular animal. A totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps.As used herein, the term “extracellular vesicles” collectively refers to biological nanoparticles derived from cells, and examples thereof include exosomes, ectosomes, exovesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, oncosomes, exomeres, and apoptotic bodies, but are not limited thereto.Extracellular vesicles can be categorized, for example, according to size. For instance, as used herein, the term “small extracellular vesicle” refers to extracellular vesicles having a diameter of between about 50-200 nm. In contrast, extracellular vesicles having a diameter of more than about 200 nm, but less than 400 nm, may be referred to as “medium extracellular vesicles,” and extracellular vesicles having a diameter of more than about 400 nm may be referred to as “large extracellular vesicles.” As used herein, the term “small extracellular vesicle fraction” (“sEV”) refers to a part, extract, or fraction, of secretome or conditioned medium, that is concentrated and / or enriched for small extracellular vesicles having a diameter of between about 50-200 nm. Such concentration and / or enrichment may be obtained using one or more of the purification, isolation, concentration, and / or enrichment, techniques disclosed herein. In some alternative embodiments herein, enrichment may not be performed, may not be achieved, or may not be possible.The term “exosome” as used herein refers to an extracellular vesicle that is released from a cell upon fusion of the multivesicular body (MVB) (an intermediate endocytic compartment) with the plasma membrane.“Exosome-like vesicles,” which have a common origin with exosomes, are typically described as having size and sedimentation properties that distinguish them from exosomes and, particularly, as lacking lipid raft microdomains. “Ectosomes,” as used herein, are typically neutrophil- or monocyte-derived microvesicles.“Microparticles” as used herein are typically about 50-1000 nm in diameter and originate from the plasma membrane. “Extracellular membranous structures” also include linear or foldedmembrane fragments, e.g., from necrotic death, as well as membranous structures from other cellular sources, including secreted lysosomes and nanotubes.As used herein, “apoptotic blebs or bodies” are typically about 1 to 5 μm in diameter and are released as blebs of cells undergoing apoptosis, i.e., diseased, unwanted and / or aberrant cells.Within the class of extracellular vesicles, important components are “exosomes” themselves, which may be between about 40 to 50 run and about 200 nm in diameter and being membranous vesicles, i.e., vesicles surrounded by a phospholipid bilayer, of endocytic origin, which result from cxocytic fusion, or “cxocytosis” of multivcsicular bodies (MVBs). In some cases, exosomes can be between about 40 to 50 nm up to about 200 nm in diameter, such as being from 60 nm to 180 nm.As used herein, the terms “secretome” and “secretome composition” interchangeably refer to one or more molecules and / or biological factors that are secreted by cells into the extracellular space (such as into a culture medium). A secretome or secretome composition may include, without limitation, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space (such as into a culture medium). A secretome or secretome composition may be left unpurified or further processed (for example, components of a secretome or secretome composition may be present within culture medium, such as in a conditioned medium; or alternatively, components of a secretome or secretome composition may be purified, isolated, and / or enriched, from a culture medium or extract, part, or fraction thereof). A secretome or secretome composition may further comprise one or more substances that are not secreted from a cell, for example, media elements (e.g., culture media, additives, nutrients, etc.). Alternatively, a secretome or secretome composition may not comprise media elements (e.g., culture media, additives, nutrients, etc.).As used herein, the term “conditioned medium” refers to a culture medium (or extract, part, or fraction thereof) in which one or more cells of interest have been cultured. Preferably, conditioned medium is separated from the cultured cells before use and / or further processing. The culturing of cells in culture medium may result in the secretion and / or accumulation of one or more molecules and / or biological factors (which may include, without limitation, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space); the medium containing the one or more molecules and / or biological factors is a conditioned medium. Examples of methods of preparing conditionedmedia have been described in, for example, U.S. Patent No. 6,372,494, which is incorporated by reference herein in its entirety.As used herein, the term “cell culture” refers to cells grown under controlled condition(s) outside the natural environment of the cells. For instance, cells can be propagated completely outside of their natural environment (in vitro) or can be removed from their natural environment and the cultured (ex vivo). During cell culture, cells may survive in a non-replicative state, or may replicate and grow in number, depending on, for example, the specific culture media, the culture conditions, and the type of cells. An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example.The term “cell line” as used herein can refer to cultured cells that can be passaged at least one time without terminating.The term “suspension” as used herein can refer to cell culture conditions in which cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using an apparatus well known to those skilled in the art.The term “monolayer” as used herein can refer to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of cells proliferating in a monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support.The term “plated” or “plating” as used herein in reference to cells can refer to establishing cell cultures in vitro. For example, cells can be diluted in cell culture media and then added to a cell culture plate, dish, or flask. Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and / or cell densities.The term “cell plating” can also extend to the term “cell passaging.” Cells can be passaged using cell culture techniques well known to those skilled in the art. The term “cell passaging” can refer to a technique that involves the steps of (1) releasing cells from a solid support or substrate and disassociation of these cells, and (2) diluting the cells in media suitable for further cell proliferation. Cell passaging may also refer to removing a portion of liquid medium containing cultured cells and adding liquid medium to the original culture vessel to dilute the cells and allow further cell proliferation. In addition, cells may also be added to a new culture vessel that has been supplemented with medium suitable for further cell proliferation.As used herein, the terms “culture medium,” “growth medium” or “medium” are used interchangeably and refer to a composition that is intended to support the growth and survival of organisms. While culture media is often in liquid form, other physical forms may be used, such as, for example, a solid, semi-solid, gel, suspension, and the like.As used herein, the term “serum-free,” in the context of a culture medium or growth medium, refers to a culture or growth medium in which serum is absent. Serum typically refers to the liquid component of clotted blood, after the clotting factors (e.g., fibrinogen and prothrombin) have been removed by clot formation. Scrum, such as fetal bovine scrum, is routinely used in the art as a component of cell culture media, as the various proteins and growth factors therein are particularly useful for the survival, growth, and division of cells.As used herein, the term “basal medium” refers to an unsupplemented synthetic medium that may contain buffers, one or more carbon sources, amino acids, and salts. Depending on the application, basal medium may be supplemented with growth factors and supplements, including, but not limited to, additional buffering agents, amino acids, antibiotics, proteins, and growth factors useful, for instance, for promoting growth, or maintaining or changing differentiation status, of particular cell types (e.g., fibroblast growth factor-basic (bFGF), also known as fibroblast growth factor 2 (FGF-2)).As used herein, the terms “wild-type,” “naturally occurring,” and “unmodified” are used herein to mean the typical (or most common) form, appearance, phenotype, or strain existing in nature; for example, the typical form of cells, organisms, polynucleotides, proteins, macromolecular complexes, genes, RNAs, DNAs, or genomes as they occur in, and can be isolated from, a source in nature. The wild-type form, appearance, phenotype, or strain serve as the original parent before an intentional modification. Thus, mutant, variant, engineered, recombinant, and modified forms are not wild-type forms.As used herein, the term “isolated” refers to material removed from its original environment, and is thus altered “by the hand of man” from its natural state.As used herein, the term “enriched” means to selectively concentrate or increase the amount of one or more components in a composition, with respect to one or more other components. For instance, enrichment may include reducing or decreasing the amount of (e.g., removing or eliminating) unwanted materials; and / or may include specifically selecting or isolating desirable materials from a composition.The terms “engineered,” “genetically engineered,” “genetically modified,” “recombinant,” ‘modified,” “non-naturally occurring,” and “non-native” indicate intentional human manipulation of the genome of an organism or cell. The terms encompass methods of genomic modification that include genomic editing, as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art.As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “oligonucleotide” all refer to polymeric forms of nucleotides. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides that, when in linear form, has one 5 ’ end and one 3’ end, and can comprise one or more nucleic acid sequences. The nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length. Polynucleotides may perform any function and may have various secondary and tertiary structures. The terms encompass known analogs of natural nucleotides and nucleotides that are modified in the base, sugar, and / or phosphate moieties. Analogs of a particular nucleotide have the same base-pairing specificity (e.g., an analog of A base pairs with T). A polynucleotide may comprise one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. Nucleotide structure may be modified before or after a polymer is assembled. Following polymerization, polynucleotides may be additionally modified via, for example, conjugation with a labeling component or target binding component. A nucleotide sequence may incorporate non-nucleotide components. The terms also encompass nucleic acids comprising modified backbone residues or linkages, that are synthetic, naturally occurring, and / or non- naturally occurring, and have similar binding properties as a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), Locked Nucleic Acid (LNA™) (Exiqon, Inc., Woburn, MA) nucleosides, glycol nucleic acid, bridged nucleic acids, and morpholino structures. Peptide-nucleic acids (PNAs) are synthetic homologs of nucleic acids wherein the polynucleotide phosphate-sugar backbone is replaced by a flexible pseudo-peptide polymer. Nucleobases are linked to the polymer. PNAs have the capacity to hybridize with high affinity and specificity tocomplementary sequences of RNA and DNA. Polynucleotide sequences are displayed herein in the conventional 5’ to 3’ orientation unless otherwise indicated.As used herein, “sequence identity” generally refers to the percent identity of nucleotide bases or amino acids comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms having various weighting parameters. Sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment by various methods and computer programs (e.g., Exonerate, BLAST, CS-BLAST, FASTA, HMMER, L- ALIGN, and the like) available through the worldwide web at sites including, but not limited to, GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk.). Sequence identity between two polynucleotides or two polypeptide sequences is generally calculated using the standard default parameters of the various methods or computer programs. A high degree of sequence identity between two polynucleotides or two polypeptides is often between about 90% identity and 100% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 90% identity or higher, about 91% identity or higher, about 92% identity or higher, about 93% identity or higher, about 94% identity or higher, about 95% identity or higher, about 96% identity or higher, about 97% identity or higher, about 98% identity or higher, or about 99% identity or higher, over the length of the reference polynucleotide or polypeptide or query sequence. Sequence identity can also be calculated for the overlapping region of two sequences where only a portion of the two sequences can be aligned.A moderate degree of sequence identity between two polynucleotides or two polypeptides is often between about 80% identity to about 90% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 80% identity or higher, about 81% identity or higher, about 82% identity or higher, about 83% identity or higher, about 84% identity or higher, about 85% identity or higher, about 86% identity or higher, about 87% identity or higher, about 88% identity or higher, or about 89% identity or higher, but less than 90%, over the length of the reference polynucleotide or polypeptide or query sequence.A low degree of sequence identity between two polynucleotides or two polypeptides is often between about 50% identity and 75% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 50% identity or higher, about 60% identity or higher, about 70% identity or higher, but less than 75% identity, over the length of the reference polynucleotide or polypeptide or query sequence.As used herein, “binding” refers to a non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between a polynucleotide and a polynucleotide, or between a protein and a protein, and the like). Such non-covalent interaction is also referred to as “associating” or “interacting” (e.g., if a first macromolecule interacts with a second macromolecule, the first macromolecule binds to second macromolecule in a non-covalent manner). Some portions of a binding interaction may be sequence-specific (the terms “sequence-specific binding,” “sequence-specifically bind,” “site-specific binding,” and “site specifically binds” are used interchangeably herein). Binding interactions can be characterized by a dissociation constant (Kd). “Binding affinity” refers to the strength of the binding interaction. An increased binding affinity is correlated with a lower Kd.“Gene” as used herein refers to a polynucleotide sequence comprising exons and related regulatory sequences. A gene may further comprise introns and / or untranslated regions (UTRs).As used herein, “expression” refers to transcription of a polynucleotide from a DNA template, resulting in, for example, a messenger RNA (mRNA) or other RNA transcript (e.g., noncoding, such as structural or scaffolding RNAs). The term further refers to the process through which transcribed mRNA is translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be referred to collectively as “gene products.” Expression may include splicing the mRNA in a eukaryotic cell, if the polynucleotide is derived from genomic DNA.A “coding sequence” or a sequence that “encodes” a selected polypeptide, is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ terminus and a translation stop codon at the 3’ terminus. A transcription termination sequence may be located 3’ to the coding sequence.As used herein, a “different” or “altered” level of, for example, a characteristic or property, is a difference that is measurably different, and preferably, statistically significant (for example, not attributable to the standard error of the assay). In some embodiments, a difference, e.g., as compared to a control or reference sample, may be, for example, a greater than 10% difference, a greater than 20% difference, a greater than 30% difference, a greater than 40% difference, a greater than 50% difference, a greater than 60% difference, a greater than 70% difference, a greater than 80% difference, a greater than 90% difference, a greater than 2-fold difference; a greater than 5-fold difference; a greater than 10-fold difference; a greater than 20-fold difference; a greater than 50-fold difference; a greater than 75 -fold difference; a greater than 100-fold difference; a greater than 250-fold difference; a greater than 500-fold difference; a greater than 750-fold difference; or a greater than 1,000-fold difference, for example.As used herein, the term “between” is inclusive of end values in a given range (e.g., between about 1 and about 50 nucleotides in length includes 1 nucleotide and 50 nucleotides).As used herein, the term “amino acid” refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, pcptidomimctics, glycine, and D or L optical isomers.As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to polymers of amino acids. A polypeptide may be of any length. It may be branched or linear, it may be interrupted by non-amino acids, and it may comprise modified amino acids. The terms also refer to an amino acid polymer that has been modified through, for example, acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and / or conjugation (e.g., with a labeling component or ligand). Polypeptide sequences are displayed herein in the conventional N-terminal to C-terminal orientation, unless otherwise indicated. Polypeptides and polynucleotides can be made using routine techniques in the field of molecular biology.A “moiety” as used herein refers to a portion of a molecule. A moiety can be a functional group or describe a portion of a molecule with multiple functional groups (e.g., that share common structural aspects). The terms “moiety” and “functional group” are typically used interchangeably; however, a “functional group” can more specifically refer to a portion of a molecule that comprises some common chemical behavior. “Moiety” is often used as a structural description.The terms “effective amount” or “therapeutically effective amount” of a composition or agent, such as a therapeutic composition as provided herein, refers to a sufficient amount of the composition or agent to provide the desired response. Such responses will depend on the particular disease in question and related conditions.“Transformation” as used herein refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion. For example, transformation can be by direct uptake, transfection, infection, and the like. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, or, alternatively, may be integrated into the host genome.As used herein, the term “hypoxia” or “hypoxic” refers to a condition where the oxygen (O2) concentration is below atmospheric O2concentration (typically 20-21%). In some embodiments, hypoxia refers to a condition with an O2concentration that is between 0% and 19%, between 2% and 18%, between 3% and 17%, between 4% and 16%, between 5% and 15%, between 5% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.As used herein, the term “normoxia” refers to a normal atmospheric concentration of oxygen, typically around 20% to 21% O2.Generation of Progenitor Cells from Stem CellsThe present disclosure relates, in part, to methods for generating a secretome containing extracellular vesicles (EVs) from progenitor cells. In certain embodiments herein, progenitor cells may be isolated from a subject or tissue and used in the methods of the present disclosure. In other embodiments, progenitor cells may be generated from pluripotent stem cells, such as from embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs).Generation of iPSC cells iPSC cells may be obtained from, for example, somatic cells, including human somatic cells. The somatic cell may be derived from a human or non-human animal, including, for example, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like.In some embodiments, the somatic cell is selected from keratinizing epithelial cells, mucosal epithelial cells, exocrine gland epithelial cells, endocrine cells, liver cells, epithelial cells, endothelial cells, fibroblasts, muscle cells, cells of the blood and the immune system, cells of the nervous system including nerve cells and glial cells, pigment cells, and progenitor cells, including hematopoietic stem cells. The somatic cell may be fully differentiated (specialized) or may be lessthan fully differentiated. For instance, undifferentiated progenitor cells that are not PSCs, including somatic stem cells, and finally differentiated mature cells, can be used. The somatic cell may be from an animal of any age, including adult and fetal cells.The somatic cell may be of mammalian origin. Allogeneic or autologous stem cells can be used, if for example, the secretome (or extracellular vesicles) from a progenitor cell thereof is used for administration in vivo. In some embodiments, iPSCs are not MHC- / HLA-matched to a subject. In some embodiments, iPSCs are MHC- / HLA-matched to a subject. In embodiments, for example, where iPSCs arc to be used to produce PSC-derived progenitor cells (to obtain a secretome, or extracellular vesicles, for therapeutic use in a subject), somatic cells may be obtained from the subject to be treated, or from another subject with the same or substantially the same HLA type as that of the subject. Somatic cells can be cultured before nuclear reprogramming, or can be reprogrammed without culturing after isolation, for example.To introduce reprogramming factors into somatic cells, for example, viral vectors may be used, including, e.g., vectors from viruses such as SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses, human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and retroviruses. Lentiviruses include, but are not limited to, Human Immunodeficiency V irus type 1 (HIV - 1 ), Human Immunodeficiency Virus type 2 (HIV-2), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), Equine Infectious Anaemia Virus (EIAV), Bovine Immunodeficiency Virus (BIV), Visna Virus of sheep (VISNA) and Caprine Arthritis-Encephalitis Virus (CAEV). Lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and in vitro gene transfer and expression of nucleic acid sequences. A viral vector can be targeted to a specific cell type by linkage of a viral protein, such as an envelope protein, to a binding agent, such as an antibody, or a particular ligand (for targeting to, for instance, a receptor or protein on or within a particular cell type).In some embodiments, a viral vector, such as a lentiviral vector, can integrate into the genome of the host cell. The genetic material thus transferred is then transcribed and possibly translated into proteins inside the host cell. In other embodiments, viral vectors are used that do not integrate into the genome of a host cell.A viral gene delivery system can be an RNA-based or DNA-based viral vector. An episomal gene delivery system can be a plasmid, an Epstein-Barr virus (EBV)-based episomalvector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentiviral vector, for example.Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art. One of skill in the art can readily produce induced pluripotent stem cells, see for example, Published U.S. Patent Application No. 2009 / 0246875, Published U.S. Patent Application No. 2010 / 0210014; Published U.S. Patent Application No. 2012 / 0276636; U.S. Pat. Nos. 8,058,065; 8,129,187; and U.S. Pat. No. 8,268,620, all of which are incorporated herein by reference.Generally, reprogramming factors which can be used to create induced pluripotent stem cells, either singly, in combination, or as fusions with transactivation domains, include, but are not limited to, one or more ofthe following genes: Oct4 (Oct3 / 4, Pou5fl), Sox (e.g., Soxl, Sox2, Sox3, Soxl8, or Soxl5), Klf (e.g., Klf4, Klf1, Klf3, Klf2 or Klf5), Myc (e.g., c-myc, N-myc or L-myc), nanog, or LIN28. As examples of sequences for these genes and proteins, the following accession numbers are provided: Mouse MyoD: M84918, NM 010866; Mouse Oct4 (POU5F1): NM 013633; Mouse Sox2: NM_011443; Mouse Klf4: NM_010637; Mouse c-Myc: NM 001177352, NM 001177353, NM 001177354 Mouse Nanog: NM 028016; Mouse Lin28: NM 145833: Human MyoD: NM 002478; Human Oct4 (POU5F1): NM 002701, NM 203289, NM 001173531; Human Sox2: NM 003106; Human Klf4: NM 004235; Human c-Myc: NM 002467; Human Nanog: NM 024865; and / or Human Lin28: NM 024674. Also contemplated are sequences similar thereto, including those having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, at least three, or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc and Klf4 are utilized.Exemplary reprogramming factors for the production of iPSCs include (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7 or Soxl8; Klf4 is replaceable with Klf1, Klf2 or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papilloma virus (HPV)16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, SV40LT; (9) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT; (10) Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb is replaceable with Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6; (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmi1; (18) Oct3 / 4, Klf4, Sox2, Lin28 (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb is replaceable with Esrrg). iPSCs typically display the characteristic morphology of human embryonic stem cells (hESCs), and express the pluripotency factor, NANOG. Embryonic stem cell specific surface antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81) may also be used to identify fully reprogrammed human cells. Additionally, at a functional level, PSCs, such as ESCs and iPSCs, also demonstrate the ability to differentiate into lineages from all three embryonic germ layers, and form teratomas in vivo (e.g., in SCID mice).Differentiating PSCs to Generate Progenitor CellsThe present disclosure further contemplates differentiating PSCs, including ESCs and iPSCs, into progenitor cells. Such progenitor cells can then be used to produce a secretome (and extracellular vesicles) of the present disclosure.Progenitor cells of the present disclosure include, for example, hematopoietic progenitor cells, myeloid progenitor cells, neural progenitor cells; pancreatic progenitor cells, cardiac progenitor cells, cardiomyocyte progenitor cells, cardiovascular progenitor cells, renal progenitor cells, skeletal myoblasts, satellite cells, intermediate progenitor cells formed in the subventricular zone, radial glial cells, bone marrow stromal cells, periosteum cells, endothelial progenitor cells, blast cells, boundary caop cells, and mesenchymal stem cells. Methods for differentiating pluripotent stem cells to progenitor cells, and for culturing and maintaining progenitor cells are described in for example, U.S. Patent Application No. 17 / 931,669, entitled “Methods for the Production of Committed Cardiac Progenitor Cells,” which is incorporated by reference herein in its entirety.Production of Secretome / Extracellular VesiclesThe present disclosure encompasses the culturing of progenitor cells for secretome / extracellular vesicle production under GMP-ready and / or GMP-compatible conditions, to produce, e.g., GMP-ready and / or GMP-compatible products. The present disclosure also encompasses the culturing of progenitor cells for secretome / extracellular vesicle production under non-GMP-ready and / or non-GMP-compatible conditions, to produce, e.g., non-GMP-ready and / or non-GMP-compatible products.In methods for generating secretomes or extracellular vesicles of the present disclosure, progenitor cells arc typically subjected to two or more culturing steps in a scrum-frcc culture medium.In a first culturing step, one or more progenitor cells are cultured in a first serum-free culture medium that comprises basal medium, human serum albumin, and one or more growth factors. This first serum-free culture medium is then replaced with a second serum-free culture medium that comprises basal medium but does not comprise human serum albumin or the one or more growth factors. In a second culturing step, the one or more progenitor cells are then cultured in the second serum-free culture medium. Following the second culturing step, the second serum- free culture medium is recovered, to thereby obtain conditioned medium containing the secretome of the one or more progenitor cells.The one or more progenitor cells can be, for example, progenitor cells that have recently been isolated or differentiated (e.g., from stem cells). Alternatively, in some embodiments, progenitor cells that have previously been refrigerated, frozen, and / or cryopreserved, may be used in the culturing methods of the present disclosure. In some embodiments, progenitor cells are thawed from a cryopreserved state (e.g., -80°C or colder) before use. In some embodiments thereof, the cells are thawed in a thawing medium. In some embodiments, the thawing medium may comprise a liquid medium (e.g., alpha-MEM, STEMdiff™ Cardiomyocyte Support Medium (StemCell, Ref: 05027)) containing one or more supplements. In some embodiments, the supplement in the thawing medium may be one or more of a carbon source (e.g., glucose), an albumin, B-27, insulin, FGF-2, FGF, and an antibiotic (e.g., gentamicin). In some embodiments, the cells may be thawed in a thawing device, such as, for example, a water bath or a water-free thawing system (e.g., ThawSTAR™ Automated Thawing System, Biolife Solutions®). Cells may be thawed, for example, within a tube or bottle (e.g., plastic, glass), or bag (e.g., an Ethyl VinylAcetate (EVA) bag), such as a 500-1000 mL volume bag (e.g., Coming, Refs: 91-200-41, 91-200- 42).The one or more growth factors may be selected based on the type of progenitor cell, for example. In some embodiments, the one or more growth factors may be selected from Adrenomedullin, Angiopoietin, Autocrine motility factor, Bone morphogenetic proteins (BMPs), Ciliary neurotrophic factor (CNTF), Leukemia inhibitory factor (LIF), Macrophage colonystimulating factor (M-CSF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Epidermal growth factor (EGF), Ephrin A1, Ephrin A2, Ephrin A3, Ephrin A4, Ephrin A5, Ephrin Bl, Ephrin B2, Ephrin B3, Erythropoietin (EPO), Fibroblast growth factor 1 (FGF-1), Fibroblast growth factor 2 (FGF-2), Fibroblast growth factor 3 (FGF-3), Fibroblast growth factor 4 (FGF-4), Fibroblast growth factor 5 (FGF-5), Fibroblast growth factor 6 (FGF-6), Fibroblast growth factor 7 (FGF-7), Fibroblast growth factor 8 (FGF-8), Fibroblast growth factor 9 (FGF-9), Fibroblast growth factor 10 (FGF-10), Fibroblast growth factor 11 (FGF-11), Fibroblast growth factor 12 (FGF-12), Fibroblast growth factor 13 (FGF-13), Fibroblast growth factor 14 (FGF-14), Fibroblast growth factor 15 (FGF-15), Fibroblast growth factor 16 (FGF-16), Fibroblast growth factor 17 (FGF-17), Fibroblast growth factor 18 (FGF-18), Fibroblast growth factor 19 (FGF-19), Fibroblast growth factor 20 (FGF-20), Fibroblast growth factor 21 (FGF-21), Fibroblast growth factor 22 (FG-F22), Fibroblast growth factor 23 (FGF-23), Foetal Bovine Somatotrophin (FBS), Glial cell line-derived neurotrophic factor (GDNF), Neurturin, Persephin, Artemin, Growth differentiation factor-9 (GDF-9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin, Insulin-like growth factor-1 (IGF-1), Insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, Keratinocyte growth factor (KGF), Migration-stimulating factor (MSF), Macrophage-stimulating protein (MSP), Myostatin (GDF-8), Neuregulin 1 (NRG1), Neuregulin 2 (NRG2), Neuregulin 3 (NRG3), Neuregulin 4 (NRG4), Brain-derived neurotrophic factor (BDNF), Nerve growth factor (NGF), Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4), Placental growth factor (PGF), Platelet- derived growth factor (PDGF), Renalase (RNLS), T-cell growth factor (TCGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-α), Transforming growth factor beta (TGF-β), Tumor necrosis factor-alpha (TNF-α), and Vascular endothelial growth factor (VEGF).The amount of growth factor may be adjusted depending on the desired culture conditions and / or need. In some embodiments, the one or more growth factors may each independently bepresent in an amount from 0.001 μg / mL - 1000 μg / mL, in an amount from 0.01 μg / mL - 100 μg / mL, in an amount from 0.1 μg / mL - 10 μg / mL, in an amount from 0.05 μg / mL - 5 μg / mL, in an amount from 0.5 μg / mL - 2.5 μg / mL, or in an amount of about 0.5 μg / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL or about 5 μg / mL.In some embodiments, the one or more growth factors comprise FGF-2. In some embodiments, the one or more growth factors consist of FGF-2.The basal medium may be any basal culture medium suitable for the cell type to be cultured, including, for example, Dulbccco’s Modified Eagle’s Medium (DMEM), DMEM F12 medium, Eagle’s Minimum Essential Medium (MEM), α-MEM, F-12K medium, Iscove’s Modified Dulbecco’s Medium, Knockout DMEM, or RPMI-1640 medium, or variants, combinations, or modifications thereof.Additional supplements can also be added to the basal medium to supply the cells with trace elements for optimal growth and expansion. Such supplements include, for example, insulin, transferrin, sodium selenium, Hanks’ Balanced Salt Solution, Earle’s Salt Solution, antioxidant supplements, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'- ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, as well as additional amino acids, and combinations thereof. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L- valine.Optionally, hormones can also be used in cell culture and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, beta-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine. Beta-mercaptoethanol can also be supplemented in cell culture media.Lipids and lipid carriers can also be used to supplement cell culture media, depending on the type of cell. Such lipids and carriers can include, but are not limited to, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, among others.In certain embodiments, an albumin, such as human serum albumin, is present in the first serum-free culture medium. The albumin, including human serum albumin, may be, for example, isolated, synthetic, recombinant, and / or modified. The amount of albumin may be adjusted depending on the desired culture conditions and / or need. In some embodiments, the albumin may be present in an amount from 0.1 μg / mL - 50 mg / mL, in an amount from 1 μg / mL - 25 mg / mL, in an amount from 10 μg / mL - 20 mg / mL, in an amount from 100 μg / mL - 10 mg / mL, in an amount from 0.5 mg / mL - 5 mg / mL, in an amount from 1 mg / mL - 3 mg / mL, or in an amount of about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL.In some embodiments, the serum-free media further comprises one or more selected from the group consisting of: glutamine; biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine, glutathione; L-camitine; linoleic acid; progesterone; putrescine; sodium selenite; triodo-I-thyronine; an amino acid; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.The basal medium may also be supplemented with one or more carbon sources. The one or more carbon sources may be selected from, for example, carbon sources such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose. In some embodiments, a carbon source, such as glucose, may be present in an amount of at least 0.01 g / mL, 0.05 g / mL, 0.1 g / mL, 0.5 g / mL, 1 g / mL, 1.5 g / mL, 2 g / mL, 2.5 g / mL, 3 g / mL, 4 g / mL, or 5 g / mL.A rock inhibitor may also be included in a culture medium, such as, for example, the rock inhibitor H1152.The first and second culturing steps may be performed for differing lengths of time. For instance, the first and second culturing steps may each independently be performed for a period of 6-96 hours, 12-72 hours, 36-60 hours, 42-56 hours, or for about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours.In some embodiments, the first culturing step is performed for a period of 42-56 hours, such as about 48 hours. In some embodiments, the second culturing step is performed for a period of 42-56 hours, such as about 48 hours.In some embodiments, the first culturing step is performed for a period of 42-96 hours, such as about 72 hours. In some embodiments, the second culturing step is performed for a period of 42-56 hours, such as about 48 hours.In some embodiments, all or a part of the first and / or second culturing step is performed under hypoxic conditions. In some embodiments, all or a part of the second culturing step is performed under hypoxic conditions. In some embodiments, the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours, of the second culturing step is performed under hypoxic conditions. In some embodiments, the hypoxic condition is an O2concentration that is between 0% and 15%, between 0% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.In some embodiments, all or a part of the first and / or second culturing step is performed under normoxic conditions. In some embodiments, all or a part of the second culturing step is performed under normoxic conditions. In some embodiments, at least the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours, of the second culturing step is performed under normoxic conditions. In some embodiments, the normoxic condition is an O2concentration that is between 20% and 21%.In some embodiments, all or a part of the first and / or second culturing step is performed in the presence of insulin. In some embodiments, all or a part of the first culturing step is performed in the presence of insulin. In some embodiments, the first culturing step comprises culturing in the presence of insulin for at least 24 hours, at least 48 hours, or at least 72 hours. In some embodiments, all or a part of the second culturing step is performed in the presence of insulin. In some embodiments, the second culturing step comprises culturing in the presence of insulin for at least 24 hours, at least 48 hours, or at least 72 hours.In some embodiments, the one or more progenitor cells are washed, using one or more washing steps, between the first and second culturing steps. In some embodiments, the washing medium may comprise a liquid medium (e.g., alpha-MEM, DMEM) optionally containing one or more supplements. In some embodiments, the supplement is a carbon source (e.g., glucose). In some embodiments, the one or more progenitor cells are not washed between the first and second culturing steps (for instance, the first culture medium is removed and the second culture medium is then added).The first and / or second culturing steps can be performed in suspension or attached to a solid support. The culturing may be two-dimensional or three-dimensional cell culturing.For instance, in some embodiments, the culture vessel used for culturing may be a flask, flask fortissue culture (e.g., T25, T75), hyperflask (e.g., CellBind surface HYPERFlask®; Coming, Ref: 10024) or hyperstack (e.g., 12 or 36 chamber, HYPERStacks®, Coming, Refs: 10012, 10036, 10013, 10037), dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers (e.g., 1ST, 2ST, 5ST, 10ST; Coming, Refs: 3268, 3269, 3313, 3319), culture bag, roller bottle, bioreactor, stirred culture vessel, spinner flask, microcarrier, or a vertical wheel bioreactor, for example. The one or more progenitor cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30, 40, 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, 550 mL, 600 mL, 800 mL, 1000 mL, 1500 mL, 1 L, 5L, 10L, 50 L, 100 L, 1000 L, 5000 L, or 10,000 L, for example.In embodiments in which culturing comprises two-dimensional cell culture, such as on the surface of a culture vessel, the culture surface (to which the cells are intended to adhere) may be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, type I, type II, and type IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly- D and poly-L-lysine, Matrigel, thrombospondin, osteopontin, poly-D-lysine, human extracellular matrix, Coming® Cell-Tak™ Cell and Tissue Adhesive, Coming PuraMatrix® Peptide Hydrogel, and / or vitronectin.In some embodiments, where culturing of cells is performed as adherent culture, e.g., where cells are adhered to a solid support, cells may be seeded at an amount of 25,000-250,000 cells per cm2; 50,000-200,000 cells per cm2; 75,000-175,000 cells per cm2; or between 100,000- 150,000 cells per cm2.In some embodiments, where culturing of cells is performed as adherent culture, e.g., where cells are adhered to a solid support, cells may be seeded to the solid support under gravitational force. In other embodiments, the cells may be seeded to the solid support under centrifugation.In some embodiments, following the second culturing step, the second serum-free culture medium used in the second culturing step is recovered to obtain a conditioned medium containing the secretome of the one or more progenitor cells.The recovered, conditioned medium may in some embodiments be subjected to one or more further processing steps. Following the second culturing step, the second serum-free culture medium used in the second culturing step may be removed, analyzed, recovered, concentrated, enriched, isolated, purified, refrigerated, frozen, cryopreserved, lyophilized, sterilized, etc.In some embodiments, the recovered, conditioned medium may be pre-cleared or clarified to remove particulates of greater than a certain size. For instance, the recovered, conditioned medium maybe pre-cleared or clarified by one or more centrifugation and / or filtration techniques. In some embodiments, in-line filters may be used, gradually stepping down the pore size to minimize clogging and loss of material. In some embodiments, such as where TFF is performed, pore sizes as low as 0.2 μm may be used to avoid clogging / high pressures at the TFF stage.In some embodiments, the recovered, conditioned medium is further processed to obtain a particular extract or fraction of the recovered, conditioned medium. For instance, the recovered, conditioned medium may be further processed to separate a small extracellular vesicle-enriched fraction (sEV) therefrom. An sEV fraction may be separated from the recovered, conditioned medium (or from a previously processed extract or fraction thereof) by one or more techniques such as centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, sonication, densitygradient ultracentrifugation, tangential flow filtration, size-exclusion chromatography, ionexchange chromatography, affinity capture, polymer-based precipitation, or organic solvent precipitation, for example.In some embodiments, conditioned medium is subjected to clarification by one or more filtration steps. In some embodiments thereof, one or more of the filtration steps utilizes a filter membrane having a particular pore size. In some embodiments thereof, a filter is used having a pore size of between 0.1 μm and 500 μm, or between 0.2 μm and 200 μm; or having a pore size less than or equal to 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 40 μm, 30 μm, 20 μm, 15 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm or 0.1 μm.In some embodiments, the clarification comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7, filtration steps. In some embodiments, the clarification comprises 4 filtration steps. In some embodiments, successive filtration steps utilize filters having increasingly smaller pores.In some embodiments thereof, a first filtration step comprises use of an approximately 200 μm filter (e.g., a 200 μm drip chamber filter; Gravity Blood set, BD careFusion, Ref: VH-22-EGA); a second filtration step comprises use of an approximately 15 μm filter (e.g., DIDACTIC, Ref: PER1FL25); a third filtration step comprises use of an approximately 0.2 μm filter, optionally containing a pre-filter, for example, an approximately 1.2 μm pre-filter (e.g., Sartoguard PES XLG MidiCaps, pore sizes: 1.2 μm + 0.2 μm, Sartorius, Ref: 5475307F7— OO-A); and a fourth filtration step comprises use of an approximately 0.22 μm filter (e.g., Vacuum Filter / Storage Bottle System, 0.22 μm pore, 33.2cm2PES Membrane, Coming, Ref: 431097), as illustrated in Example 5 and in FIG. 13.In other embodiments thereof, a first filtration step comprises use of an approximately 5 μm filter (e.g., Sartopure PP3 MidiCaps, pore size: 5 μm, Sartorius, Ref: 5055342P9-OO-A); a second filtration step comprises use of an approximately 0.2 μm filter, optionally containing a prefilter, for example, an approximately 1.2 μm pre-filter (e.g., Sartoguard PES MidiCaps, pore sizes: 1.2 μm + 0.2 μm, Sartorius, Ref: 5475307F9— OO— A, and a third filtration step comprises use of an approximately 0.2 μm filter, optionally containing a pre-filter, for example, an approximately 0.45 μm pre-filter (e.g., Sartopure 2 MidiCaps, pore sizes: 0.45 μm + 0.2 μm, Sartorius, Ref: 5445307H8— OO— A), as illustrated in Example 12 and in FIG. 29.In some embodiments, conditioned medium may be subjected to clarification by one or more centrifugation steps. In some embodiments, conditioned medium may be subjected to clarification by a combination of centrifugation and filtration step(s).In some embodiments, one or more additives are added to the conditioned medium, such as before clarification, and / or after clarification. In some embodiments, an additive is added that reduces aggregation. In some embodiments thereof, the additive is one or more selected from trehalose, histidine (e.g., L-histidine), arginine (e.g., L-arginine), citrate-dextrose solution, a Dnase (e.g., Dnase I), ferric citrate, or Anti-Clumping Agent (Gibco / Life technologies, Ref: 01-0057; Lonza, Ref: BE02-058E).In some embodiments, conditioned medium or sEV may be subjected to isolation, enrichment, and / or concentration step(s) using tangential flow filtration (TFF). In some embodiments, the conditioned medium or sEV is subjected to TFF after clarification that employed one or more clarification steps (e.g., such as after one or more filtration and / or centrifugation steps). TFF is a rapid and efficient method for separating, enriching and purifying biomolecules. In some embodiments, TFF can be used, e.g., for concentrating (e.g., concentrating small extracellular vesicles from conditioned media); for diafiltration; and for concentrating and diafiltration. Diafiltration is a type of ultrafiltration process in which the retentate (the fraction that docs not pass through the membrane) is diluted with buffer and re-ultrafiltered, to reduce the concentration of soluble permeate components and increase further the concentration of retained components.In some embodiments, TFF is used for enriching, concentrating and diafiltration of conditioned medium or sEV (e.g., for concentration and diafiltration of EV secretome). In some embodiments, TFF is first used to concentrate conditioned medium or sEV, and is subsequently used for diafiltration. In some embodiments, a TFF process may comprise a further step of concentrating after diafiltration. In some embodiments, TFF is used for diafiltration but not concentrating. In some embodiments, TFF is used for concentrating but not diafiltration.In some embodiments, the TFF membrane has a cut-off value of or less than 10 kDa, of or less than 20 kDa, of or less than 30 kDa, of or less than 40 kDa, of or less than 50 kDa, of or less than 60 kDa, of or less than 70 kDa, of or less than 80 kDa, of or less than 90 kDa, of or less than 100 kDa, or of or less than 150 kDa. In some embodiments, the TFF membrane has a cut-off value of about 10 kDa, about 30 kDa, about 100 kDa, or about 500 kDa. In some embodiments, the TFF membrane has a cut-off value of 30 kDa or about 30 kDa.In some embodiments, the TFF membrane comprises cellulose. In some embodiments, the TFF membrane comprises regenerated cellulose. In some embodiments, the TFF membrane comprises a polyethersulfone (PES) membrane.In some embodiments, a TFF pressure of less than 0.1 bar, 0.5 bar, 1 bar, 1.5 bar, 2 bar, 2.5 bar, 3 bar, 3.5 bar, 4 bar, 4.5 bar, or 5 bar, may be used. In some embodiments, a TFF pressure of less