Production of secretome-containing compositions and methods of using and analyzing same

Abstract

The present disclosure provides methods for producing and / or purifying secretomes, extracellular vesicles, and fractions thereof from progenitor cells, and compositions containing such produced secretomes, extracellular vesicles, and fractions thereof. The present disclosure further provides methods for analyzing the activity, functionality, and efficacy of such secretomes, extracellular vesicles, and fractions thereof. The present disclosure also relates to therapeutic uses of secretomes, extracellular vesicles, and fractions thereof. The present disclosure further relates to a scalable, Good Manufacturing Practice (GMP)-compliant culture protocol for releasing clinically usable secretomes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63 / 417,887, filed October 20, 2022, and U.S. Provisional Patent Application No. 63 / 469,359, filed May 26, 2023, the entire disclosures of which are incorporated herein by reference.

[0002] Incorporation by Reference of Sequence Listing The contents of the electronically submitted sequence listing, file name: F285249_Sequence listing as filed.xml; size: 13,305 bytes; creation date: October 20, 2023, filed herewith, are incorporated herein by reference in their entirety.

[0003] The present disclosure generally relates to the production, purification, isolation, and / or enrichment of secretomes from cells (e.g., but not limited to, progenitor cells), secretome-containing compositions containing such produced, purified, isolated, and / or enriched secretomes, and methods for analyzing one or more activities, properties, and / or characteristics of such secretome-containing compositions. The present disclosure also relates to therapeutic uses of secretome-containing compositions containing secreted bioactive molecules produced, purified, isolated, and / or enriched by one or more methods disclosed herein. The present disclosure further relates to Good Manufacturing Practice (GMP)-compliant, scalable culture protocols, compositions, and uses thereof for the release, purification, isolation, and / or enrichment of clinically usable secretomes. [Background technology]

[0004] Cells, including in vitro or ex vivo cultures, secrete a variety of molecules and biological factors (collectively known as the 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 in membrane-bound extracellular vesicles, such as exosomes. Extracellular vesicles can alter the biology of other cells by signaling or delivering their cargo (e.g., including proteins, lipids, and nucleic acids). The cargo of extracellular vesicles is enveloped in a membrane that allows, among other things, specific targeting (e.g., to target cells) via specific markers on the membrane and increased stability during transport in body fluids, such as through the bloodstream or the blood-brain barrier (BBB).

[0005] Exosomes exert a wide range of important physiological functions, for example, by acting as molecular messengers transporting information between different cell types. For example, depending on their source, exosomes deliver proteins, lipids, and soluble factors, including RNAs and microRNAs, involved in signaling pathways that can affect apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, and immunity by directing T cells toward immune activation, immune suppression, growth, division, survival, differentiation, stress response, apoptosis, and more. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). Extracellular vesicles can contain combinations of molecules that can act in concert to exert specific biological effects. Exosomes incorporate a wide range of cytoplasmic and membrane components that reflect the properties of the parent cell. Therefore, the term applied to the cell of origin can sometimes be used simply to refer to the secreted exosomes.

[0006] Progenitor cells have the ability to proliferate and differentiate into mature cells, making them attractive for therapeutic applications, such as regenerative medicine in the treatment of myocardial infarction and congestive heart failure. Extracellular vesicles secreted by human embryonic stem cell-derived cardiovascular progenitor cells have been reported to produce therapeutic effects similar to those of their secreted counterparts in a mouse model of chronic heart failure (see Kervadec et al. (J. Heart Lung Transplant, 2016;35:795-807)). This suggests that a significant mechanism of action of transplanted progenitor cells is the release of biological factors after transplantation (e.g., stimulating endogenous regenerative or repair pathways). This raises the possibility of effective cell-free therapies, which offer advantages such as convenience, stability, and improved operator handling. However, there is currently a need for improved production methods for generating, purifying, isolating, and / or enriching extracellular vesicles and their compositions for allogeneic or autologous human administration and use.

[0007] Established techniques for producing extracellular vesicles typically use reagents and / or conditions that are not compatible with clinical or therapeutic use or GMP standards. Furthermore, extracellular vesicles produced by one method will have different functions and properties than extracellular vesicles or secretomes produced by another similar method. See Thery et al. (J Extracell Vesicles. 2018 Nov 23;7(1):1535750).