than or equal to 3.5 bar is used, to address filter clogging and slow filtration rates (e.g., when using large scale (>5L) media processing of spent medias using a low cut-off, such as 30kDa).In some embodiments, conditioned media or sEV subjected to TFF can be further purified, isolated, and / or enriched (after TFF) using one or more purification, isolation, and / or enrichment,techniques. For instance, the resulting product from TFF can be subjected to a chromatography step, such as an ion exchange chromatography step or a steric exclusion chromatography step, to even further purify small extracellular vesicles. In some embodiments, conditioned media subjected to TFF, with or without further purification, isolation, and / or enrichment, may be further concentrated, such as by ultracentrifugation.Any of the above-described processing techniques can be performed on recovered, conditioned medium (or a previously processed extract or fraction thereof) that is fresh, or has previously been frozen and / or refrigerated, for example.In some embodiments, secretome-, extracellular vesicle-, and sEV -containing compositions produced by the methods herein may have added thereto at least one additive to prevent aggregation. The additive may be one or more selected from trehalose, histidine (e.g., L- histidine), arginine (e.g., L-arginine), citrate-dextrose solution, a Dnase (e.g., Dnase I), ferric citrate, or Anti-Clumping Agent (Gibco / Life technologies, Ref: 01-0057; Lonza, Ref: BE02-058E). In some embodiments, trehalose is added. In some embodiments, trehalose or L-histidine is added.In some embodiments, the sEV fraction is CD63+, CD81+, and / or CD9+. The sEV fraction may contain one or more extracellular vesicle types, such as, for example, one or more of exosomes, microparticles, and extracellular vesicles. The sEV fraction may also contain secreted proteins (enveloped and / or unenveloped). Extracellular vesicles within conditioned media or sEV fractions of the present disclosure may contain, for example, one or more components selected from tetraspanins (e.g., CD9, CD63 and CD81), ceramide, MHC class I, MHC class II, integrins, adhesion molecules, phosphatidylserine, sphingomyelin, cholesterol, cytoskeletal proteins (e.g., actin, gelsolin, myosin, tubulin), enzymes (e.g., catalase, GAPDH, nitric oxide synthase, LT synthases), nucleic acids (e.g., RNA, miRNA), heat shock proteins (e.g., HSP70 and HSP90), exosome biogenesis proteins (ALIX, Tsg101), LT, prostaglandins, and S100 proteins.In some embodiments, the presence of desired extracellular vesicle types in a fraction can be determined, for example, by nanoparticle tracking analysis (to determine the sizes of particles in the fraction); and / or by confirming the presence of one or more markers associated with a desired extracellular vesicle type. For instance, a fraction of recovered, conditioned media can be analyzed for the presence of desired extracellular vesicle types by detecting the presence of one or more markers in the fraction, such as, for example, CD9, CD63 and / or CD81.In some embodiments, an sEV formulation or composition is positive for CD9, CD63 and CD81 (canonical EV markers), and is positive for the cardiac-related markers CD49e, R0R1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29 and CD142. In some embodiments, an sEV formulation or composition contains a lesser amount of one or more markers selected from the group consisting of CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CDlc, CD25, CD40, CDl lc, CD86, CD31, CD20, CD19, CD209, HLA-ABC, CD62P, CD42a and CD69, as compared to the amount of CD9, CD63 and / or CD81 in the sEV formulation or composition. In some embodiments, an sEV formulation or composition contains an undetectable amount of (e.g., by MACSPlex assay, by immunoassay, etc.), or is negative for, one or more markers selected from the group consisting of CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69.In some embodiments, the sEV formulation or composition is at least one of the following: an sEV formulation or composition that has been enriched for extracellular vesicles having a diameter of between about 50-200 nm or between 50-200 nm; an sEV formulation or composition that has been enriched for extracellular vesicles having a diameter of between about 50-150 nm or between 50-150 nm; an sEV formulation or composition that is substantially free or free of whole cells; and an sEV formulation or composition that is substantially free of one or more culture medium components (e.g., phenol-red).In some embodiments, such as, for example, some GMP-compatible processes, testing panels are conducted to analyze and / or determine one or more properties of the processes, products thereof, or intermediate products, etc.For instance, during the vesiculation stage (including, e.g., thawing, plating, culturing and / or harvesting steps), one or more properties of the cells may be examined (including, for example: the number of viable cells, the percentage viability of the cells; morphologies of the cells; identity of the cells; karyotype of the cells; and / or transcriptome of the cells).Additionally, or alternatively, one or more properties of a secretome and / or extracellular vesicle-containing fraction, extract, or composition can be analyzed using one or more tests (including, e.g., particle concentration and / or particle size distribution; protein concentration; protein profile concentration; RNA profile; potency; marker identity; host cell protein assessment; residual DNA quantification and / or characterization; sterility; mycoplasma; endotoxin; appearance; pH; osmolarity; extractable volume; hemolytic activity; complement activation;platelet activation; and / or genotoxicity), to determine one or more properties of the secretome / extracellular vesicles. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., may be analyzed by electron microscopy.In some embodiments, RNA may be extracted from EVs to analyze the RNA transcriptome of an EV composition, formulation, fraction, or secretome, etc. In some embodiments thereof, microRNA is analyzed, such as, for example, by generating a cDNA library from extracted RNA; and sequencing all or a part of the library. In some embodiments, the sequence analysis comprises sorting the scqucnccd RNAs into different biotypcs. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains all or some of the miRNAs depicted in FIG. 51 or TABLE 9. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the miRNAs depicted in FIG. 51 or TABLE 9. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains all or some of the miRNAs. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 miRNAs. In some embodiments, an EV composition, formulation, or fraction, etc., contains at least one of miR-302, miR-16, miR-126 and miR-93.In some embodiments, proteomic analysis of an EV composition, formulation, fraction, or secretome, etc., may be conducted. In some embodiments, proteins may be isolated from an EV composition, formulation, fraction, or secretome, etc., and analyzed by mass spectrometry, such as, for example, nano-LC-MS / MS and HPLC-MS / MS analysis. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25 proteins.Additionally, one or more of the above properties can be assessed on conditioned media before clarification; on conditioned media after clarification; on isolated and / or concentrated secretome / extracellular vesicles; and / or on final formulations.In some embodiments, final formulations may be tested immediately after production and / or 1-week, 2-weeks, 1 -month, 2 -months, 3-months, 6-months, 1-year, 18 months and several years, after being formulated.An exemplary process / product testing panel is shown in TABLE 49. This exemplary process is in addition to the description above. This panel was developed to characterize the process described herein and ensure reproducibility, which further led to the development of CTC1-EV.TABLE 49: An exemplary secretome / extracellular vesicle process / product testing panelTherapeutic Compositions and ApplicationsThe present disclosure contemplates the generation of secretome-, extracellular vesicle-, and sEV -containing compositions useful as therapeutic agents. In some embodiments, the methods of the present disclosure comprise administering an effective amount of a secretome-, extracellular vesicle-, and / or sEV -containing composition to a subject in need thereof.Tissues treated according to the methods of the present disclosure include, without limitation, cardiac tissue, brain or other neural tissue, skeletal muscle tissue, pulmonary tissue, arterial tissue, capillary tissue, renal tissue, hepatic tissue, tissue of the gastrointestinal tract, epithelial tissue, connective tissue, tissue of the urinary tract, etc. The tissue to be treated may be damaged or fully or partly non-functional due to an injury, age-related degeneration, acute or chronic disease, cancer, or infection, for example. Such tissues may be treated, for example, by intravenous administration of a secretome-, extracellular vesicle-, and / or sEV-containing composition.In some embodiments, compositions of the present disclosure may be used to treat diseases such as myocardial infarction, stroke, heart failure, and critical limb ischemia, for example. In some embodiments, compositions of the present disclosure may be used to treat heart failure which has one or more of the following characteristics: is acute, chronic, ischemic, non-ischemic, with ventricular dilation, without ventricular dilation, with reduced left ventricular ejection fraction, or with preserved left ventricular ejection fraction. In some embodiments, compositions of the present disclosure may be used to treat heart failure selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy induced heart failure. In some embodiments, compositions of the present disclosure may be used to treat diseases such as congestive heart failure, heart disease, ischemic heart disease, valvular heart disease, connective tissue diseases, viral or bacterial infection, myopathy, dystrophinopathy, liver disease, renal disease, sickle cell disease, diabetes, ocular diseases, and neurological diseases. In some embodiments, compositions of the present disclosure may be used to treat chemotherapy-induced cardiomyopathy (e.g., caused by anthracycline administration). It will be recognized that a suitable progenitor cell type(s) may be selected depending on the disease to be treated, or the tissue to be targeted.For example, in some embodiments, a subject with a cardiac disease, such as acute myocardial infarction, chemotherapy-induced cardiomyopathy, or heart failure, can be treated with a secretome-, extracellular vesicle-, and / or sEV-containing composition, produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells.Additionally, a secretome-, extracellular vesicle-, and / or sEV -containing composition produced from an appropriate progenitor cell type can also be used to improve the functioning or performance of a tissue. For instance, an improvement in angiogenesis, or an improvement in cardiac performance, may be effected by delivering a secretome-, extracellular vesicle-, and / or sEV -containing composition, produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells, to a subject in need thereof.In some embodiments, the administration comprises administration at a tissue or organ site that is the same as the target tissue. In some embodiments, the administration comprises administration at a tissue or organ site that is different from the target tissue. Such administration may include, for example, intravenous administration.A secretome-, extracellular vesicle-, and / or sEV -containing composition may contain, or be administered with, a pharmaceutically-acceptable diluent, carrier, or excipient. Such a composition may also contain, in some embodiments, pharmaceutically acceptable concentrations of one or more of a salt, buffering agent, preservative, or other therapeutic agent. Some examples of materials which can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical formulations. For instance, in some embodiments, a secretome-, extracellular vesicle-, and / or sEV-containing composition, may be formulated with a biomaterial, such as an injectable biomaterial. Exemplary injectable biomaterials are described, for example, in WO 2018 / 046870, incorporated by reference herein in its entirety.The secretome-, extracellular vesicle-, and / or sEV-containing compositions of the present disclosure may be administered in effective amounts, such as therapeutically effective amounts, depending on the purpose. An effective amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses,and individual patient parameters including age, physical condition, size, weight, and the stage of disease. These factors are well known to those of ordinary skill in the art.Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intramyocardial, intra-coronary, aerosol, suppository, epicardial patch, oral administration, or by perfusion. For instance, therapeutic compositions for parenteral administration may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. For instance, in some embodiments, a subject with a cardiac disease, such as acute myocardial infarction or heart failure, can be treated with a secretome-, extracellular vesicle-, and / or sEV-containing composition, produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells, wherein the composition is administered intravenously.In some embodiments, a single dose of a secretome-, extracellular vesicle-, and / or sEV- containing composition may be administered. In other embodiments, multiple doses, spanning one or more doses per day, week, or month, are administered to the subject. In some embodiments, single or repeated administration of a secretome-, extracellular vesicle-, and / or sEV-containing composition, including two, three, four, five or more administrations, may be made. In some embodiments, the secretome-, extracellular vesicle-, and / or sEV-containing composition may be administered continuously. Repeated or continuous administration may occur over a period of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6 days, or 1-7 days) or several weeks (e.g., 1-2 weeks, 1-3 weeks, or 1-4 weeks) or months, depending on the nature and / or severity of the condition being treated. If administration is repeated but not continuous, the time in between administrations may be hours (e.g., 4 hours, 6 hours, or 12 hours), days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days), or weeks (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks). The time between administrations may be the same or they may differ. As an example, if symptoms worsen, or do not improve, the secretome-, extracellular vesicle-, and / or sEV-containing composition, may be administered more frequently. Contrarily, if symptoms stabilize or diminish, the secretome-, extracellular vesicle-, and / or sEV-containing composition may be administered less frequently.In some embodiments, a secretome-, extracellular vesicle-, and / or sEV -containing composition is administered in several doses, for example three, on or about several days, weeks, or months apart, for example two weeks apart, by intravenous administration. In some embodiments thereof, the composition may be diluted with, formulated with, and / or administered together with, a carrier, diluent, or suitable material (e.g., saline).Assays for Determining Secretome and Extracellular Vesicle Activity, Functionality, and / or PotencyThe present disclosure also encompasses methods for analyzing the activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV- containing composition.The activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition, can be assessed by various techniques, depending on, for example, the type of progenitor cells used to produce the conditioned media or composition, and the desired use of the conditioned media or composition.For instance, the activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition, can be assessed by administering the conditioned media, secretome-, extracellular vesicle-, and / or sEV-containing composition, to target cells in vitro, ex vivo, or in vivo. One or more properties of the target cells can then be analyzed, such as, for example, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology, to determine the activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition.In some embodiments, assays known in the art may be used to determine the activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition.For instance, for conditioned media; or for a secretome-, extracellular vesicle-, and / or sEV- containing composition, obtained from cardiovascular progenitor cells or cardiomyocyte progenitor cells, the activity, functionality, and / or potency, thereof may be measured using a known cardiomyocyte viability assay, such as described in El Harane et al. (Ear. Heart J., 2018, 39(20): 1835-1847).Specifically, serum-deprived cardiac myoblasts (e.g., H9c2 cells) may be contacted with conditioned media; or a secretome-, extracellular vesicle-, and / or sEV-containing composition, and the viability of the cells measured thereafter. In some embodiments of this assay, the cells are deprived of serum before administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition. In other embodiments, the cells are deprived of serum after administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition. In some embodiments, the cells are deprived of serum before and after administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV- containing composition.In other embodiments, the angiogenic activity of a conditioned media or a secretome-, extracellular vesicle-, and / or sEV-containing composition, can be measured, for example, using a HUVEC scratch wound healing assay. In HUVEC scratch wound healing assays, HUVEC cells are cultured on a culture surface, and the cultured cell layer(s) is then scratched; angiogenic activity of a conditioned media or a secretome-, extracellular vesicle-, and / or sEV-containing composition, can then be determined by the capacity of the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition, to produce closure of the wound under serum-free conditions.In some embodiments, the activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition, may be analyzed using a HUVEC (Human umbilical vein endothelial cells) plating assay. In some embodiments thereof, HUVEC cells are cultured in basal medium in the presence of conditioned media; or a secretome-, extracellular vesicle-, and / or sEV-containing composition, to analyze the effect of the conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition, on HUVEC viability. In some embodiments thereof, the conditioned media; secretome-, extracellular vesicle-, and / or sEV-containing composition, improves cell seeding, survival, and / or proliferation, of the HUVEC cells.Cell viability (in cell viability assays) may be measured using, for example, a DNA- labeling dye or a nuclear-staining dye. The dye may be used with live cell imaging. Cell viability may also be measured by microscopy, such as fluorescence microscopy, using such a DNA- labeling dye or a nuclear-staining dye. Cell viability may also be measured, in a HUVEC plating assay for example, by analyzing ATP content.In some embodiments, a conditioned media; or a secretome-, extracellular vesicle-, and / or sEV-containing composition, may be analyzed in an anti-fibrosis assay, to determine the effect of the conditioned media; or the secretome-, extracellular vesicle-, and / or sEV-containing composition, on treating or reducing fibrosis. In some embodiments thereof, cells are stimulated to induce a fibrotic state. In some embodiments, the cells induced to a fibrotic state are fibroblasts, such as, for example, cardiac fibroblasts. In some embodiments, the cells are stimulated with at least one stimulating agent to induce a fibrotic state. In some embodiments, the stimulating agent is a TGF-β (e.g., TGF-β1, TGF-β2 and / or TGF-β3), and / or bleomycin. In some embodiments, the induction, treatment, and / or reduction, of fibrosis is determined by analyzing one or more markers of fibrosis. In some embodiments, the one or more markers of fibrosis are analyzed by quantifying the amount of transcript encoding a marker of fibrosis. In some embodiments, the amount of transcript is quantified by quantitative reverse transcription polymerase chain reaction. In some embodiments, the amount of transcript is quantified using an array or next generation sequencing. In some embodiments, the expression of at least one of MMP2 and Periostin are analyzed.An activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition, may also be determined with reference to one or more control samples. For instance, control cells may be one or more of: serum-deprived control cells which are not administered the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition; control cells which are not serum-deprived; or serum-deprived control cells which are administered a mock conditioned media or mock secretome-, extracellular vesicle-, and / or sEV-containing composition.In some methods of the present disclosure, an activity, functionality, and / or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and / or sEV-containing composition, can be assessed by a method comprising administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition, to target cells cultured under at least one stress-inducing condition, and analyzing at least one property of the cells. The one or more properties of the target cells that may be analyzed can be selected from, for instance, cell migration, cell survival, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology. In some embodiments, the at least one property measured is cell adhesion, cell number, cell growth, and / or cell morphology, and wherein the cell adhesion,cell number, cell growth, and / or cell morphology, is determined by measuring electrical impedance across a culture vessel surface in the culture.In a first method thereof, target cells are cultured in a pre-treatment medium under at least one stress-inducing condition, followed by administering a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition, to the cell culture. The target cells are then cultured in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, and at least one property of the cultured cells is measured one or more times during the culturing. In some embodiments, the at least one property is measured multiple times during the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition (such as, for example, 5 minutes to 10 hours apart from each other; 10 minutes to 4 hours apart from each other; or 30 minutes to 2 hours apart from each other).In some embodiments of this first method, the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, occurs in the presence of the at least one stress-inducing condition. In other embodiments of this first method, the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, occurs in the absence of the at least one stressinducing condition.In some embodiments of this first method, the pre-treatment medium is removed from the cells before the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition. Thus, in embodiments of the first method where the at least one stress-inducing condition is provided by the pre-treatment medium (e.g., by a stress-inducing agent present in the pre-treatment medium), the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, occurs in the absence of the at least one stress-inducing condition.In other embodiments of this first method, the pre-treatment medium is not removed from the cells before the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition. Thus, in embodiments of the first method where the at least one stress-inducing condition is provided by the pre-treatment medium (e.g., by a stress-inducing agent present in the pre-treatment medium), the culturing in the presenceof the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, occurs in the presence of the at least one stress-inducing condition.In a second method, target cells are cultured in a pre-treatment medium, followed by