[0008] Therefore, the therapeutic administration, efficacy, and safety of extracellular vesicle-containing compositions may depend on the method and process. For example, regulatory approval for the manufacture of pharmaceuticals and biological substances requires strict adherence to laws and regulations promulgated for the purpose of establishing safe and effective manufacturing facilities and products. As a non-limiting example, "Good Manufacturing Practices" (GMP) and "Good Laboratory Practices" (GLP) have been established and implemented for drugs and biological products by regulations of the FDA (U.S. Food and Drug Administration), CDER (Center for Drug Evaluation and Research), and CBER (Center for Biological Products Evaluation and Research). Similar GMP and / or GLP methods are implemented worldwide, for example, in the EMEA.

[0009] For example, the use of serum in culture protocols raises reliability and biosafety concerns, especially when serum obtained from animals can be contaminated with infectious agents such as viruses or prions. Although fetal bovine serum (FBS) is a widely used growth supplement for cell and tissue culture media, FBS is less suitable for clinical or therapeutic use for these reasons.

[0010] In contrast, the use of serum-free media offers many advantages, including formulation consistency and safety. However, the use of serum-free media alone can adversely affect cell metabolism and growth, and there is a need for Good Manufacturing Practice (GMP) compliant compositions and methods for generating, purifying, isolating, and / or enriching secretome compositions.

[0011] Additionally, improved treatments are needed for patients, such as cancer survivors, who are treated with anthracyclines and are at risk of developing left ventricular (LV) dysfunction, sometimes 10 to 20 years after the end of cancer treatment. Several factors, such as cumulative dose, age, and cardiovascular risk, increase the probability of developing anthracycline-induced cardiotoxicity, but in either case, patients require careful monitoring, sometimes a reduction in the dosing regimen, or the use of classical neurohormonal blockade prophylaxis, the benefits of which remain unclear.

[0012] Anthracycline treatment causes DNA damage, oxidative and energetic stress, leading to inflammation, extracellular matrix remodeling and defects in cardiac contractility, which in the long term lead to LV dysfunction.

[0013] In order to develop, optimize, and release a product for human therapeutic use, it is important to establish its safety and efficacy in appropriate models. A combined approach of testing a product in in vitro human cells and animal studies provides a powerful data set that describes the efficacy and safety of a product and predicts its efficacy, safety, and use in human subjects. Summary of the Invention [Means for solving the problem]

[0014] The present disclosure addresses the above-mentioned limitations in the art by providing methods for producing, purifying, isolating and / or enriching secretomes using serum-free media, thereby enabling GMP-compatible, scalable, quality-controlled culture protocols for the release of clinically usable secretomes.

[0015] The present disclosure also provides methods for producing, purifying, isolating, and / or enriching secretomes, extracellular vesicles, and fractions thereof from cells (e.g., but not limited to, progenitor cells), and provides compositions containing such produced, 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 therapeutic uses of secretomes, extracellular vesicles, and fractions thereof.

[0016] 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 produced, purified, isolated, and / or enriched secretomes, extracellular vesicles, and fractions thereof for the treatment and / or prevention of chemotherapy-induced cardiomyopathy in a subject.

[0017] Non-limiting embodiments of the present disclosure include the following.

[0018] [1] A method for generating a secretome, the method comprising: (a) culturing one or more progenitor cells in a first serum-free culture medium, the first serum-free culture medium comprising a basal medium, human serum albumin, and one or more growth factors; (b) removing the first serum-free culture medium from the one or more progenitor cells; (c) culturing the one or more progenitor cells in a second serum-free culture medium, the second serum-free culture medium comprising a basal medium but not comprising human serum albumin or growth factors; and (d) recovering the second serum-free culture medium after the culturing of step (c), thereby obtaining a conditioned medium comprising the secretome of one or more progenitor cells.

[0019] [2] The method according to [1], wherein one of the one or more growth factors is fibroblast growth factor 2 (FGF-2).

[0020] [3] The method according to [1] or [2], wherein the first and second serum-free media are supplemented with a carbohydrate source.

[0021] [4] The method according to [3], wherein the carbohydrate source is glucose.

[0022] [5] The method according to any one of [1] to [4], wherein the first and second serum-free media are supplemented with an antibiotic.

[0023] [6] The method according to [5], wherein the antibiotic is gentamicin.

[0024] [7] The method according to any one of [1] to [6], wherein the first serum-free medium further contains 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; triiodo-I-thyronine; amino acids; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.

[0025] [8] The method according to any one of [1] to [7], wherein the basal medium is a minimum essential medium (MEM).

[0026] [9] The method according to [8], wherein the MEM is α-MEM.

[0027]

[10] The method according to any one of [1] to [9], wherein the culture in step (a) is for 6 to 96 hours.

[0028]

[11] The method according to

[10] , wherein the culturing in step (a) is for 12 to 96 hours.

[0029]

[12] The method according to

[11] , wherein the culture in step (a) is for 36 to 84 hours.

[0030]

[13] The method according to

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

[0031]

[14] The method according to any one of [1] to

[13] , wherein the culturing in step (c) is for 6 to 96 hours.

[0032]

[15] The method according to

[14] , wherein the culturing in step (c) is for 12 to 72 hours.

[0033]

[16] The method according to

[15] , wherein the culturing in step (c) is for 36 to 60 hours.

[0034]

[17] The method according to

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

[0035]

[18] The method according to

[14] , wherein the final 12 to 36 hours of the culture in step (c) are carried out under hypoxic conditions.

[0036]

[19] The method according to

[18] , wherein the culture conditions include culturing in an atmosphere having 1 to 21% oxygen.

[0037]

[20] The method according to any one of [1] to

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

[0038]

[21] The method according to any one of [1] to

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

[0039]

[22] The method according to any one of [1] to

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

[0040]

[23] The method according to any one of [1] to [4] and [7] to

[22] , wherein the first and second serum-free media do not contain antibiotics.

[0041]

[24] The method according to any one of [1] to

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

[0042]

[25] The method according to

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

[0043]

[26] The method according to

[25] , wherein the surface of the culture vessel is coated with a substance for promoting cell adhesion.

[0044]

[27] The method according to

[26] , wherein the substance for promoting cell adhesion is vitronectin or fibronectin.

[0045]

[28] The method according to any one of [1] to

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

[0046]

[29] The method of

[28] , 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.

[0047]

[30] The method according to any one of [1] to

[29] , further comprising pre-clarifying the medium recovered in step (d) by centrifugation, filtration, or a combination of centrifugation and filtration.

[0048]

[31] The method according to any one of [1] to

[30] , further comprising freezing the medium collected in step (d).

[0049]

[32] The method according to any one of [1] to

[31] , wherein the one or more progenitor cells cultured in step (a) have been frozen in advance.

[0050]

[33] The method described in any one of [1] to

[32] , further comprising concentrating and / or enriching small extracellular vesicles (sEVs) from the medium recovered in step (d).

[0051]

[34] The method according to

[33] , wherein the sEVs are concentrated and / or enriched from the collected culture medium by at least one process selected from the group consisting of ultracentrifugation, filtration, ultrafiltration, tangential flow filtration, size exclusion chromatography, and affinity capture.

[0052]

[35] The enrichment is characterized by: (a) CD63 + , CD81 + and / or CD9 +The method of

[33] , wherein the method enriches extracellular vesicles having one or more of the following characteristics: (a) a diameter of 50 to 200 nm; (b) a diameter of 50 to 200 nm; (c) a positivity 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) a negativity for one or more of CD19, CD4, CD209, HLA-ABC (human leukocyte antigen-ABC), CD62P, CD42a, and CD69.

[0053]

[36] The method according to

[33] , wherein the sEVs include one or more of exosomes, microparticles, extracellular vesicles, and secreted peptides / proteins.

[0054]

[37] A secretome-containing composition obtained by the method according to any one of [1] to

[32] .

[0055]

[38] An sEV-containing composition obtained by the method described in any one of

[33] to

[36] .

[0056]

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

[32] .

[0057]

[40] The method of

[39] , further comprising purifying, concentrating, isolating and / or enriching the secretome-containing composition by one or more purification, concentration, isolation and / or enrichment steps.

[0058]

[41] The method of

[39] , further comprising adding a pharmaceutically acceptable excipient or carrier to the secretome-containing composition.

[0059]

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

[33] to

[36] .

[0060]

[43] The method described in

[42] , further comprising purifying, concentrating, isolating and / or enriching the sEV-containing composition by one or more purification, concentration, isolation and / or enrichment steps.

[0061]

[44] The method according to

[42] , further comprising adding a pharmaceutically acceptable excipient or carrier to the sEV-containing composition.

[0062]

[45] A therapeutic composition comprising the secretome-containing composition according to

[37] and a pharmaceutically acceptable excipient or carrier.

[0063]

[46] A therapeutic composition comprising the sEV-containing composition described in

[38] and a pharmaceutically acceptable excipient or carrier.

[0064]

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

[0065]

[48] ​​An sEV-containing composition obtained by the method described in

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

[0066]

[49] A therapeutic composition comprising the composition according to

[47] and a pharmaceutically acceptable excipient or carrier.

[0067]

[50] A therapeutic composition comprising the composition according to

[48] and a pharmaceutically acceptable excipient or carrier.