administering a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition (and optionally thereafter, culturing the target cells in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition). The target cells are then cultured under at least one stress-inducing condition, and at least one property of the cultured cells is measured one or more times during the culturing under the at least one strcssinducing condition (which also occurs in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition). In some embodiments, the at least one property is measured multiple times during the culturing under the at least one stress-inducing condition (and in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition), such as, for example, 5 minutes to 10 hours apart from each other; 10 minutes to 4 hours apart from each other; or 30 minutes to 2 hours apart from each other.In some embodiments of this second method, the target cells are cultured in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, before being cultured under the at least one stress-inducing condition. In other embodiments of this second method, the target cells are not cultured in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, before being cultured under the at least one stress-inducing condition. In some embodiments of this second method, the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV- containing composition, is removed from the target cells before the target cells are cultured in the presence of the at least one stress-inducing condition.In some embodiments of the above first and second methods, the stress-inducing condition is culturing in the presence of a cellular stress agent. In some embodiments of the second method, the cellular stress agent is co-administered to the target cells with the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition.In some embodiments of the above first and second methods, the cellular stress agent is one or more apoptosis-inducing agents.The one or more apoptosis-inducing agents may be selected from, for example, doxorubicin, staurosporine, etoposide, camptothecin, paclitaxel, vinblastine, gambogic acid,daunorubicin, tyrphostins, thapsigargin, okadaic acid, mifepristone, colchicine, ionomycin, 24(S)- hydroxycholesterol, cytochalasin D, brefeldin A, raptinal, carboplatin, C2 ceramide, actinomycin D, rosiglitazone, kaempferol, berberine chloride, bioymifi, betulinic acid, tamoxifen, embelin, phytosphingosine, mitomycin C, birinapant, anisomycin, genistein, cycloheximide, and the like.In some embodiments, the apoptosis-inducing agent is an indolocarbazole. In some embodiments, the apoptosis-inducing agent is an indolo (2,3-a) pyrrole (3,4-c) carbazole. In some embodiments, the apoptosis-inducing agent is staurosporine, or a derivative thereof. In other embodiments, the apoptosis-inducing agent is doxorubicin, or a derivative thereof.In some embodiments of the above first and second methods, the stress-inducing condition is culturing in the presence of a chemotherapeutic agent; and the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, is analyzed in a chemotherapy-induced cardiomyopathy assay. In some embodiments thereof, the chemotherapeutic agent is an anthracycline. In some embodiments thereof, the anthracycline is one more of aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments thereof, the chemotherapeutic agent is or comprises doxorubicin.In some embodiments thereof, the chemotherapy-induced cardiomyopathy assay comprises treating cells, such as cardiomyocytes, with a chemotherapeutic agent to induce cardiomyopathy, before culturing the treated cells in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition. In some embodiments thereof, the induction of the chemotherapy-induced cardiomyopathy; and / or the treating or reducing of the chemotherapy-induced cardiomyopathy, is measured by analyzing ATP content. In some embodiments, the induction of the chemotherapy-induced cardiomyopathy; and / or the treating or reducing of the chemotherapy-induced cardiomyopathy, is measured by analyzing mitochondrial function, for example, using a Seahorse method (e.g., Seahorse Mito Stress Test (Seahorse XFp Cell Mito Stress Test Kit, Agilent)).In some embodiments of the first and second methods, at least one property measured is viability of the cultured cells. The viability may be measured, for example, using a DNA-labeling dye or a nuclear-staining dye. In some embodiments thereof, the DNA-labeling dye or the nuclear- staining dye is a fluorescent dye, such as a far-red fluorescent dye.In some embodiments, a conditioned media; or a secretome-, extracellular vesicle-, and / or sEV-containing composition, maybe analyzed in a chemotherapy-induced cardiomyopathy animalmodel. In some embodiments thereof, the animal model is a rat model of chemotherapy-induced cardiomyopathy. In some embodiments thereof, the chemotherapeutic agent is an anthracycline. In some embodiments thereof, the anthracycline is one more of aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments thereof, the chemotherapeutic agent is or comprises doxorubicin. In some embodiments, the induction, treatment, or reduction, of chemotherapy-induced cardiomyopathy may be measured by one or more of: echocardiography (to determine LVESV and LVEDV, for example); electrocardiography; blood pressure (systolic, diastolic, etc.) measurements; functional status assessed by an NYHA score; quality of life; measurements of LVEF and LV volumes; maximum oxygen consumption during exercise; immune response by detection of antibodies specific to donor cells after each infusion; and assay of pro- and anti-inflammatory cytokines. In some embodiments, a conditioned media; or a secretome-, extracellular vesicle-, and / or sEV-containing composition, may have the capacity to counter the energetic stress (a metabolic / energy-related pathology) induced by the chemotherapy (e.g., mitochondrial damage, insufficient energy production).In some embodiments of the first and second methods, one or more of the culturing of the target cells with: (a) the pre-treatment medium; (b) the conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition; and (c) at least one stress-inducing condition, may occur in the absence of serum. In some embodiments, the target cells may be deprived of serum before administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition. In other embodiments, the target cells may be deprived of serum after administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition. In some embodiments, the cells are deprived of serum before and after administering the conditioned media or the secretome-, extracellular vesicle-, and / or sEV-containing composition.In embodiments of the first and second methods, the target cells can be cultured in the pretreatment medium for differing lengths of time. For instance, the target cells can be cultured in the pre-treatment medium for 30 minutes to 10 hours, 1 hour to 5 hours, or more than, less than, or about, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.In embodiments of the first and second methods, the target cells are cultured with the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition,for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 48 hours.In some embodiments of the first and second methods, the target cells are cultured in vitro prior to culturing in the pre-treatment medium. For instance, the target cells may be cultured in vitro for between 1-21 days, between 3-17 days, between 5-14 days, or less than 20 days, less than 18 days, less than 16 days, less than 14 days, less than 12 days, less than 10 days, less than 8 days, less than 6 days, less than 4 days, or less than 2 days, prior to culturing in the pre-treatment medium. In certain embodiments in which the target cells arc cultured in vitro prior to culturing in the pretreatment medium, the target cells are supplied with fresh culture medium prior to culturing in the pre-treatment medium. For instance, the target cells may be supplied with fresh culture medium 6-72 hours, 8-60 hours, 10-48 hours, 12-36 hours, prior to culturing in the pre-treatment medium.In embodiments of the first and second methods, the culturing of the target cells may be two-dimensional or three-dimensional cell culturing. For instance, in some embodiments, the culture vessel used for culturing may be a flask, flask for tissue culture, hyperflask, dish, petri dish, dish fortissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, culture bag, roller bottle, bioreactor, stirred culture vessel, spinner flask, microcarrier, or a vertical wheel bioreactor, for example.In embodiments in which culturing comprises two-dimensional cell culture, such as on the surface of a culture vessel, the culture surface (to which the cells are intended to adhere) may be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, type I, type II, and type IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly- D and poly-L-lysine, Matrigel, thrombospondin, and / or vitronectin.In embodiments of the first and second methods, the at least one property may also be analyzed with reference to one or more control samples.For instance, the first and second methods may further comprise culturing positive control cells in parallel, wherein the positive control cells are not administered the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, and are not cultured under the at least one stress-inducing condition. Thus, in embodiments in which the stress inducing condition is the presence of an apoptosis-inducing agent, the positive control cells are not administered the apoptosis-inducing agent.The first and second methods may comprise culturing negative control cells in parallel, wherein the negative control cells are not administered the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition. In some embodiments, the negative control cells comprise negative control cells subjected to the same steps as the target cells, except that they are not administered the secretome.In certain embodiments, the negative control cells comprise negative control cells cultured in the pre-treatment medium under the at least one stress-inducing condition. The at least one property measured in the target cells may also then be measured in the negative control cells, cither during or after they are cultured in the pre-treatment medium under the at least one stress-inducing condition.In some embodiments, the negative control cells comprise negative control cells to which a mock conditioned medium or a mock secretome-, extracellular vesicle-, and / or sEV-containing composition is added. In specific embodiments thereof, the mock conditioned medium or the mock secretome-, extracellular vesicle-, and / or sEV-containing composition is produced by omitting cells from the process of producing a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition, such as a process of the present disclosure.The use of such a negative control(s) allows an activity, functionality and / or potency, of a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition, to be evaluated. For instance, where the at least one property measured is viability of the cultured cells, a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition, may be determined to have an activity, functionality, potency (and / or exhibit a therapeutic effect), when the viability of the target cells is higher than the viability of the negative control cells.Alternatively, for instance, where the at least one property measured is cell adhesion, cell growth, and / or cell number, and wherein the cell adhesion, cell growth, and / or cell number is determined by measuring electrical impedance across a culture vessel surface in the culture, a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition may be determined to have an activity, functionality, potency (and / or exhibit a therapeutic effect), when the electrical impedance across a culture vessel surface in the culture is higher than the electrical impedance across a culture vessel surface in a culture of negative control cells.Any one or more samples, and / or any one or more positive and / or negative controls, may be performed in replicate, such as, for example, in duplicate, in triplicate, etc. In some embodiments thereof in which cell viability is measured, and where replicate cultures are performed, the number of positive control cells in the replicate cultures may be averaged to produce an average maximum cell number (and the number of target cells in each replicate test culture may be normalized to the average maximum cell number, to calculate cell viability).To more accurately compare an activity, functionality, and / or potency, between different conditioned media or secretome-, extracellular vesicle-, and / or sEV-containing compositions, it may be beneficial to determine the amount of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition, added to target cells. This can be determined, for example, based on one or more of: the amount of secreting cells that produced the secretome; the protein content of said secretome; the RNA content of said secretome; the exosome amount of said secretome; and particle number.Methods of treatment using the compositions of the present disclosureThe present disclosure further contemplates using the compositions of the present disclosure for the treatment or prevention of various diseases and conditions in a subject in need thereof. The methods of treatment or prevention contemplated herein include, for example, treatment or prevention of cardiovascular diseases and conditions, such as myocardial infarction, heart failure, myocarditis, cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, post-chemotherapy induced heart failure, ventricular dysfunction, atrial dysfunction, or arrhythmia.In some embodiments, the heart failure is acute heart failure, chronic heart failure, ischemic heart failure, non-ischemic heart failure, heart failure with ventricular dilation, heart failure without ventricular dilation, heart failure with reduced left ventricular ejection fraction, or heart failure with preserved left ventricular ejection fraction.In some embodiments cardiomyopathy is chemotherapy-induced cardiomyopathy. In some embodiments the chemotherapy-induced cardiomyopathy is anthracycline-induced cardiomyopathy. In some embodiments, anthracycline is doxorubicin.In some embodiments, the methods of the present disclosure improve cardiac performance, fitness, endurance or recovery of a subject.In some embodiments, the methods of the present disclosure improve angiogenesis, improve cardiomyocyte viability, improve endothelial cell survival, health and function, reduce fibrosis in cardiac fibroblasts, improve survival of stressed cardiomyocytes, improve survival, viability and proliferation of stressed endothelial cells, improve cell migration and / or wound healing capabilities in a subject, for example, by improving migration and / or wound healing capabilities of stressed endothelial cells. In some embodiments the cardiac fibroblasts in which fibrosis is reduced by the methods of the present disclosure are stimulated cardiac fibroblasts, such as cardiac fibroblasts stimulated by TGF-β1. In some embodiments, the methods reduce the expression of the pro-fibrotic marker, POSTN, in TGF-β1-stimulated cardiac fibroblasts to level prior to stimulation with TGF-β1 or below.In some embodiments, the methods of the present disclosure improve or maintain Left Ventricular End Systolic Volume (LVESV). In some embodiments, the methods maintain LVESV within 20%, within 15%, within 10%, within 5%, or within 2% of the pre-treatment LVESV.In some embodiments, the methods of the present disclosure do not induce an allogeneic inflammatory response in a subject, do not induce an allogeneic peripheral blood mononuclear cell (PBMC) activation, do not induce a significant increase in the percentage of IFNg or IL-2 expressing PBMCs, do not induce allogeneic natural killer (NK) cell degranulation, or do not significantly increase the percentage of CD 107 expressing NK cells.In some embodiments, the compositions of the present disclosure are administered to a subject by intravenous infusion, direct cardiac injection, intra-arterially or by interventional cardiology methods, such as by a catheter-based administration.The dose, route of administration, frequency of administration and duration of treatment with the compositions of the present invention may be determined by considering the disease or condition for which the treatment is administered, severity and duration of the disease or condition, medical history and overall health of the subject being treated, tolerability of the composition, adverse effects and other factors.The compositions of the present disclosure may be administered, for example, at a dose containing secretome obtained from 0.1 to 10 million cells per kg weight of the subject being treated, from 0.5 to 5 million cells per kg weight of said subject, from 1 to 3 million cells per kg weight of said subject, from 1 to 2 million cells per kg weight of said subject, or 1 million cells per kg weight of said subject. The dose may be administered at one time, e.g., per each intravenousinfusion, cardiac injection or intra-arterial administration, or over several administrations. In some embodiments, the cells are cardiomyocyte progenitor cells, cardiac progenitor cells, cardiovascular progenitor cells, or mixtures thereof.The compositions of the present disclosure may be administered, for example, at a dose containing from 1 x 109to 60 x 109particles per kg weight of the subject, from 10 x 109to 60 x 109particles, per kg weight of the subject, or from 10 x 109to 40 x 109particles per kg weight of the subject, from 20 x 109to 40 x 109particles per kg weight of the subject, 20 x 109particles per kg weight of the subject, or 10 x 109particles per kg weight of the subject. The number of particles may be measured, for example, by Nanoparticle Tracking Analysis (NTA). The dose may be administered at one time, e.g., per each intravenous infusion, direct cardiac injection or intraarterial administration or over several administrations.The compositions of the present disclosure may be administered, for example, at a cumulative daily dose containing from 20 x 109to 200 x 109particles per kg weight of the subject, from 30 x 109to 100 x 109particles per kg weight of the subject, 60 x 109particles per kg weight of the subject, 50 x 109particles per kg weight of the subject, 40 x 109particles per kg weight of the subject, 30 x 109particles per kg weight of the subject, 20 x 109particles per kg weight of the subject, or 10 x 109particles per kg weight of the subject. The number of particles maybe measured by NTA. The cumulative daily dose may be administered at one time, e.g., one intravenous infusion, direct cardiac injection or intra-arterial administration or over several administrations.The compositions of the present disclosure may be administered, for example, 1 to 10 times per day, 3 to 6 times per day, 1 to 5 times per day, 3 times per day, 2 times per day, or once per day.The duration of treatment may be, for example, 65 days or less, 5 to 50 days, 10 to 50 days, 20 to 45 days, 42 days, 21 days, 14 days, or 7 days.During the course of the treatment, the compositions of the present disclosure may be administered, for example, every day, every other day, at a frequency of from every day to every 30 days, from every 7 days to every 21 days, every 21 days, every 14 days, every 7 days.Testing products on non-human mammalian species is important for modeling complex diseases that affect the biology and / or physiology of multiple cells, tissues, organs, and systems. Using animal models enables testing the effect of products on physiology of tissues, organs, and organisms. A new animal model of heart failure is described herein. While the term ‘heart failure’is typically used for human subjects, its use herein is extended to the animal models. Two different heart failure models are described. One is a post-ischemia chronic heart failure model in mice which is induced by surgical means (permanent occlusion of the left ventricular coronary artery), and the other is a non-ischemic, chemotherapy drug-induced cardiomyopathy with left ventricular dysfunction and other signs of heart failure. Both models involve left ventricular dysfunction.In the chemotherapy-induced cardiomyopathy (CCM) model described herein, left ventricular dysfunction is induced in rats through the administration of doxorubicin, which belongs to the anthracyclinc class of anticanccr therapies and is one of the most widely used antincoplastic drugs, thanks to its broad spectrum of activity. Anthracyclines are chemotherapy agents known to induce heart failure in some patients. The model described herein effectively recapitulates many physiological features of chemotherapy-induced cardiomyopathy (CCM) in humans, such as progressively larger Left Ventricular End Systolic Volume (LVESV) and Left Ventricular End Diastolic Volume (LVEDV), decreasing ejection fraction (LVEF), decreased systolic elastance, and a slower LV-depolarization (increased QTc at EKG). In human patients, these characteristics are features of degrading cardiac function and are associated with worse prognosis. The model described herein is useful for determining the effect of extracellular vesicle (EV)-containing compositions on heart physiology. Beneficial effects of the EV-containing compositions in this non-ischemic model are expected to be predictive of the beneficial effects of the EV-containing compositions on the function of hearts in human subjects in non-ischemic heart failure, including subjects with chemotherapy-induced heart failure.The beneficial effects of the EV-containing compositions in the post-ischemic model of chronic heart failure are expected to be predictive of the beneficial effects of the EV-containing compositions on the function of hearts in human subects in post-ischemic heart failure, such as patients who have had a myocardial infaction, for example.Using the presented rat model of CCM, it is shown herein that CTC 1-EV final formulation, has a favorable effect on the physiology of the failing heart. The present inventors have demonstrated that the animals treated with the EV-containing compositions have reduced progression of left ventricualr dysfunction as compared to controls as shown by less heart volume enlargement over the study period. The controls, by contrast, continue to deteriorate over the study period, as shown by the increase in systolic and diastolic heart volumes.In addition to testing efficacy of a product in animal models, it is useful to test products in an in vitro model of human disease using human cells. In vitro models are key to understanding the specific effects a product has on specific cell types by enabling direct and specific analysis of a given cell type. The most useful in vitro cell models are on human cells, as this most closely matches the intended use of a product designed for treating human subjects. If desired, in vitro models can be limited to a single cell type, so that the biological effects of the EV-containing compositions on that specific cell type can be interrogated. Additionally, co-culture models and mixed cell models arc also useful for exploring the interplay of different cell types. In this specification, the effect of the EV-containing compositions on four human cell types in monoculture, and one mixed peripheral blood mononuclear cell (PBMC) model is described. All five of these cell types / mixtures are relevant to the pathology of heart failure, including ischemic and non-ischemic heart failure, including chemotherapy-induced heart failure. Inventors found positive biological effects of the EV-containing compositions on human cardiomyocytes, human endothelial cells, human cardiac fibroblasts, and a lack of allogeneic activation of human NK cells or human PBMCs.Cardiomyocytes are stressed in heart failure and can be in programmed cell death (apoptosis). Preservation of cardiomyocyte health and survival will have a beneficial effect on hearts in failure or hearts in ventricular dysfunction. The present inventors have shown that the EV-containing compositions promote survival of human cardiomyocytes when under apoptosisinducing stress. The EV-containing compositions are expected to promote cardiomyocyte health and survival in human subjects in heart failure. The EV-containing compositions are expected to improve cardiomyocyte-related functions of a heart in failure in a human subject.Endothelial cells are integral components of blood vessels and lymphatic tissues of the heart. A lack of sufficient circulation and drainage into and out of the heart tissues contributes to deteriorating heart function in failing hearts. Promoting endothelial cell survival, proliferation and migration under stress will encourage beneficial remodeling of the heart tissue and or reduce negative remodeling of the heart tissues. Together this will improve or help preserve heart function. The present inventors have shown that the EV-containing compositions support in vitro human endothelial cell survival in two different forms of stress, in vitro human endothelial cell proliferation and in vitro human endothelial cell migration, when these cells are under stress. The EV-containing compositions are expected to promote endothelial cell survival, proliferation andmigration in human subjects in heart failure, in which vessel health and wound healing capabilities are compromised. Increasing vascularization of the failing heart tissue would support maintaining the health of the heart tissue. The EV-containing compositions are expected to improve endothelial cell-related functions of a heart in failure in a human subject.In heart failure, there is increased fibrosis. Fibrosis contributes to the negative remodeling of a failing heart. Decreasing fibrosis will support better heart function in a failing heart. The present inventors have shown that the EV-containing compositions reduce signs of fibrosis in human cardiac fibroblast cells that have been stressed into a state of increased fibrosis. The EV- containing compositions are expected to reduce fibrosis in human subjects in heart failure and to reduce the negative effects of fibrosis on the functioning of the heart.Heart failure leads to a pro-inflammatory state in humans, which contributes to the deterioration of heart function. The present inventors have shown in vitro and in vivo, that the EV- containing compositions of the present invention do not stimulate allogeneic NK degranulation. The present inventors have also shown that the EV-containing compositions do not induce allogenic PBMC activation in vitro. The EV-containing compositions of the current invention does not promote NK degranulation in human subjects in heart failure, and to not induce PBMC activation in human subjects in heart failure. The EV-containing compositions of the present invention are immunologically neutral or anti-inflammatory when administered to human patients in heart failure.In vivo pre-clinical safety testing was performed on the EV-containing compositions (refer to clinical trial NCT05774509, which is incorporated by reference hereby in its entirety). GLP mouse and GLP rat models were used to show pre-clinical safety aspects of the EV-containing compositions of the present invention. Immunocompetent animal models selected showed no signs of acute immune reaction after repeated administrations, even with this being a xenograft. Both the mouse and rat models showed no toxicity of the EV-containing compositions when administered at high doses. Furthermore, a GLP immunocompromised mouse model showed no tumorigenicity of the EV-containing compositions. It is important to prove in an animal model, which is more complex than an in vitro model, as it has a functional immune system, circulatory system, etc. The animal model test results demonstrated that the EV-containing compositions of the present invention have a good safety profile, which is necessary to establish prior to testing in human subjects.The EV-containing compositions of the present invention are non-toxic and not tumorigenic when administered to human subjects.Another aspect of the EV-containing compositions of the present invention is the combination of multiple, parallel, beneficial biological effects expected to affect the biology of multiple cell types in a beneficial way in a patient in need of treatment, together with a positive safety profile. The EV-containing compositions of the present invention are a complex mixture of biological molecules, enabling the simultaneous protective, therapeutic, or regenerative properties described above, supporting the health, survival and function of multiple cell types in concert, contributing to the physiological effects observed in the animal models of chemotherapy-induced cardiomyopathy (CCM) as shown here as the therapeutic effects when used to treat humans.In addition to novel and unexpected biological and therapeutic effects of the EV-containing compositions disclosed of the present invention, the manufacturing process is designed, optimized, and tested at phase 1 clinical manufacturing scale, using GMP compatible methods, materials and reagents, for the manufacturing of a EV-containing compositions for use in human subjects. The use of multi-layer cell stacks, in-line clarification process, TFF (tangential flow filtration), ability to work from frozen cells if desired, ability to freeze TFF retentates for future pooling if needed, and lack of centrifugation steps, make the process defined herein are scalable to phase 2 and phase 3 manufacturing and to commercial manufacturing scales.Additionally, the inventors have disclosed a novel, groundbreaking, inventive and comprehensive panel of in-process quality control tests, and release tests for Quality Control, which ensure reproducibility, stability, safety, and potency of the EV-enriched secretome or EVs and compositions comprising thereof as a therapeutic. Features of the panel described herein may be applied to the quality control of other EV-enriched therapeutics, ensuring process control, the reproducibility of EV-containing compositions, the safety of EV-containing compositions, the potency of EV-containing compositions, and the stability of EV-containing compositions, to enable their use in a human subject.EXAMPLESNon-limiting embodiments of the present invention are illustrated in the following Examples. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts,concentrations, percent changes, and the like), but some experimental errors and deviations should be accounted for. It should be understood that these Examples are given by way of illustration only and are not intended to limit the scope of what the inventor regards as various embodiments of the present invention. Not all of the following steps set forth in each Example are required nor must the order of the steps in each Example be as presented.Example 1Generation of Cardiovascular Progenitor Cells from iPSCsHuman iPS cells (iPSCs) were expanded and differentiated into cardiovascular progenitor cells (CPCs) by suspension culture in PBS-mini vessels (PBS MINI 0.5 L Bioreactor Single Use Vessels; PBS Biotech ref: 1A-0.5-D-001), using the process depicted in FIG. 1. At the end of the CPC differentiation period, cells were counted as follows. A small sample (5-10 mL) of cell aggregates in suspension was removed from the suspension culture vessels, cell aggregates were gravity settled, supernatant removed and aggregates were resuspended in 3-5 mL of room temperature TrypLE Select (Invitrogen ref: 12563029), and incubated for 3-10 min at 37°C. Digestions were stopped using double the volume of RPMI-B27 Quench media (RPMI 1640 Medium (Gibco ref: 118875-085) supplemented with B-27 XenoFree, CTS grade 50x (Gibco ref: A14867-01, fc = 1x), filter sterilized using a 0.2 μm filter (ThermoScientific ref 567-0020)). Cell suspensions were then centrifuged at 300 x g for 5 minutes, and the resulting supernatant was discarded. The remaining cell pellets were delicately loosened, and the cells were resuspended in 5-10 mL of MEM alpha media base (MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561 -.37). Of these resuspended cells, one or two 500 μL samples were counted using a ViCell XR cell viability analyzer (Beckman Coulter), according to the manufacturer’s directions. The viable cells per mL were noted. Two distinct differentiation runs were performed, as depicted in FIG. 2 and 3, and similar yields of CPC per input iPSC were obtained.To confirm that the resulting cells were indeed CPCs, RNA expression by the resulting cells was analyzed. Specifically, between 1 and 2 million cells from the cell samples were removed and lysed in RLT plus buffer (Qiagen 1030963) for RNA extraction. RNA was extracted from the lysates using the Qiasymphony RNA kit (Qiagen, Ref: 931636), following the manufacturer’s directions. mRNA levels for 48 custom selected genes were evaluated using the qPCR Fluidigm platform. Unsupervised hierarchical clustering was performed on raw data using the “SINGULARAnalysis Toolset” package in R v3.1.1 (FIG. 4). Unsupervised hierarchical clustering was also performed on gene z-scores using JMP software vl7 (method=ward, unstandardized) (FIG.5). RNA expression by the resulting cells was compared to RNA expression by iPSC and cardiomyocyte control cells, confirming that gene expression by the resulting cells was consistent with them being CPCs (FIG. 4, FIG. 5 and TABLE 1). TABLE 1 presents the Ct data needed to produce FIG. 4 and FIG. 5.To dissociate CPC aggregates to single cells, 300-800 mL of the aggregate suspension of CPCs were collected from the differentiation suspension cultures and allowed to settle for approximately 5 minutes in 500 mL conical tubes. Spent media was then removed, and cell aggregates were washed in DPBS - / -. The washed cell aggregates were then resuspended in room temperature TrypLE (in approximately 25 mL TrypLE for 100 mL original aggregate suspension volume) and were allowed to dissociate for 10 min at 37°C. The cell aggregate dissociations were quenched with an equal volume of RPMI-B27 Quench media, and the dissociated cells were spun at 400 x g for 5 minutes. The resulting cell pellets were resuspended in RPMI-B27 Quench media, and then strained (Falcon 100 μm Cell strainer, Coming ref: 352360) into conical tubes and counted using a ViCell XR cell viability analyzer (Beckman Coulter).A subset of these cells were re-spun at 300 x g for 5 minutes, resuspended for fresh CPC plated vesiculation culture in alpha-MEM complete media (MEM alpha media base (MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561 -.37); Gentamicin (Gibco ref 15750060, final concentration (fc) = 0.025 mg / mL); glucose supplement (Gibco ref A2494001, at a ratio of 1:200); Flexbumin (with 25% w / vol human serum albumin, Baxter ref: NDC0944-0493-02 code 2G0012, fc HSA = 2mg / mL); B27 (minus insulin) (50x, Gibco ref Al 895601, fc = 1x); Human FGF-2 Premium grade (Miltenyi Biotec ref: A12873-01, fc= 1μg / mL); filter sterilized using a 0.2 μm filter (ThermoScientific ref 567-0020); media were used the same day). The freshly harvested single cells were again counted using a ViCell XR cell viability analyzer (Beckman Coulter) and plated (see Example 2). The remainder of the single cell suspensions were spun at 400 x g for 5 minutes, and the cells were resuspended in cryopreservation media (CryoStor CS-10, BioLife Solution ref: 210102) at 25 million cells / mL, frozen at -80°C, and then stored in liquid nitrogen for later use in thawed CPC plated vesiculation culture.Ct data that was used to produce FIG. 4 and FIG. 5 are shown in TABLE 1.TABLE 1. Ct values for genes of interest as used to generate FIG. 4 and FIG. 5 heatmaps.Example 2Vesiculation Culture of Cardiovascular Progenitor CellsCPCs were cultured in the vesiculation process as fresh aggregates in suspension culture, as fresh single cells plated onto hyperflasks, or as thawed single cells plated onto hyperflasks after having been cryopreserved and maintained at -80°C or less until time of use. Specifically, CPCs produced in Example 1 were used in suspension vesiculation culture and in adherent vesiculation culture in hyperflasks as described below.For suspension vesiculation culture, the volumes of aggregates in PBS-mini vessels at the end of the CPC differentiation process were noted (300-400 mL per vessel; “day+0” volumes). The cell aggregates underwent a 100% media exchange according to the following steps: (1) cell aggregates were transferred from PBS-mini vessels to conical tubes and allowed to settle for approximately 15 min; (2) PBS-mini vessels were rinsed three times with MEM alpha media base (MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561 -.37); (3) spent media was removed from settled cell aggregates; (4) cell aggregates were washed three times with an appropriate volume of MEM alpha media base; and (5) washed cell aggregates were re-seeded into their original (washed) PBS-mini vessels in alpha-MEM complete media (as described above) at their day+0 volumes to maintain cell density.The seeded cell aggregates were then cultured in suspension (37°C, 5% CO2, at atmospheric oxygen) with agitation at 40 rpm for 2 days (until “day+2”). At day+2, cell aggregates underwent a 100% media exchange following three rinses in MEM alpha media base. For this day+2 media exchange, the cell aggregates were re-seeded into their original PBS-mini vessel in alpha-MEM poor media (MEM alpha media base (MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561 -.37), supplemented with Gentamicin (Gibco ref 15750060, final concentration (fc) = 0.025 mg / mL), and glucose supplement (Gibco ref A2494001, at a ratio of 1:200), filter sterilized using a 0.2 μm filter (ThermoScientific ref 567-0020)), at the same volumes as their day+0 volumes. The cell aggregates were then cultured (37°C, 5% CO2, at atmospheric oxygen) in suspension, with agitation at 40 rpm for another 2 days, until the end of the vesiculation period (“day+4”).For Hyperflask adherent culture, fresh single cell CPCs were seeded at 100,000 cells / cm2onto vitronectin-coated hyperflasks in alpha-MEM complete media (“day+0”). In addition, cryopreserved CPCs were thawed at 37°C for 3 min, transferred to an empty conical tube, thenresuspended (dropwise) in alpha-MEM complete media. The thawed cell suspensions were centrifuged, and the cell pellets were resuspended in alpha-MEM complete media. The thawed CPCs were seeded at 100,000 cells / cm2onto vitronectin-coated hyperflasks in alpha-MEM complete media (“day+0”). The seeded cells for both fresh and thawed CPCs were then cultured (37ºC, 5% CO2, at atmospheric oxygen) for 2 days (until “day+2”). At day+2, spent media was removed, and the flasks were rinsed three times with 50-100 mL of pre- warmed MEM alpha media base. The culture vessels were then filled with alpha-MEM poor media, according to the manufacturer’s directions, and incubated for 2 more days (37°C, 5% CO2, at atmospheric oxygen) until the end of the vesiculation period (“day+4”).At day+2 and day+4, cells in the suspension cultures were counted as described above in Example 1. At day+4, cells in the adherent cultures were harvested by 1 / rinsing the cells with DPBS, 2 / incubating cells with 100mL of pre-warmed 0.05% Trypsin-EDTA (Gibco, 15400-054, diluted in DPBS) for 2-3 minutes at room temperature, 3 / quenching the harvest with 100mL aMEM + glutamax supplemented with B27 (minus insulin) (f.c. 1x) , 4 / collecting the bulk cell suspension into a 500mL conical centrifuge tube, 5 / rinsing harvested flasks with basal aMEM media to recover any remaining cells and adding this rinse to the bulk cell suspension. The concentration of cells in the suspensions were determined using the ViCell Automated Cell Counter, and the cells per cm2from the harvested vessels were back-calculated.In addition to the CPC adherent and suspension vesiculation cultures, virgin media controls were also performed for adherent and suspension cultures.For the suspension vesiculation culture virgin media controls, new 0.5 L PBS-mini vessels were filled with 400 mL alpha-MEM complete media (at “day+0”), and incubated for 2 days (37°C, 5% CO2, at atmospheric oxygen), with agitation at 40 rpm. After the two days (“day+2”), the spent culture media was removed, and vessels were rinsed thoroughly (three times each with 50- 100 mL of pre- warmed MEM alpha media base). The PBS-mini vessels were then filled with 400 mL alpha-MEM poor media, and incubated for 2 more days (37°C, 5% CO2, at atmospheric oxygen), until “day+4.”For the adherent vesiculation culture virgin media controls, vitronectin-coated hyperflasks were filled with alpha-MEM complete media and incubated for 2 days (37°C, 5% CO2, at atmospheric oxygen). After these two days (“day+2”), the spent culture media was removed, and the vessels were rinsed thoroughly (three times each with 50-100 mL of pre- warmed MEM alphamedia base). The hyperflasks were then filled with alpha-MEM poor media, and incubated for 2 more days (37°C, 5% CO2, at atmospheric oxygen), until “day+4.”At day+4, media from the suspension and adherent cell cultures (conditioned media, MC), as well as day+4 media from the virgin control vessels (virgin media, MV), were collected, and pre-cleared by serial centrifugation (400 × g for 10 minutes at 4°C, then 2000 × g for 30 minutes at 4°C). The pre-cleared media was then aliquoted into conical tubes, and frozen at -80°C. FIG. 6 depicts a process flow diagram for the generation of conditioned media and virgin media controls.Example 3Preparation of Small Extracellular Vesicle-Enriched Fraction (sEV)To validate the vesiculation process, samples of the conditioned and control media were subjected to ultracentrifugation, in order to generate sEV and MV preparations for molecular characterization and in vitro functional analyses. Two biological replicates of each sample type were prepared. FIG. 7 depicts a process flow diagram for the isolation of sEV or mock (virgin media) control samples.MC and MV were thawed at room temperature for 1-4 hours, or overnight at 4°C. After thawing, MC and MV were ultracentrifuged at 100,000 × g for 16 hours at 4°C (wX+ Ultra Series Centrifuge, ThermoScientific; rotor: F50L-8x39; Acceleration: 9; Deceleration: 9), and the resulting supernatants were removed. The bottom of each tube was rinsed twice with 100 μL volumes of 0.1 μm filtered DPBS- / - (0.1μm PES Filter Unit, ThermoFisher 565-0010) without disturbing the pellet, and then each pellet was resuspended in 0.1 μm filtered DPBS- / - by gentle agitation of the solvent with a sterilized glass stir bar. sEV preparations were collected, and tubes were rinsed with 0.1 μm filtered DPBS- / - for maximum product recovery (to a total resuspension plus rinse target volume as calculated based on the number of secreting cells giving rise to the conditioned media). 45μL were targeted for every 1.4 x 106day+4 secreting cells as calculated by the following formula:Target sEV Resuspension Volume = (Total Viable Cells at day+4 ÷ Total Volume Conditioned Media at day+4) x Volume MC Centrifuged × (45 μL 1.4 x 106Viable Cells).Target resuspension volumes for MV controls were matched to the relevant MC target resuspension volumes. For MC and MV generated in PBS-mini vessels, sEV preparations werefiltered at 0.65 μm (Ultrafree 0.65μm DV Durapore, Millipore ref: UFC30DV05) to remove large particulates. sEV and MV control preparations were aliquoted and frozen at -80°C. sEV and MV control preparations were further analyzed, as described below.First, the particle concentration and size distribution in sEV and MV control preparations were determined by nanoparticle tracking analysis (NTA; NanoSight). The nanoparticle tracking analysis confirmed the presence of particles of the size of exosomes and microparticles in the sEV prepared from CPC conditioned media, but not in MV controls. FIG. 8 depicts representative size distribution curves from two sEVs and two control MV samples. Observable particle sizes ranged from approximately <30 nm to 300 nm or so, with a peak generally between 50-150 nm, corresponding to the size of exosomes or small microparticles.Second, the presence of the exosome-associated vesicle surface marker CD63 was also analyzed using the PS Capture Exosome ELISA Kit (Wako Chemicals, ref: 293-77601), with the primary antibody being an anti-CD63 antibody (Wako Chemicals, ref: 292-79251), and the secondary antibody being an HRP-conjugated Anti-mouse IgG antibody (Wako Chemicals, ref: 299-79261). Input volumes were set such that 400 ng protein from sEV and MV control preparations was added to each well. This anti-CD63 ELISA evaluation confirmed the presence of exosome-associated CD63 surface antigen in each of the sEV samples, but in none of the MV controls (FIG. 9). CD63 signal was higher in the aggregate sample than in the plated samples, although the CD63 signal was consistent between replicates of plated samples. The protein content of sEV and MV control preparations was determined by BCA analysis, using the Pierce Micro BCA kit (ThermoScientific ref: 23235).Example 4In vitro analysis of sEV FunctionalityTo analyze the functionality of the sEV preparations, three in vitro assays were used: a HUVEC scratch wound healing assay; a cardiomyocyte viability assay using serum-deprived H9c2 cells; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed by Essen BioSciences, for the Incucyte) was employed, according to the manufacturer’s directions. Briefly, HUVEC cells were expanded using HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C-22210), supplemented with the Endothelial Cell Growth MediumSupplement Pack (PromoCell, Ref: C-39210). After expansion, the cells were cryopreserved in CS10 (Cryostore, ref: 210102) at 1-2 x 106cells per aliquot (enough for between a half to a full 96-well plate). Two days prior to assay, HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells / well, and grown in HUVEC Complete media for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout maintenance and assay process. Wells were scratched using a Wound Maker (EssenBio, Ref: 4493) according to the manufacturer’s directions, and cells were then rinsed with Endothelial Cell Basal Media and cultured overnight (cither in HUVEC Complete Media alone, as a positive control; in Endothelial Cell Basal Media alone, as a negative control; or in Endothelial Cell Basal Media supplemented with sEV or MV preparations). Using an Incucyte with the Scratch Wound Healing Module, plates were imaged every three hours for a total of 18 hours. Wound closure was determined using the manufacturer’s software, and values were baseline (negative control) subtracted, and normalized to the positive control. FIG. 10 depicts that the sEV preparations, but not the control MV preparation, promoted wound healing, indicating the functionality of the sEV preparation.For the cardiomyocyte viability assay using serum-deprived H9c2 cells, the assay was performed essentially as described in El Harane et al. (Eur. Heart J., 2018; 39:1835-1847). In this assay, H9c2 cardiomyocytes are proliferative when culture media is rich in serum (e.g., cultured in H9c2 Complete Media), but cease to proliferate and lose viability when they are deprived of serum (e.g., cultured in H9c2 Poor Media). The capacity of sEV and MV preparations to promote H9c2 cardiomyocyte viability was determined by supplementing the H9c2 Poor Media with increasing concentrations of sEV and MV control preparations. FIG. 11 depicts that the sEV preparations, but not the control MV preparation, improved H9c2 cardiomyocyte viability in the absence of serum, indicating the functionality of the sEV preparation.For the cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes, iCell Cardiomyocytes2(Fujifilm Cellular Dynamics, Inc., ref: CMC-100-012-001) were plated at 50,000 cells / well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001), and cultured for 4 hours. The media was then exchanged for iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., ref: M1003), and cells were cultured for up to 7 days, with full media exchanges every 2-3 days. After a minimum of 4 days, cells were exposed to iCMM with NucSpot Live 650 dye(Biotium, ref: 40082) (this served as a viable cell control); or to iCMM with NucSpot Live 650 dye, and staurosporine (Abeam, ref: abl46588) at a final in-well concentration of 2 μM (this also served as an apoptotic cell control). Dye, PBS, and DMSO concentrations, and final well volumes, were equivalent in all wells. Cells were cultured in these pre-incubation media for four hours. After this incubation, the pre-incubation media was removed, and the wells were rinsed with iCMM. Cells were then fed with iCMM with NucSpot Live 650 dye and PBS, or iCMM with NucSpot Live 650 dye supplemented with increasing concentrations of sEV or MV control preparations while maintaining PBS final volumes. Wells were imaged in an Incucytc every hour for 24 hours, and nuclei counts were determined. FIG. 12 depicts that the sEV preparations, but not the control MV preparation, improved cardiomyocyte survival, indicating the functionality of the sEV preparation. The results depicted in FIG. 12 are detailed in TABLE 2.TABLE 2. 