[0068]

[51] 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 described in

[49] or

[50] .

[0069]

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

[49] or

[50] .

[0070]

[53] A method for improving cardiac function, comprising administering to a subject in need thereof the therapeutic composition according to

[49] or

[50] .

[0071]

[54] The method according to

[11] , wherein the culturing in step (a) is for 60 to 84 hours.

[0072]

[55] The method according to

[14] , wherein the final 12 to 36 hours of the culture in step (c) are carried out under normoxic conditions.

[0073]

[56] The method according to

[55] , wherein the normoxic conditions include culturing in an atmosphere containing 20 to 21% oxygen.

[0074]

[57] The method of

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

[0075]

[58] The method of

[39] , further comprising cryopreserving, freezing or lyophilizing the secretome-containing composition.

[0076]

[59] The method described in

[42] , further comprising cryopreserving, freezing or lyophilizing the sEV-containing composition.

[0077]

[60] The method according to [2], wherein the first serum-free medium contains 0.1 to 10 μg / mL of FGF-2.

[0078]

[61] The method according to

[60] , wherein the first serum-free medium contains 0.5 to 5 μg / mL of FGF-2.

[0079]

[62] The method according to

[61] , wherein the first serum-free medium contains 0.5 to 2.5 μg / mL of FGF-2.

[0080]

[63] The method according to

[62] , wherein the first serum-free medium contains about 1 μg / mL of FGF-2.

[0081]

[64] The method according to any one of [1] to

[36] ,

[39] to

[44] and

[54] to

[63] , which complies with Good Manufacturing Practice (GMP).

[0082]

[65] The secretome-containing composition according to

[37] , which corresponds to GMP.

[0083]

[66] The sEV-containing composition according to

[38] , which corresponds to GMP.

[0084]

[67] The method according to

[14] , wherein the final 12 to 36 hours of the culture in step (c) are carried out under normoxic conditions.

[0085]

[68] The method according to

[67] , wherein the normoxic conditions include culturing in an atmosphere containing 20 to 21% oxygen.

[0086]

[69] The method of

[30] , wherein the pre-clarification comprises at least three filtration steps.

[0087]

[70] The method according to

[34] , wherein the separation of the sEVs from the collected medium comprises tangential flow filtration.

[0088]

[71] The secretome-containing composition according to

[37] , which contains trehalose and L-histidine.

[0089]

[72] The sEV-containing composition according to

[38] , which comprises trehalose and L-histidine.

[0090]

[73] The secretome-containing composition according to

[37] or

[65] , which is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

[0091]

[74] The sEV-containing composition according to

[38] or

[66] , which is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

[0092]

[75] The secretome-containing composition according to

[37] or

[65] , which is at least one of a composition enriched in extracellular vesicles having a diameter of about 50 to 200 nm or 50 to 200 nm, preferably about 50 to 150 nm or 50 to 150 nm; a composition that is substantially free of or does not contain whole cells; and / or a composition that is substantially free of one or more culture medium components.

[0093]

[76] The sEV-containing composition according to

[38] or

[66] , which is at least one of: a composition enriched in extracellular vesicles having a diameter of about 50 to 200 nm or 50 to 200 nm, preferably about 50 to 150 nm or 50 to 150 nm; a composition that is substantially free of or does not contain whole cells; and / or a composition that is substantially free of one or more culture medium components.

[0094]

[77] The method according to

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

[0095]

[78] The method according to

[77] , 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.

[0096]

[79] The secretome-containing composition according to

[37] or

[65] , which is capable of promoting cardiomyocyte viability in an in vitro chemotherapy-induced cardiomyopathy viability assay.

[0097]

[80] The sEV-containing composition according to

[38] or

[66] , which is capable of promoting cardiomyocyte viability in an in vitro chemotherapy-induced cardiomyopathy viability assay.

[0098]

[81] The secretome-containing composition according to

[79] , wherein in the in vitro chemotherapy-induced cardiomyopathy survival assay, the chemotherapy is an anthracycline.

[0099]

[82] The secretome-containing composition according to

[81] , wherein the anthracycline is doxorubicin.

[0100]

[83] The sEV-containing composition described in

[80] , wherein in the in vitro chemotherapy-induced cardiomyopathy survival assay, the chemotherapy is an anthracycline.

[0101]

[84] The sEV-containing composition described in

[83] , wherein the anthracycline is doxorubicin.

[0102]

[85] The method according to

[51] , wherein the chemotherapy-induced cardiomyopathy is caused by an anthracycline.