18-hour time point data points as depicted in FIG. 12Example 5Exemplary Good Manufacturing Practices (GMP)-Compatiblc Process for Producing Small Extracellular Vesicle-Enriched Fraction (sEV) Formulations of CTC1-EVA first exemplary GMP-compatible process for producing sEV-containing formulations was developed. The production process included four main stages: vesiculation; conditioned media clarification; enrichment and concentration of small EV-enriched secretome; andproduction of the final sEV formulation. Flow diagrams outlining the GMP-compatible process that was performed are depicted in FIGS. 13 and 14.VesiculationFor the vesiculation step, cardiovascular progenitor cells (CPCs) that had been cryopreserved and stored under vapor-phase liquid nitrogen (or within a -150°C freezer) were initially thawed for two minutes at 37°C in a thawing medium (MEM alpha (MEM α , GlutaMAX™ Supplement, no nucleosides; Gibco / Life Technologies; ref: 32561-029); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL; Ydralbum®(LFB), at a final concentration of 20 mg / mL; B-27™ Supplement (50x, Life Tech Ref: 17504001 at a final concentration of 1x); and Rock Inhibitor H1152 (Sigma Ref: 555550, at a final concentration of 0.392 μg / mL), within an EVA bag (Coming). 18 mL of thawing medium was used per 1 mL of CPCs.After thawing, CPCs were seeded onto vitronectin (Life Tech Ref: VTN-N; recombinant human protein, truncated (Ref: A31804); 5 μg / mL, sterilized using a 0.22 μm filter (syringe filter 0.2 μm polyethersulfone (PES) membrane) coated culture flasks (8 x 10ST CellStack Culture Chambers, tissue culture (TC)-treated (Corning Ref: 3271); as well as 2 x TC-treated, vitronectin- coated T75 flasks), at a seeding density of about 100,000 cells per cm2, using 0.2 mL / cm2of complete medium (MEM α, GlutaMAX™ Supplement, no nucleosides; Gibco / Life Technologies; ref: 32561-029; glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL; Ydralbum®(LFB; 200 g / L); B-27™ Supplement (50x, Life Tech Ref: 17504001 or 17504044, at a final concentration of 1x); Gentamicin (Panpharma, at a final concentration of 25 μg / mL); and Human FGF-2 Premium grade (Miltenyi Biotec ref: A12873-01, at a final concentration of 1 μg / mL)). Seeding was performed without prior centrifugation of the cell suspension. The seeded CPCs were then cultured in complete medium for three days at 37°C, in the presence of 5% CO2and atmospheric oxygen.Immediately prior to seeding (“D+0”), cells were analyzed to determine the number and percentage of viable cells (see TABLE 5, column 2 , *1 (Test 20)) using a NucleoCounter NC- 200 (Chemometec) with DAPI / AO staining (Ph. Eur. 2.7.29); to determine their identity (see FIG. 15 and Example 7) by flow cytometry using a MACSQuant 10 Flow Cytometer; and to analyze their transcriptome (see FIG. 16 and Example 8).After the 3 -day culturing (“D+3”), the cells from one of the cultured T75 flasks were harvested. These harvested cells were analyzed to determine the number and percentage of viable cells (see TABLE 5, column 3, *2 (Test 20)) using a NucleoCounter NC-200 (Chemometec) with DAPI / AO staining (Ph. Eur. 2.7.29); to determine their identity (see FIG. 15 and Example 7) by flow cytometry using a MACSQuant 10 Flow Cytometer; and to analyze their transcriptome (see FIG. 16 and Example 8). Spent media from the 10ST CellStack Culture Chambers was also tested for sterility, and for the presence of mycoplasma and endotoxin.For the remaining flasks (8 x 10ST CellStack Culture Chambers; and 1 x T75), the cells were visualized by microscopy to determine their morphology (see FIG. 18), and washed twice with a wash medium (MEM alpha (Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL), before being cultured for 2 days at 37°C, in the presence of 5% CO2, in a starvation media (poor media) (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL). After this 2-day incubation (“D+5”), the culture media (conditioned media) was collected, and the cells from the 10ST CellStack Culture Chambers and the remaining T75 flask were harvested.As with the cells at D+3, the cells at D+5 were again visualized by microscopy to determine their morphology (see FIG. 18); and the cells harvested at D+5 were further analyzed to determine the number and percentage of viable cells (see TABLE 5, column 4, *3 (Test 20)); to determine their identity (see FIG. 15 and Example 7) by flow cytometry using a MACSQuant 10 Flow Cytometer; and to analyze their transcriptome (see FIG. 16 and Example 8). The collected conditioned media was tested for sterility, and for the presence of mycoplasma and endotoxin, before further processing.Conditioned Media ClarificationClarification of the conditioned media was conducted via a series of four filtration steps. First, filtration was performed using a 200 μm drip chamber filter (Gravity Blood Set, BD careFusion Ref: VH-22-EGA). The resulting filtrate was then filtered with an infuser, using a 15 μm filter (DIDACTIC, Ref: PER1FL25). The resulting filtrate was then filtered using Sartoguard PES XLG MidiCaps (Pore sizes (prefilter + filter): 1.2 μm + 0.2 μm, size 7 (0.065 m2); Sartorius Ref: 5475307F7— OO— A). Next, the resulting filtrate was further filtered using a Vacuum Filter / Storage Bottle System (0.22 μm, Pore 33.2cm2, PES Membrane; Coming Ref: 431097).Enrichment and ConcentrationFollowing clarification of the conditioned media, the conditioned media was subjected to enrichment and concentration of the small EV secretome.First, the clarified conditioned media was subjected to Tangential Flow Filtration (TFF), using a TFF Allegro™ CM150 (PALL / Sartorius). For the TFF manifold, a sterile single-use Flow Path Manual Valve P&F (PALL / Sartorius, reference: 744-69N) was used, together with a 5 L Retentate Assembly (sterile, single use; PALL / Sartorius Ref: 744-69L). For the TFF cassette, sterile single-use regenerated cellulose filters (30 kDa cut-off; 0.14 m2; Sartorius Ref: Opta filter assembly + 3D51445901MFFSG) were used. For recovery of the retentate (i.e., what is retained in the TFF), a Bench Top TFF IL Bag was used (PALL / Sartorius, reference: 7442-0303P).Initially, the TFF device was washed with 10L of H2O, and 1 L of 1 x PBS (filter sterilized using a 0.2 μm filter) before operation. Next, after administration of the clarified conditioned media to the TFF device, the retentate was concentrated (to 500 mL; not exceeding 3 bars of pressure). After this initial concentration step, the retentate was subjected to diafiltration (6 diafiltration volumes; using 1 x DPBS, filter sterilized using a 0.2 μM filter). After diafiltration, the retentate was further concentrated, to produce a total volume of at least 100 mL. The parameters of the TFF process were as follows: feed manifold pressure (PT01) - 0.86-2.1 bars; retentate manifold pressure (PT02) - 0.11-0.14 bars; retentate manifold flow rate (FT01) - 0.03- 0.32 L / min; transmembrane pressure (TMP01) - 0.4-1.1 bars; and quattroflow pump (P01) - 18- 23%.Example 6 Formulation / CompositionAfter enrichment and concentration by TFF, retentate was processed as depicted in FIG. 14. Briefly, retentate alone, retentate including 25 mM trehalose, and retentate including 5 g / L L- histidine, were each stored in glass vials (2 mL, bromobutyl cap; Adelphi Ref: VCDIN2RDLS1) and stored at -80°C. Quality control testing was performed on these samples (the different stages at which quality control testing was undertaken are indicated with a "*," e.g., *6, *7, etc.). Additionally, final sEV formulations were also prepared by filter sterilizing retentate (with or without 25 mM trehalose) using a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm,Millipore, Ref: SVGPL10RC). After the sterilization step, the final formulations (with or without the addition of 25 mM trehalose) were bottled into glass vials (2 mL, bromobutyl cap; Adelphi Ref: VCDIN2RDLS1). In addition, any pharmaceutically suitable carrier may be utilized. Final formulations were stored at -80°C for future use or testing.The final formulations, therefore, were in PBS (with or without trehalose), and were positive for CD9, CD63 and CD81 (canonical EV markers), as well as positive for the cardiac- related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29 and CD142, as detected by MACSPlex (as shown in FIGS. 21, 23, 25 and 26).Example 7Characterization of the Identity of CPCs During Vesiculation in the GMP-Compatible ProcessTo assess the identity of the cells during the vesiculation process in Example 5, the D+0 CPCs, as well as the harvested cells at D+3 and D+5, were analyzed by flow cytometry. iPSCs and cardiomyocyte (CM) cells were included as controls. As shown in FIG. 15, flow cytometry analysis, performed using a MACSQuant 10 Flow Cytometer with iPSC-, CPC- and cardiacmarkers, demonstrated that the CPCs became more mature over the five-day vesiculation period. Specifically, the CPCs maintained little to no NANOG or SOX2 protein expression, and exhibited a continued increase in CD56, cTNT, and aMHC, protein expression (however, they did not reach expression levels of CD56, cTNT, and aMHC similar to cardiomyocytes, indicating that they remained progenitors throughout the process). iPSC and CM control cells were analyzed separately, and the average values are presented in FIG. 15 for comparative purposes.Example 8Transcriptome Analysis of CPCs During Vesiculation in the GMP-Compatible ProcessTo assess the transcriptome of the cells during the vesiculation process in Example 5, RNA was extracted from the CPCs at D+0, and from the harvested cells at D+3 and D+5 of the vesiculation process. RNA was also extracted from iPSCs (pluripotent cell controls), and from iPSC-derived cardiomyocytes (differentiated cardiomyocyte controls). Total RNA was sequenced on the Illumina NovaSeq 6000 platform, and differential gene expression was determined on normalized data.The heatmaps depicted in FIG. 16 and FIG. 17 were generated based on hierarchical clustering analysis using the UPGMA clustering method, with correlation distance metric in TIBCO Spotfire software v11.2.0. The genes included in the panel are expressed at different stages of differentiation (from iPSC through to beating cardiomyocytes), as well as related off-target cells. The gene expression analysis results depicted in FIG. 16 and FIG. 17 thus confirmed that the cells retained the characteristics of cardiovascular progenitors throughout the vesiculation process. The data used to generate the heatmaps for FIG. 16 and FIG. 17 are presented in TABLE 3.TABLE 3. Gene names and logzFPKM values corresponding to the data presented in the heatmap depicted in FIG. 16 and FIG. 17.Example 9Analysis of EV Particle Concentration and EV Particle Size Distribution in the GMP-CompatibleProcessTo assess the particle concentration and size distribution of EVs produced in Example 5, the clarified conditioned media (before TFF), and the final formulations (with and without trehalose), were analyzed by nanoparticle tracking analysis (NTA; NanoSight). FIG. 19 depicts representative size distribution curves for each sample. The overall size distributions, means and modes, were similar between samples. A peak was observed generally between about 50-150 nm, corresponding to the size of exosomes or small microparticles. The TFF step resulted in an approximately 32-fold concentration of particles. Similar experiments were also conducted on the stored retentate samples depicted in FIG. 14 (with and without trehalose or histidine) which were not filter sterilized (“*6,” samples a-c). The results of these experiments are shown in FIG. 20.Example 10Analysis of EV Markers in CTC1-EV produced by the GMP-Compatible ProcessTo assess the presence of EV markers in the clarified conditioned media (before TFF) and the final formulations (with and without trehalose) in Example 5, a MACSPlex Exosome Kit human (Miltenyi Ref: 130-108-813) was used to identify and quantify the presence of EV markers. As shown in FIG. 21, the analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81 and CD63) in both the conditioned media (before TFF), and in the final formulation (with and without trehalose). Further still, as shown in FIG. 22, the MACSPlex analysis also revealed a variety of markers that were found to be present either in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CDlc, CD25, CD40, CDl lc, CD86, CD31 and CD20); or were substantially absent (CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69), in the conditioned media (before TFF), and / or in the final formulation (with and without trehalose). Similar experiments were also conducted on the stored retentate samples depicted in FIG. 14 (with and without trehalose or histidine) which were not filter sterilized (“*6,” samples a-c, Test 20). The results of these experiments are shown in FIGS. 23 and 24.Additionally, as shown by FIG. 25, additional cardiac-related markers were also observed in the conditioned media (before TFF), and in the final formulation (with and without trehalose). Similar experiments were also conducted to confirm the presence of these additional cardiac-related markers in the stored retentate samples depicted in FIG. 14 (with and without trehalose or histidine) which were not filter sterilized (“*6,” samples a-c, Test 20). The results of these experiments are shown in FIG. 26.Example 11In vitro analysis of the Potency of CTC1-EV Produced by the GMP-Compatible ProcessTo analyze the functionality and potency of the final formulations produced by the GMP- compatiblc process in Example 5, two in vitro assays were used: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed by Essen BioSciences, for the Incucyte) was employed, according to the manufacturer’s directions. Briefly, HUVEC cells were expanded using HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C-22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210). After expansion, the cells were cryopreserved in CS10 (Cryostore, ref: 210102) at 1-2 x 106cells per aliquot (enough for between a half to a full 96-well plate). Two days prior to assay, HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells / well, and grown in HUVEC Complete media for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. Wells were scratched using a Wound Maker (EssenBio, Ref: 4493) according to the manufacturer’s directions, and cells were then rinsed with Endothelial Cell Basal Media and cultured overnight (either in HUVEC Complete Media and PBS, as a positive control; in Endothelial Cell Basal Media and PBS, as a negative control; or in Endothelial Cell Basal Media supplemented with sEV preparations in PBS). Using an Incucyte with the Scratch Wound Healing Module, plates were imaged at 21 hours after treatment. Wound closure was determined using the manufacturer’s software, and values were baseline (negative control) subtracted, and normalized to the positive control. FIG. 27 depicts that the final formulations with and without trehalose (sample b and a, respectively) promoted wound healing.For the cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes, iCell Cardiomyocytes2(Fujifilm Cellular Dynamics, Inc., ref: CMC- 100-012-001) were plated at 50,000 cells / well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001), and cultured for 4 hours. The media was thenexchanged for iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., ref: Ml 003), and cells were cultured for up to 7 days, with full media exchanges every 2-3 days. After a minimum of 4 days, cells were exposed to iCMM with NucSpot Live 650 dye (Biotium, ref: 40082) (this served as a viable cell control); or to iCMM with NucSpot Live 650 dye, and staurosporine (Abeam, ref: abl46588) at a final in-well concentration of 2 μM (this also served as an apoptotic cell control). Dye, PBS, and DMSO concentrations, and final well volumes, were equivalent in all wells. Cells were cultured in these pre-incubation media for four hours. After this incubation, the pre-incubation media was removed, and the wells were rinsed with iCMM. Cells were then fed with iCMM with NucSpot Live 650 dye and PBS, or iCMM with NucSpot Live 650 dye supplemented with increasing concentrations of sEV preparations (sample a and b) while maintaining PBS final volumes. Wells were imaged in an Incucyte at 24 hours, and nuclei counts were determined. FIG. 28 depicts that the final formulations with and without trehalose promoted cardiomyocyte survival.The testing panel used with respect to the processes / products of Example 5, and as embodied, e.g., in Examples 6-11, is shown in TABLE 4. The results therefore are shown in TABLE 5. Additionally, TABLE 6 depicts the degree of enrichment, as compared to conditioned media after clarification, for the retentates and final formulations produced in Example 6.TABLE 4. The secretome / extracellular vesicle process / product testing panel relating to Examples 5-17.TABLE 5. The results for certain criteria shown in the testing panel in TABLE 49, with respect to Examples 5-11.TABLE 6. The degree of enrichment (as calculated by the increase of particles per unit protein), as compared to conditioned media after clarification, for the retentates and final formulations produced in Example 6.Example 12Second Exemplary Good Manufacturing Practices (GMP)-Compatible Process for ProducingSmall Extracellular Vesicle-Enriched Fraction (sEV) Formulations of CTC1-EVA second exemplary GMP-compatible process for producing sEV-containing formulations was developed. The production process included four main stages: vesiculation; conditioned media clarification; enrichment and concentration of small EV-enriched secretome; and production of the final sEV formulation. Flow diagrams outlining the GMP-compatible process that was performed are depicted in FIGS. 29 and 30.VesiculationFor the vesiculation step, cardiovascular progenitor cells (CPCs) that had been cryopreserved and stored under vapor-phase liquid nitrogen (or within a -150°C freezer) were initially thawed for 2.5 minutes at 37°C in a thawing medium (MEM alpha (1000 mL ofMacopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL; Ydralbum®(LFB), at a final concentration of 20mg / mL; B-27™ Supplement (50x, Life Tech Ref: 17504001 at a final concentration of 1x); and Rock Inhibitor Hl 152 (Sigma Ref: 555550, at a final concentration of 0.392 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter), within an EVA bag (Coming). 18 mL of thawing medium was used per 1 mL of CPCs.After thawing, CPCs were seeded onto vitronectin (Life Tech Ref: VTN-N; recombinant human protein, truncated (Ref: A31804); 5 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter) coated culture flasks (12 x 10ST CellStack Culture Chambers, tissue culture (TC)-treated (Coming Ref: 3271); as well as 2 x TC-treated, vitronectin-coated T75 flasks), at a seeding density of about 100,000 cells per cm2, using 0.2 mL / cm2of complete medium (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL; Ydralbum®(LFB; 200 g / L); B- 27™ Supplement (50x, Life Tech Ref: 17504001 or 17504044, at a final concentration of 1x); Gentamicin (Panpharma, at a final concentration of 25 μg / mL); and Human FGF-2 Premium grade (Miltenyi Biotec ref: A12873-01, at a final concentration of 1 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter)). Seeding was performed without prior centrifugation of the cell suspension. The seeded CPCs were then cultured in complete medium for three days at 37°C, in the presence of 5% CO2and atmospheric oxygen.Immediately prior to seeding (“D+0”), cells were analyzed to determine the number and percentage of viable cells (see TABLE 7, column 1 (“D+0 cells") using a NucleoCounter NC-200 (Chemometec) with DAPI / AO staining (Ph. Eur. 2.7.29); to determine their identity (see FIG. 31 and Example 14) by flow cytometry using a MACSQuant 10 Flow Cytometer.After the 3 -day culturing (“D+3”), the cells from one of the cultured T75 flasks were harvested. These harvested cells were analyzed to determine the number and percentage of viable cells (see TABLE 7, column 2 (“D+3 material”) using a NucleoCounter NC-200 (Chemometec) with DAPI / AO staining (Ph. Eur. 2.7.29); and to determine their identity (see FIG. 31 and Example 14) by flow cytometry using a MACSQuant 10 Flow Cytometer. Spent media from the 10ST CellStack Culture Chambers was also tested for sterility, and for the presence of mycoplasma and endotoxin.For the remaining flasks (12 x 10ST CellStack Culture Chambers; and 1 x T75), the cells were visualized by microscopy to determine their morphology (see FIG. 32), and washed twice with a wash medium (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%)supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL), before being cultured for 2 days at 37°C, in the presence of 5% CO2and atmospheric oxygen, in a starvation media (poor media) (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg / mL). After this 2-day incubation (“D+5”), the culture media (conditioned media) was collected, and the cells from the 10ST CellStack Culture Chambers and the remaining T75 flask were harvested.As with the cells at D+3, the cells at D+5 were again visualized by microscopy to determine their morphology (see FIG. 32); and the cells harvested at D+5 were further analyzed to determine the number and percentage of viable cells (see TABLE 7, column 3 (“D+5 cells”); and to determine their identity (see FIG. 31 and Example 14) by flow cytometry using a MACSQuant 10 Flow Cytometer. The collected conditioned media was tested for sterility, and for the presence of mycoplasma and endotoxin, before further processing.TABLE 7. The results for certain criteria shown in the testing panel in TABLE 49, with respect to Examples 12-17.Conditioned Media ClarificationClarification of the conditioned media was conducted via a series of three filtration steps. First, filtration was performed using a Sartopure PP3 MidiCaps 5 μm PES filter (Sartorius, Ref: 5055342P9— OO— A (Sartorius)). The resulting filtrate was then filtered using a Sartoguard PES MidiCaps filter (Pore sizes (prefilter + filter): 1.2 μm + 0.2 μm; Sartorius Ref: 5475307F9— OO— The resulting filtrate was then filtered using a Sartopure 2 MidiCaps filter (Pore sizes (prefilter + filter): 0.45 μm + 0.2 μm; Sartorius Ref: 5445307H8— OO— A).Enrichment and ConcentrationFollowing clarification of the conditioned media, the conditioned media was subjected to enrichment and concentration of the small EV secretome.First, the clarified conditioned media was subjected to Tangential Flow Filtration (TFF), using a TFF Allegro™ CM150 (PALL / Sartorius). For the TFF manifold, a sterile single-use Flow Path Manual Valve P&F (PALL / Sartorius, reference: 744-69N) was used, together with a 10 L Retentate Assembly (sterile, single use; PALL / Sartorius Ref: 744-69M). For the TFF cassette, sterile single-use regenerated cellulose filters (30 kDa cut-off; 0.14 m2; Sartorius Ref: Opta filter assembly + 3D51445901MFFSG) were used. For recovery of the retentate (i.e., what is retained in the TFF), a Bench Top TFF IL Bag was used (PALL / Sartorius, reference: 7442-0303P).Initially, the TFF device was washed with 10L of H2O, and 2 L of 1 x PBS before operation. Next, after administration of the clarified conditioned media to the TFF device, the retentate was concentrated (to 500 mL; not exceeding 3 bars of pressure). After this initial concentration step, the retentate was subjected to diafiltration (6 diafiltration volumes; using 1 x DPBS). After diafiltration, the retentate was further concentrated, to produce a total volume of at least 100 mL. The parameters of the TFF process were as follows: feed manifold pressure (PT01) - 0.94-2.1 bars; retentate manifold pressure (PT02) - 0.12-0.13 bars; retentate manifold flow rate (FT01) - 0.012- 0.58 L / min; transmembrane pressure (TMP01) - 0.53-1.11 bars; and quattroflow pump (P01) - 14-20%.Example 13 Formulation / CompositionAfter enrichment and concentration by TFF, the final sEV formulation was then prepared by filter sterilizing the resulting retentate using a 0.22 μm filter (Sterivex™-GP Pressure FilterUnit, 0.22 μm, Millipore, Ref: SVGPL10RC). In some experiments, 25 mM trehalose was added before this sterilization step to avoid aggregation. After the sterilization step, the final formulation (with or without the addition of 25 mM trehalose) was bottled into glass vials (2 mL, bromobutyl cap; Adelphi Ref: VCDIN2RDLS1). Final product formulation was then stored at -80°C for future use or testing. Additionally, final formulations were also tested in which the retentate was first frozen and stored at -80°C before sterilizing filtration using either a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, Ref: SVGPL10RC), or a Sartopure 2 filter (Pore sizes (prefilter + filter): 0.45 μm + 0.2 μm; Sartorius Ref: 5441307H4— OO--B) to produce final formulations thereof, as shown in FIG. 30.The final formulations, therefore, were in PBS (with or without trehalose), and were positive for CD9, CD63 and CD81 (canonical EV markers), as well as positive for the cardiac- related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29 and CD142, as detected by MACSPlex (as shown in FIGS. 35 and 37).Example 14Characterization of the Identity of CPCs During Vesiculation in the GMP-Compatible ProcessTo assess the identity of the cells during the vesiculation process in Example 12, the D+0 CPCs, as well as the harvested cells at D+3 and D+5, were analyzed by flow cytometry. iPSCs and cardiomyocyte (CM) cells were included as controls. As shown in FIG. 31, flow cytometry analysis, performed using a MACSQuant 10 Flow Cytometer with iPSC-, CPC- and cardiacmarkers, demonstrated that the CPCs became more mature over the five-day vesiculation period. Specifically, the CPCs maintained little to no Nanog or SOX2 protein expression, and exhibited a continued increase in CD56, cTNT, and aMHC, protein expression (however, they did not reach expression levels of CD56, cTNT, and aMHC similar to cardiomyocytes, indicating that they remained progenitors throughout the process). iPSC and CM control cells were analyzed separately, and the average values are presented in FIG. 31 for comparative purposes.Example 15Analysis of EV Particle Concentration and EV Particle Size Distribution in CTC1-EV in theGMP-Compatible ProcessTo assess the particle concentration and size distribution of EVs produced in Example 12and Example 13, conditioned media prior to clarification (*4 (Test 22)) and after clarification (*5 (Test 22)), and the final formulations (with and without trehalose, samples b and a, respectively), were analyzed by nanoparticle tracking analysis (NTA; NanoSight). FIG. 33 depicts representative size distribution curves for each sample. The overall size distributions, means and modes, were similar between samples. A peak was observed generally between 50-150 nm, corresponding to the size of exosomes or small microparticles. The TFF step resulted in an approximately 32-fold concentration of particles. Similar experiments were also conducted on the previously-frozen retentate and final formulation samples (filtered with STcrivcx-GP or Sartoporc 2) depicted in FIG. 30 (*6, sample a (Test 22); *7, sample c (Test 22); and *7, sample d (Test 22)). The results of these experiments are shown in FIG. 34. The TFF step resulted in an approximately 20-fold concentration of particles, even though particles were lost during final sterilizing filtration (especially for the final formulations produced from thawed retentate).Example 16Analysis of EV Markers in CTC1-EV Final Formulation produced by the GMP-CompatibleProcessTo assess the presence of EV markers in the clarified conditioned media (before TFF) and the final formulations (with and without trehalose) in Example 12, a MACSPlex Exosome Kit human (Miltenyi Ref: 130-108-813) was used to identify and quantify the presence of EV markers. As shown in FIG. 35, the analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81 and CD63) in both the conditioned media (before TFF), and in the final formulation (with and without trehalose). Further still, as shown in FIG. 36, the MACSPlex analysis also revealed a variety of markers that were found to be present either in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CDlc, CD25, CD40, CDllc, CD86, CD31 and CD20); or were substantially absent (CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69), in the conditioned media (before TFF), and / or in the final formulation (with and without trehalose).Additionally, as shown by FIG. 37, additional cardiac-related markers were also observed in the conditioned media (before TFF), and in the final formulation (with and without trehalose).Example 17In vitro analysis of the Potency of CTC1-EV Final Formulation Produced by the GMP-Compatible ProcessTo analyze the functionality and potency of the final formulations produced by the GMP- compatible process in Example 12, two in vitro assays were used: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed by Essen BioScicnccs, for the Incucytc) was employed, according to the manufacturer’s directions. Briefly, HUVEC cells were expanded using HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C-22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210). After expansion, the cells were cryopreserved in CS10 (Cryostore, ref: 210102) at 1-2 x 106cells per aliquot (enough for between a half to a full 96-well plate). Two days prior to assay, HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells / well, and grown in HUVEC Complete media for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. Wells were scratched using a Wound Maker (EssenBio, Ref: 4493) according to the manufacturer’s directions, and cells were then rinsed with Endothelial Cell Basal Media and cultured overnight (either in HUVEC Complete Media with PBS, as a positive control; in Endothelial Cell Basal Media with PBS, as a negative control; or in Endothelial Cell Basal Media supplemented with sEV preparations in PBS). Using an Incucyte with the Scratch Wound Healing Module, plates were imaged at 18 hours after treatment. Wound closure was determined using the manufacturer’s software, and values were baseline (negative control) subtracted, and normalized to the positive control. FIG. 38 depicts that the final formulations with and without trehalose (*7, samples b and a, (Test 22) respectively) promoted wound healing. FIG. 39 depicts that the previously-frozen final formulations without trehalose (*7, samples c and d, (Test 22)) promoted wound healing.For the cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes, iCell Cardiomyocytes2(Fujifilm Cellular Dynamics, Inc., ref: CMC- 100-012-001) were plated at 50,000 cells / well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001), and cultured for 4 hours. The media was then exchanged for iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics,Inc., ref: Ml 003), and cells were cultured for up to 7 days, with full media exchanges every 2-3 days. After a minimum of 4 days, cells were exposed to iCMM with NucSpot Live 650 dye (Biotium, ref: 40082) (this served as a viable cell control); or to iCMM with NucSpot Live 650 dye, and staurosporine (Abeam, ref: abl46588) at a final in-well concentration of 2 μM (this also served as an apoptotic cell control). Dye, PBS, and DMSO concentrations, and final well volumes, were equivalent in all wells. Cells were cultured in these pre-incubation media for four hours. After this incubation, the pre-incubation media was removed, and the wells were rinsed with iCMM. Cells were then fed with iCMM with NucSpot Live 650 dye and PBS, or iCMM with NucSpot Live 650 dye supplemented with increasing concentrations of sEV preparations while maintaining PBS final volumes. Wells were imaged in an Incucyte at 24 hours, and nuclei counts were determined. FIG. 40 depicts that the final formulations with and without trehalose (*7, samples b and a, respectively, (Test 22)) promoted cardiomyocyte survival. FIG. 41 depicts that the previously-frozen final formulations without trehalose (*7, samples c and d, (Test 22)) promoted cardiomyocyte survival.The testing panel used with respect to the processes / products of Example 12, and as embodied, e.g., in Examples 13-17, is shown in TABLE 4. The results therefore are shown in TABLE 7. Additionally, TABLE 8 depicts the degree of enrichment (as calculated by the increase of particles per unit protein), as compared to conditioned media after clarification, for the retentates and final formulations produced in Example 12.TABLE 8. The degree of enrichment (as calculated by the increase in particles per unit protein), as compared to conditioned media after clarification, for the retentates, and final formulations produced in Example 12 and Example 13.Example 18 nalysis of the Effect of Cardiovascular Progenitor Cell (CPC) EVs on Cardiac Function in a Mouse Heart Failure ModelTo analyze the in vivo functionality and potency of sEV preparations produced in accordance with methods described in the present disclosure, a mouse model was used to determine the effect of sEV preparations on cardiac function (in mice in which heart failure had been induced).Heart failure was induced in C57BL / 6 mice essentially as described in Kervadec et al. (J. Heart Lung Transplant, 2016, 35(6): 795-807; incorporated by reference herein in its entirety). Briefly, surgical occlusion of the left coronary artery was performed in 42 mice in total, to induce chronic heart failure (CHF). At three weeks post-occlusion, 22 of the mice were treated with either PBS vehicle control (60 μL, n=l l) or sEV (60 μL, n=l l), delivered by percutaneous injections under echocardiographic guidance into the peri-infarct myocardium (as described in Kervadec et al.). The administered sEV was produced in accordance with the “sEV 5.3” scheme depicted in FIGS. 2 and 3 (whereby the sEV was prepared by ultracentrifugation from clarified “MC5”), and the resulting EV were resuspended in half the typical PBS volume (to generate a 2-fold concentrated sEV preparation, containing the secretome from 6.22E+04 cells per μL of sEV preparation).At four weeks post-occlusion, cardiac function was assessed by echocardiography. The results thereof are shown in FIG. 42. Amongst the CHF mice, significantly fewer sEV-treated mice (as compared to the PBS-treated mice) had severely progressive LVESV hypertrophy (defined here as an equal to or greater than 9. luL increase in Left Ventricular End Systolic Volume, LVESV; p<0.05). Amongst the CHF mice, significantly fewer sEV-treated mice (as compared to the PBS-treated mice) had severely progressive LVEDV hypertrophy (defined here as equal to orgreater than a 4 uL increase in Left Ventricular End Diastolic Volume, LVEDV; p<0.05). And although not significant, there was a strong trend that fewer sEV-treated CHF mice (as compared to the PBS-treated mice) had severely progressive loss of Ejection Fraction, EF, (as defined here as a loss of 5.5% or more in EF; p<0.056). Further, although not statistically significant, the Average Ejection Fraction of the PBS group deteriorated 2.5-fold more than the sEV-treated group (-4% vs -1.6%, respectively; ns). The results confirmed the ability of the sEV preparation to improve cardiac function in vivo.Example 19Production of Small Extracellular Vesicle-Enriched Fraction (sEV) Clinical Candidate (CTC1-EV) Formulations; CTC1-EV Final FormulationThree CPC sEV clinical candidate formulations (Test Examples 25, 26 and 27 herein) were generated for further analysis. Test Examples 25 and 26 were generated from CPCs essentially as described in Example 12 herein, however the TFF used a total volume of 15L; a TFF cassette having a 0.28 m2size filter (30 KDa cut-off; 0.28 m2; Sartorius ref: Opta filter assembly + SFM- OP- 1445921) was used; and a TFF feed pressure of 3.5 bars was employed.FIG. 105 illustrates the process used to generate Test Example 25. In this example, FCDI CTC1 cardiovascular progenitor cells were thawed and plated onto vitronectin coated flasks. This lot of cells is referred to as “Clin001”. After thawing and prior to plating, a sample of cells was taken (“*1 (Test 25)”). Twelve 10-layer cell stacks (CS10) and two t-75 flasks were seeded with FCDI CTC 1 cells at a density of 100 thousand cells per square centimeter in complete media. They were cultured in a humidified incubator for three days at 37 degrees Celsius, at 5% CO2. After three days of expansion, cells from one of the T-75 flasks were harvested for in-process characterization. This sample is called “*2 (Test 25)”. After three days of expansion, the spent media was removed from all of the remaining vessels, and “Poor Media” was added back to the vessel (“change to poor media”) on the third day, post plating (“D+3”). The cells were maintained in culture in this poor media for two more days, at 37 degrees Celsius, in a humidified incubator with 5% CO2. This is what is referred to as the vesiculation media. Five days after plating the cells, the spent media was collected from the twelve CS10 flasks. This media is referred to as “*4 (Test 25)”. An aliquot of this material was used for in-process testing. Five days after plating the cells, cells from one of the T-75 flasks were harvested for-in process testing. Cells from three of thetwelve 10-layer cell stacks were harvested for in-process testing. Cells harvested at this stage of the process (five days after plating, Day + 5, “D+5") are collectively referred to as “*3 (Test 25)". The *4 (Test 25) material then underwent clarification.To clarify the spent media, the media was filtered three times using first the Sartopure®PP3 filter with a filter size of 5 μm; next with a Sartoguard PES filter with 0.2 um nominal filter size, and lastly with a Sartopore®2 filter with a filter size of “(0.45 + 0.2 um)”. The resulting filtrate after these three filtrations is the “Conditioned media after clarification" or “Conditioned Media" for short. Fifteen liters of Conditioned Media were produced in this Test Example 25. This Conditioned Media is referred to as “*5 (Test 25)". An aliquot of this material was used for in- process testing. An aliquot of 30 mL of this material was submitted to ultracentrifugation to produce an EV-enriched secretome for additional testing. This material is referred to as “*5a.uc (Test 25)”.The Conditioned media after clarification was then processed by tangential flow filtration (“TFF”) using regenerated cellulose filters with a 30 kilodalton cut-off. The filter used for Test Example 25 was 0.28 square meters in surface area. The TFF process concentrated the retentate first and then six volumes of DPBS was used to perform a diafiltration of the retentate. The diafiltered retentate was then again concentrated. Aliquots of the resulting retentate (referred to as “*6 (Test 25)") was analyzed by in-process controls. For Test Example 25, the final retentate was concentrated 5 Ox. The remaining Retentate was stored in a IL bag over night at four degrees Celsius, and then placed in a minus 80 degree freezer until needed for Test Example 27.FIG. Ill illustrates the process used to generate Test Example 26. In this example, FCDI CTC1 cardiovascular progenitor cells were thawed and plated onto vitronectin coated flasks. This lot of cells is referred to as “Clin002”. After thawing and prior to plating, a sample of cells was taken (“*l(Test 26)”). Twelve 10-layer cell stacks (CS10) and two t-75 flasks were seeded with FCDI CTC1 cells at a density of 100 thousand cells per square centimeter in complete media. They were cultured in a humidified incubator for three days at 37 degrees Celsius, at 5% CO2. After three days of expansion, cells from one of the T-75 flasks were harvested for in-process characterization. This sample is called “*2(Test 26)". After three days of expansion the spent media was removed from all of the remaining vessels, and “Poor Media" was added back to the vessel (“change to poor media) on the third day, post plating (“D+3”). The cells were maintained in culture in this poor media for two more days, at 37 degrees Celsius, in a humidified incubatorwith 5% CO2. This is what is referred to as the vesiculation media. Five days after plating the cells, the spent media was collected from the twelve CS10 flasks. This media is referred to as “*4 (Test 26)”. An aliquot of this material was used for in-process testing. Five days after plating the cells, cells from one of the T-75 flasks were harvested for in-process testing. Cells from three of the twelve 10-layer cell stacks were harvested for in-process testing. Cells harvested at this stage of the process (five days after plating, Day + 5, “D+5”) are collectively referred to as “*3(Test 26)”. The *4 (Test 26) material then underwent clarification.To clarify the spent media, the media was filtered three times using first the Sartopurc®PP3 filter with a filter size of 5 um; next with a Sartoguard PES filter with 0.2 um nominal filter size, and lastly with a Sartopore®2 filter with a filter size of “(0.45 + 0.2 um)”. The resulting filtrate after these three filtrations is the “Conditioned media after clarification” or “Conditioned Media” for short. Fifteen liters of Conditioned Media were produced in this Test Example 26. This Conditioned Media is referred to as “*5 (Test 26).” An aliquot of this material was used for in- process testing. An aliquot of 30 mL of this material was submitted to ultracentrifugation to produce an EV-enriched secretome for additional testing. This material is referred to as “*5b.uc (Test 26)”.The Conditioned media after clarification was then processed by tangential flow filtration (“TFF”) suing regenerated cellulose filters with a 30 kilodalton cut-off. The filter used for Test Example 26 was 0.28 square meters in surface area. The TFF process concentrated the retentate first and then six volumes of DPBS was used to perform a diafiltration of the retentate. The diafiltered retentate was then again concentrated. Aliquots of the resulting retentate (referred to as “*6 (Test 26)”) was analyzed by in-process controls.For Test Example 26, the final retentate was concentrated 46x;The remaining Retentate was stored in a IL bag over night at four degrees Celsius until needed for Test Example 27.FIG. 112 illustrates the process used to generate Test Example 27. To produce Test Example 27, The frozen retentate obtained at the end of Test Example 25 was thawed overnight at four degrees Celsius. This material is referred to as “Thawed Retentate Test 25." A sample of the Thawed Retentate Test Example 25 was collected for in-process testing. This material is referred to as “*7 (Test 25).” The retentate obtained at the end of the Test Example 26 was retrieved from the four-degree equipment where it had been stored overnight. This material is referred to as“Retentate fresh Test 26”. A sample of the Retentate fresh Test 26 was taken for in-process testing. This material is referred to as “*7 (Test 26).” The Thawed Retentate Test 25 and the Retentate fresh Test 26 were pooled (combined) together (referred to as “Pool”). A sample of the pool was taken for in-process testing. This material is referred to as “*8 (Test 27)”. The Pool was sterile filtered using a Sartopore® 2, Sterile Capsule (Pore size (prefilter + filter): 0.45 μm + 0.2 μm; Sartorius Ref: 5441307H4— OO--B) to produce the final formulation (without trehalose). The sterilized material was vialed into glass cryopreservation tubes and stored at -80 degrees Celsius under further use. The material in the glass cryoprcscrvation tubes is referred to as the “CTC1-EV Final Formulation”. It is also referred to as “*9 (Test 27).Example 20 Transcriptomic Analysis of CTC1-EV Final FormulationTo assess the RNA transcriptome of the CTC1-EV Final Formulation, RNA was extracted from the CPCs (during the vesiculation phase) that were used to produce Test Examples 25 and 26 at D+3 (samples *2 (Test 25), *2 (Test 26)) and D+5 (samples *3 (Test 25) and *3 (Test 26)); and from the CTC1-EV composition in the *9 (Test 27) whose preparation is describes in detail in Example 19.For the RNA extraction from CPCs (Test Examples 25 and 26), cell lysates (from 1 million cells per 450 μL RLT buffer (Qiagen, USA)) were obtained at D+3 and D+5, from the CPCs used to produce Test Examples 25 and 26. Small RNA was enriched from the CPCs using the mirVana RNA Isolation kit (Thermofisher, Ref: AM1561), according to the manufacturer’s protocol, and the resulting RNA was eluted with 100μL of Nuclease-Free Water (Teknova, Ref: W3330). 2μL of this RNA preparation was then used for assessing RNA concentration, using the Lunatic (Unchained Labs). The results of the Lunatic analysis for the cellular RNA extracted from Test Examples 25 and 26 (D+5), showing the RNA concentration, are depicted in FIG. 43. Additionally, an aliquot of each of the D+3 and D+5 RNA samples was sent to the University of Wisconsin Gene Expression Center for quality control (QC) testing and sequencing. For the QC testing, High Sensitivity RNA ScreenTape (Agilent Technologies) was used. The results of the QC testing for Test Example 25 (D+5) are depicted in FIG. 44, and the results of the QC testing for Test Example 26 (D+5) are depicted in FIG. 45, confirming the quality of the extracted cellular RNA.RNA extraction was performed on 200μL of the *9 (Test 27) using the Wako microRNA Extractor SP kit (Wako, Ref: 295-71701), according to the manufacturer’s protocol. The extracted RNA was eluted with 50μL of Nuclease-Free Water (Teknova, Ref: W3330). 2μL of this RNA preparation was then used for assessing RNA concentration, using the Lunatic (Unchained Labs). The results of the Lunatic analysis for the EV RNA extracted from *9 (Test 27), showing the RNA concentration, are depicted in FIG. 46. Additionally, an aliquot was sent to the University of Wisconsin Gene Expression Center for QC testing and sequencing. For the QC testing, a 2100 Bioanalyzer was used with the Eukaryote Total RNA Pico Assay (Agilent Technologies). The results of the QC testing for *9 (Test 27) are depicted in FIG. 47, confirming the quality of the extracted EV RNA.Additionally, small RNA libraries were prepared. The small RNA libraries were prepared using a QIAseq miRNA Library kit (Qiagen, USA), using 5μL of input RNA for each sample (namely, the extracted RNA from the cells from Test Examples 25 and 26; and from *9 (Test 27), as discussed above). To each sample, 3’ and 5’ adaptors (ata 1:5 dilution) and reverse transcriptase initiator were added, and the adapter-ligated RNA was then reverse transcribed. The resulting cDNA was purified using QMN beads (Qiagen, USA), and the cDNA libraries were then amplified for 16 cycles, and purified twice. The amplified libraries were resuspended in 19.5μL of nuclease- free water, and 17 μL was recovered. The libraries were quantified with Qubit in singlet, using a 1:100 dilution, and QC tested using an Agilent Bioanalyzer HS DN chip (Agilent Technologies, USA). The results of the QC testing for Test Examples 25 (D+5), 26 (D+5) and from EVs (*9 (Test 27)) are depicted in FIG. 48, confirming the quality of the cDNA libraries. Sequencing was performed using NovaSeq6000 on the Illumina NGS Systems.For bioinformatic analysis of the sequencing data, FastQC v0.11.9 was used to determine the quality of the raw read fastq files. Trimmomatic v0.39 was used to trim the adaptors from the raw reads. The trimmed reads were used for read mapping, and quantification was performed using miRge3.0 pipeline implemented in python with default settings (miRge 3.0 uses Bowtie vl.3.0 and SAMtools v1.7 for read mapping and quantification). FIG. 49 depicts the results of the analysis of the sequencing read lengths f...