[0103]

[86] The method according to

[85] , wherein the anthracycline is doxorubicin.

[0104]

[87] A method for maintaining physiological cardiac volume in a subject by administering to the subject a therapeutic composition according to any one of

[45] ,

[46] ,

[49] and

[50] .

[0105]

[88] The method according to any one of

[51] to

[53] and

[87] , wherein the left ventricular end-systolic volume (LVESV) is maintained within 15% of the pre-treatment LVESV.

[0106]

[89] The method according to any one of

[51] to

[53] and

[87] , wherein the LVEDV is maintained within 2% of the pre-treatment volume.

[0107]

[90] The method according to any one of

[51] to

[53] and

[87] , for preventing progressive post-ischemic heart failure.

[0108]

[91] The method according to any one of

[51] to

[53] and

[87] , which improves the survival, health and function of endothelial cells in the subject.

[0109]

[92] The method according to any one of

[51] to

[53] and

[87] , which reduces fibrosis in stimulated cardiac fibroblasts.

[0110]

[93] The method according to

[92] , which reduces the expression of the pro-fibrotic marker POSTN in TGF-β1-stimulated cardiac fibroblasts to below the level before stimulation with TGF-β1.

[0111]

[94] The method according to any one of

[51] to

[53] and

[87] , which does not induce an allogeneic inflammatory response in a subject.

[0112]

[95] The method according to any one of

[51] to

[53] and

[87] , which does not induce activation of allogeneic peripheral blood mononuclear cells (PBMCs).

[0113]

[96] The method according to any one of

[51] to

[53] and

[87] , which does not induce a significant increase in the percentage of IFNg or IL-2 expressing PBMCs.

[0114]

[97] The method according to any one of

[51] to

[53] and

[87] , which does not induce allogeneic natural killer (NK) cell degranulation.

[0115]

[98] The method according to any one of

[51] to

[53] and

[87] , which does not induce a significant increase in the percentage of CD107-expressing NK cells.

[0116]

[99] A method of improving cardiac function in a patient experiencing heart failure by administering a therapeutic composition according to any one of

[45] ,

[46] ,

[49] and

[50] .

[0117]

[0100] The method described in

[99] improves survival of stressed cardiomyocytes.

[0118]

[0101] The method according to

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

[0119]

[0102] The method described in

[99] , which improves cell migration and / or wound healing capacity in stressed endothelial cells.

[0120]

[0103] A method according to any one of

[99] to

[0102] , which improves wound healing in the subject.

[0121]

[0104] A method according to any one of

[99] to

[0103] , which reduces signs of fibrosis in the subject's fibroblasts.

[0122]

[0105] The method described in

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

[0123]

[0106] A method according to any one of

[99] to

[0105] , which does not stimulate activation of allogeneic human PBMCs.

[0124]

[0107] A method according to any one of

[99] to

[0106] , which does not induce NK degranulation of allogeneic human NK cells.

[0125] The composition exhibited a potency of 4×10 in mice and rats. 11 The method according to any one of

[99] to

[0107] , which is non-toxic at a dose of particles / kg.

[0126] The composition was found to be effective in mice at 4×10 11 The method according to any one of

[99] to

[0108] , wherein the compound is not tumorigenic at a dose of particles / kg.

[0127]

[0110] A method according to any one of

[99] to

[0109] , wherein the composition does not contain DNA fragments in the range of 179 to 742 pb at a concentration in the μg / mL range.

[0128]

[0111] The method according to any one of

[51] to

[53] and

[87] to

[0110] , wherein the therapeutic composition is administered as an intravenous infusion, a direct cardiac injection, or administered intra-arterially.

[0129]

[0112] The method described in

[0111] , wherein the therapeutic composition is administered at a dose containing secretomes obtained from 10 to 10 million cells per kg of the subject's body weight per administration.

[0130]

[0113] The method described in

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

[0131]

[0114] The method described in

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

[0132]

[0115] The method described in

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

[0133] The therapeutic composition is administered at a concentration of 1×10 nanoparticles per kg of subject body weight as measured by nanoparticle tracking analysis (NTA). 9 ~60×10 9 The method described in

[0111] , wherein the compound is administered in a dose containing particles.

[0134] The therapeutic composition is administered at a dose of 10×10 per kg of subject body weight as measured by NTA. 9 ~60×10 9 The method described in

[0111] , wherein the compound is administered in a dose containing particles.

[0135] The therapeutic composition is administered at a dose of 10×10 per kg of subject body weight as measured by NTA. 9 ~40×10 9 The method described in

[0111] , wherein the compound is administered in a dose containing particles.