Claims

What is claimed is:

1. A method for treating acute myocardial infarction, chemotherapy-induced cardiomyopathy, heart failure, myocarditis, ischemic cardiomyopathy, cardiomyopathy, ventricular dysfunction, atrial dysfunction, or arrhythmia in a subject in need thereof, comprising administering to the subject a therapeutic composition comprising a secretome of one or more progenitor cells.

2. A method for improving angiogenesis comprising administering to a subject in need thereof a therapeutic composition comprising a secretome of one or more progenitor cells.

3. A method for improving cardiac performance comprising administering to a subject in need thereof a therapeutic composition comprising a secretome of one or more progenitor cells.

4. The method of claim 1 , wherein the heart failure is acute heart failure, chronic heart failure, ischemic heart failure, non-ischemic heart failure, heart failure with ventricular dilation, heart failure without ventricular dilation, heart failure with reduced left ventricular ejection fraction, or heart failure with preserved left ventricular ejection fraction.

5. The method of claim 4, wherein the heart failure is selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy induced heart failure.

6. The method according to claim 1, wherein said chemotherapy-induced cardiomyopathy is caused by an anthracyclinc.

7. The method according to claim 6, wherein said anthracyclinc is doxorubicin.

8. A method of maintaining physiological heart volume in a subject by administering to the subject a therapeutic composition comprising a secretome of one or more progenitor cells.

9. The method of any one of claims 1-8, wherein said method maintains Left Ventricular End Systolic Volume (LVESV) within 15% of the pre-treatment LVESV.

10. The method of any one of claims 1-9, wherein said method maintains LVEDV within 2% of the pre-treatment volume.

11. The method of any one of claims 1-10, wherein the method prevents progressive post-ischemic heart failure.

12. The method of any one of claims 1-11, wherein the method improves endothelial cell survival, health and function in said subject.

13. The method of any one of claims 1-12, wherein said method reduces fibrosis in stimulated cardiac fibroblasts.

14. The method of claim 13, wherein said method reduces the expression of the pro- fibrotic marker, POSTN, in TGF-β1 -stimulated cardiac fibroblasts to level prior to stimulation with TGF-β1 or below.

15. The method of any one of claims 1-14, wherein the method does not induce an allogeneic inflammatory response in a subject.

16. The method of any one of claims 1-15, wherein said method does not induce an allogeneic peripheral blood mononuclear cell (PBMC) activation.

17. The method of any one of claims 1-16, wherein said method does not induce a significant increase in the percentage of IFNg or IL-2 expressing PBMCs.

18. The method of any one of claims 1-17, wherein the method does not induce allogeneic natural killer (NK) cell degranulation.

19. The method of any one of claims 1-18, wherein the method does not induce a significant increase in the percentage of CD 107 expressing NK cells.

20. A method of improving heart function in a subject experiencing heart failure by administering to the subject a therapeutic composition comprising a secretome of one or more progenitor cells.

21. The method of claim 20, wherein the method improves survival of stressed cardiomyocyte cells.

22. The method of claim 20, wherein the method improves one or more of the seeding, survival, viability and proliferation of stressed endothelial cells in vitro.

23. The method of claim 20, wherein the method improves cell migration and / or wound healing capabilities in stressed endothelial cells.

24. The method of any one of claims 20-23, wherein the method improves wound healing in said subject.

25. The method of any of one of claims 20-24, wherein the method reduces signs of fibrosis in fibroblast cells of said subject.

26. The method of claim 25, wherein the fibroblasts are activated with TGF-β1.

27. The method of any one of claims 20-26, wherein the method does not stimulate allogeneic human PBMC activation.

28. The method of any of claims 20-27, wherein the method does not induce NK degranulation of allogeneic human NK cells.

29. The method of any of claims 20-28, wherein the composition is non-toxic in mice and rats at a dose of 4 x 1011particles / kg.

30. The method of any of claims 20-29, wherein the composition is not tumorigenic in mice at dose of 4 x 1011particlcs / kg.

31. The method of any of claims 20-30, wherein the composition does not contain DNA fragments ranging from 179 to 742 pb, at concentrations in the μg / mL range.

32. The method of any of claims 1-31, wherein the therapeutic composition is administered as an intravenous infusion, direct cardiac injection or is administered intra-arterially.

33. The method of claim 32, wherein the therapeutic composition is administered at a dose containing secretome obtained from 0.1 to 10 million cells per kg weight of said subject per administration.

34. The method of claim 32, wherein the therapeutic composition is administered at a dose containing secretome obtained from 0.5 to 5 million cells per kg weight of said subject per administration.35.. The method of claim 32, wherein the therapeutic composition is administered at a dose containing secretome obtained from 1 to 3 million cells per kg weight of said subject.

36. The method of claim 32, wherein the therapeutic composition is administered at a dose containing secretome obtained from 1 to 2 million cells per kg weight of said subject.

37. The method of claim 32, wherein the therapeutic composition is administered at a dose containing from 1 x 109to 60 x 109particles, as measured by Nanoparticle Tracking Analysis (NTA), per kg weight of the subject.

38. The method of claim 32, wherein the therapeutic composition is administered at a dose containing from 10 x 109to 60 x 109particles, as measured by NTA, per kg weight of the subject.

39. The method of claim 32, wherein the therapeutic composition is administered at a dose containing from 10 x 109to 40 x 109particles, as measured by NTA, per kg weight of the subject.

40. The method of claim 32, wherein the therapeutic composition is administered at a dose containing from 20 x 109to 40 x 109particles, as measured by NTA, per kg weight of the subject.

41. The method of claim 32, wherein the therapeutic composition is administered at a cumulative daily dose containing from 20 x 109to 200 x 109particles, as measured by NTA, per kg weight of the subject.

42. The method of claim 32, wherein the therapeutic composition is administered at a cumulative daily dose containing from 30 x 109to 100 x 109particles, as measured by NTA, per kg weight of the subject.

43. The method of claim 32, wherein the therapeutic composition is administered at a cumulative daily dose containing 60 x 109particles, as measured by NTA, per kg weight of the subject.

44. The method of claim 32, wherein the therapeutic composition is administered at a cumulative daily dose containing 40 x 109particles, as measured by NTA, per kg weight of the subject.

45. The method of any one of claims 32-44, wherein the therapeutic composition is administered 1 to 10 times per day.

46. The method of any one of 32-44, wherein the therapeutic composition is administered 3 to 6 times per day.

47. The method of any one of 32-44, wherein the therapeutic composition is administered 1 to 5 times per day.

48. The method of any one of 32-44, wherein the therapeutic composition is administered 3 times per day.

49. The method of any one of 32-44, wherein the therapeutic composition is administered 2 times per day.

50. The method of any one of claims 1-49, wherein the duration of treatment is 60 days or less.

51. The method of claim 50, wherein the duration of treatment is from 5 to 50 days.

52. The method of claim 50, wherein the duration of treatment is from 10 to 50 days.

53. The method of claim 50, wherein the duration of treatment is from 20 to 45 days.

54. The method of claim 50, wherein the duration of treatment is 42 days.

55. The method of any one of claims 32-54, wherein the therapeutic composition is administered every day.

56. The method of any one of claims 32-54, wherein the therapeutic composition is administered every other day.

57. The method of any one of claims 32-54, wherein the therapeutic composition is administered at a frequency of from every day to every 30 days.

58. The method of any one of claims 32-54, wherein the therapeutic composition is administered at a frequency of from every 7 days to every 21 days.

59. The method of any one of claims 32-54, wherein the therapeutic composition is administered every 21 days.

60. The method of any one of claims 32-59, wherein the therapeutic composition is formulated in a solution comprising one or more pharmaceutically acceptable excipient.

61. The method of any one of claims 1-60, wherein the subject has a mutation in the tropomyosin gene.

62. The method of any one of claims 1-60, wherein the subject has a mutation in theMYH7 gene.

63. The method of any one of claims 1-60, wherein the subject has a mutation in the LMNA II gene.

64. The method of any one of claims 1-60, wherein maximal oxygen consumption at exercise is increased in the subject.

65. The method of claim 64, wherein maximal oxygen consumption at exercise is increased by 0.1% to 50%.

66. The method of claim 64, wherein maximal oxygen consumption at exercise is increased by 1% to 20%.

67. The method of claim 64, wherein maximal oxygen consumption at exercise is increased by 5% to 10%.

68. The method of claim 64, wherein maximal oxygen consumption at exercise is increased by 0.1 to 6.0 mL / kg / min.

69. The method of claim 64, wherein maximal oxygen consumption at exercise is increased by 0.5 to 5.0 mL / kg / min.

70. The method of claim 64, wherein maximal oxygen consumption at exercise is increased by 1.0 to 3.0 mL / kg / min.

71. The method of any one of claims 1-70, wherein the secretome of the one or more progenitor cells is generated by the following steps:(a) culturing one or more progenitor cells in a first serum-free culture medium, wherein said first serum-free culture medium comprises basal medium, human serum albumin, and one or more growth factors;(b) removing said first serum-free culture medium from said one or more progenitor cells;(c) culturing said one or more progenitor cells in a second serum-free culture medium, wherein said second serum-free culture medium comprises basal medium, but does not comprise human serum albumin or growth factors; and(d) recovering the second scrum-free culture medium after the culturing of step (c), to thereby obtain conditioned medium comprising the secretome of the one or more progenitor cells.

72. The method of any one of claims 1-71, wherein said one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, vascular progenitor cells and cardiovascular progenitor cells.

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