[0136] The therapeutic composition contains 20×10 per kg of subject body weight as measured by NTA. 9 ~40×10 9 The method described in

[0111] , wherein the compound is administered in a dose containing particles.

[0137] The therapeutic composition contains 20×10 per kg of subject body weight as measured by NTA. 9 ~200×10 9 The method of

[0111] , wherein the compound is administered in a cumulative daily dose containing particles.

[0138] The therapeutic composition contains 30×10 per kg of subject body weight as measured by NTA. 9 ~100×10 9 The method of

[0111] , wherein the compound is administered in a cumulative daily dose containing particles.

[0139] The therapeutic composition contains 60×10 per kg of subject body weight as measured by NTA. 9 The method of

[0111] , wherein the compound is administered in a cumulative daily dose containing particles.

[0140] The therapeutic composition contains 40×10 per kg of subject body weight as measured by NTA. 9 The method of

[0111] , wherein the compound is administered in a cumulative daily dose containing particles.

[0141]

[0124] A method according to any one of

[0111] to

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

[0142]

[0125] A method according to any one of

[0111] to

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

[0143]

[0126] A method according to any one of

[0111] to

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

[0144]

[0127] A method according to any one of

[0111] to

[0123] , wherein the composition is administered three times per day.

[0145]

[0128] A method according to any one of

[0111] to

[0123] , wherein the composition is administered twice per day.

[0146]

[0129] A method according to any one of

[51] to

[53] and

[87] to

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

[0147]

[0130] The method described in

[0129] , wherein the treatment period is 5 to 50 days.

[0148]

[0131] The method described in

[0129] , wherein the treatment period is 10 to 50 days.

[0149]

[0132] The method described in

[0129] , wherein the treatment period is 20 to 45 days.

[0150]

[0133] The method described in

[0129] , wherein the treatment period is 42 days.

[0151]

[0134] A method according to any one of

[0111] to

[0133] , wherein the therapeutic composition is administered daily.

[0152]

[0135] A method according to any one of

[0111] to

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

[0153]

[0136] A method according to any one of

[0111] to

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

[0154]

[0137] A method according to any one of

[0111] to

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

[0155]

[0138] A method according to any one of

[0111] to

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

[0156]

[0139] A method according to any one of

[0111] to

[0138] , wherein the therapeutic composition is formulated in a solution containing one or more pharmaceutically acceptable excipients.

[0157]

[0140] A secretome-containing composition obtained by the method according to any one of [1] to

[32] , the secretome-containing composition containing extracellular vesicles secreted from the progenitor cells.

[0158]

[0141] The extracellular vesicles may contain 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, h sa-miR-191-5p, hsa-miR-26b-5p, hsa-miR-151a-3p, hsa-miR-103a-3p / 107, hsa-miR-660-5p, hsa-m iR-320a-3p / 320b / 320c / 320d / 320e, hsa-miR-130a-3p, hsa-miR-19b-3p, hsa-miR-27a-3p / 27b-3p, hs A secretome-containing composition described in

[0140] , comprising one or more miRs selected from a-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.

[0159]

[0142] The extracellular vesicles may contain miR-1-5p, miR-11401, miR-1263-3p, miR-3085-3p, miR-3161-5p, miR-3678-3p, miR-3942-5p, miR-4652-5p, miR-4758-5p, and miR-4760-5p. The secretome-containing composition described in

[0140] , which contains one or more miRs selected from 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.

[0160]

[0143] The extracellular vesicles may contain any of 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, and hsa-miR-4760-5p. The secretome-containing composition described in

[0140] , comprising at least five miRs selected from 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.

[0161]

[0144] A secretome-containing composition described in any one of

[0141] to

[0143] , wherein the expression level of one or more miRs is in the range of -5 to +5 units.

[0162]

[0145] A secretome-containing composition comprising a secretome derived from a progenitor cell, wherein the secretome comprises extracellular vesicles secreted from the progenitor cell.

[0163]

[0146] A secretome-containing composition described in

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

[0164]

[0147] The extracellular vesicles contain 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, and hsa-miR-1 01-3p, hsa-miR-302d-3p, hsa-miR-25-3p, hsa-miR-126-5p, hsa-miR-423-5p, hsa-miR-532-5p, hsa-mi R-1246, hsa-miR-302a-3p, hsa-miR-20b-5p, hsa-miR-148b-3p, hsa-miR-34a-5p, hsa-miR-1-3p, hsa-m iR-191-5p, hsa-miR-26b-5p, hsa-miR-151a-3p, hsa-miR-103a-3p / 107, hsa-miR-660-5p, hsa-miR-32 0a-3p / 320b / 320c / 320d / 320e, hsa-miR-130a-3p, hsa-miR-19b-3p, hsa-miR-27a-3p / 27b-3p, hsa-miR- The secretome-containing composition according to

[0145] or

[0146] , comprising one or more miRs selected from 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.

[0165]

[0148] The extracellular vesicles may contain 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-4761-5p, hsa-miR-4762-5p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4765-5p, hsa-miR-4765-5p, hsa-miR-4760-5p, hsa-miR-4761-5p, hsa-miR-4762-5p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4765-5p, hsa-miR-4760-5p, hsa-miR-4761-5p, hsa-miR-4761-5p, hsa-miR-4762-5p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4765-5p, hsa-miR-4761 ... A secretome-containing composition according to

[0145] or

[0146] , comprising one or more miRs selected from a-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.

[0166]

[0149] The extracellular vesicles may contain 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 The secretome-containing composition according to

[0145] or

[0146] , comprising at least five miRs selected from 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.

[0167]

[0150] A secretome-containing composition described in any one of

[0147] to

[0149] , wherein the expression level of one or more miRs is in the range of -5 to +5 units.

[0168] Incorporation by Reference All patents, publications, and patent applications cited in this specification are herein incorporated by reference to the same extent 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.

[0169] The 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. [Brief explanation of the drawings]

[0170] [Figure 1] A process flow diagram from iPSCs to CPCs is shown, demonstrating the generation of cardiovascular progenitor cells from hiPSCs (steps 1-4). After CPC generation, cells were either maintained as fresh aggregates (5a) or dissociated into single cells for the vesicle formation process (step 5b). Single cells were plated fresh or cryopreserved and plated after thawing for the vesicle formation process (steps 6-7). [Figure 2A-2B] This is a flowchart showing the materials produced in Example 1. As shown in Figures 2A and 2B, two batches of CPC (CPC1 and CPC2) were produced and each was divided into three vesicle formation conditions: aggregate vesicle formation, fresh CPC plate vesicle formation, and thawed CPC plate vesicle formation. Conditioned medium from each condition was collected, pre-clarified, and frozen (MC1-6). Cells at the end of the 4-day vesicle formation process (day +4) were also collected and analyzed (C+4#1-6). Conditioned medium was subjected to ultracentrifugation (UC) to isolate vesicle fractions (sEV1-6). For MC5, three separate rounds of UC were performed on separate aliquots of MC5. In parallel, a vessel containing medium but without cells was incubated under the same conditions as the cell-containing vessel. The medium produced by this process (referred to as virgin medium) was collected (virgin medium 1-3). Subsequently, mock EV (also called MV) controls were generated from virgin medium via the same UC protocol (MV1.1-3) as above. [Figure 3A-3B]Gene expression heatmaps of 48 relevant genes for CPC differentiation and potential off-targets are shown. Data were generated using a custom Fluidigm qPCR panel. (A) Heatmap generated by calculating global z-scores using the "SINGuLAR Analysis Toolset" package in R3.1.1. (B) Heatmap generated by calculating gene z-scores and then performing hierarchical clustering in JMP software version 17 (non-standardized forward method). Ct values ​​are shown in Table 1. Data are shown from iPSC and cardiomyocyte (CM) controls, as well as from CPCs at the end of the differentiation process and CPCs 4 days into the vesicle formation process (C+4). Under these conditions, CPCs cluster together and are separated from C+4 cells, which are more mature than CPCs but less mature than CMs. Aggregates on day 4 of vesicle formation (Agg+4) differ from HyperFlask-plated cells on day 4 (HF+4). Both conditions show increased cTNT (cardiac troponin T) and α-MHC (α-myosin heavy chain) expression compared to CPCs, supporting the idea that CPCs undergoing vesiculation remain in the cardiac lineage but do not achieve a CM differentiated state, as indicated by the persistence of CPC marker expression such as PDGFRa, ISL-1, and KDR. [Figure 4] FIG. 1 shows a process flow diagram for the production of conditioned media and virgin media controls. [Figure 5] A process flow diagram for the isolation of sEVs or mock (virgin medium) control samples is shown. [Figure 6] Representative size distribution curves from two sEV and two control MV samples are shown. Suspension cultures yielded higher particle concentrations than plate cultures, and both were significantly higher than the controls. The modal particle sizes of sEV1 and sEV2 (74 nm and 99 nm, respectively) are consistent with exosomes or small microparticles. [Figure 7]ELISA results for the detection of CD-63 are shown. Bars are numbered 1–9 from left to right. sEVs (bars 1, 4, 5, 6, and 7) and MV controls (bars 2, 3, 8, and 9) were analyzed by the FUJIFILM Wako Elisa kit for the detection of CD-63, a protein found on the surface of EVs, particularly exosomes. The results show that for a given protein input, MVs do not contain a CD-63 signal, whereas sEVs from both aggregate and plated cultures contain a CD-63 signal. Aggregated sEVs (bar 1) produced a greater CD-63 / protein signal than sEVs from the plated vesicle formation protocol (bars 4–7). Duplicate preparations of sEVs from the same MCs (bars 5.1, 5.2, and 5.3; bars 5, 6, and 7) yielded similar CD63 signals. Furthermore, sEVs isolated from different MCs produced from separate lots also yielded similar CD-63 / µg protein (sEV2 (bar 4) vs. sEV5.1 / .2 / .3). Protein signals are given as absorbance 450–absorbance 620 minus the blank result (abs 450–620, blank adj). [Figure 8] Relative scratch wound closure in a HUVEC scratch wound healing assay is shown. Bars are numbered 1 to 7 from left to right. sEVs from the suspension and plating vesicle formation processes (bars 4 and 6) and their corresponding mock EV controls (MVs) (bars 5 and 7) were tested in a HUVEC scratch wound healing assay. Controls were complete HUVEC medium (positive control, "positive," bar 1), poor HUVEC medium (no supplements, negative control, "negative," bar 2), and poor medium + UC sEVs isolated from fetal bovine serum ("FBS-EVs," additional positive control, bar 3). sEVs from the suspension (bar 4) and plating (bar 6) vesicle formation processes showed improved wound healing compared to the negative and MV controls. [Figure 9]Results of the H9c2 viability assay are shown. Bars are labeled 1–7 from left to right. The results of the H9c2 cell viability assay demonstrate that sEVs from suspension cultures (bar 3) and plate cultures (bars 5 and 7) improve H9c2 survival in the serum deprivation assay. MVs (bars 4 and 6) showed minimal or no positive effect in this assay. sEVs generated from the suspension vesiculation method showed improved fold change relative to the negative control over the positive control, suggesting increased cell proliferation in addition to sustained survival. [Figure 10] Figure 10 shows the time course of cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay. Conditioning lines are labeled A-F from top to bottom according to the last data point. sEVs from plated cultures (line C) and aggregate cultures (line B) 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 to its corresponding MV control. The 18-h data points in Figure 10 are listed in Table 2. [Figures 11A-11B] FIG. 11 shows a flow chart illustrating the manufacturing steps (vesicle formation, conditioned medium clarification, and TFF in Example 20, FIG. 11A; followed by final formulation, FIG. 11B) in the first GMP-compliant process described in Example 5 and Example 6. The final formulation in this example was manufactured with and without trehalose addition prior to sterile filtration. The various steps where in-process and quality control testing was performed are indicated with an "*" (e.g., *1, *2, *3, etc.). [Figure 12] Figure 1 shows the results of flow cytometry experiments to analyze the cell marker expression profile of CPCs at different times during the vesicle formation process (D+0, D+3, and D+5). iPSCs and cardiomyocytes (CMs) were used as control cells and analyzed separately. Values ​​shown are mean values. [Figure 13]The results of transcriptome analysis of CPCs at different times during the vesicle formation process (D+0, D+3, and D+5) are shown. RNA was extracted from CPCs at D+0 and from cells at D+3 and D+5 of the vesicle formation process. RNA was also extracted from iPSCs (pluripotent cell control) and iPSC-derived cardiomyocytes (differentiated cardiomyocyte control; CM). Total RNA was sequenced on the Illumina NovaSeq 6000 platform, and differential gene expression was determined on normalized data. The heatmaps in Figures 13 and 13B were generated based on hierarchical clustering analysis using the UPGMA clustering method and the correlation distance metric in TIBCO Spotfire software v11.2.0. The heatmap in Figure 13 has a blue-red color scale, with dark blue representing low expression and dark red representing high expression. The heatmap in Figure 13B is grayscale, with white representing low expression and dark gray / black representing high expression. The data (log2FPKM) used to generate both heatmaps is shown in Table 3. [Figure 14] Figure 1 shows the morphology of CPCs during the vesicle formation process, as observed under a light microscope. Cell morphology was analyzed in both T75 and selected CS10 flasks. The image on the left is a representative image showing the typical D+3 morphology observed in all vessels analyzed on D+3. The image on the right is a representative image showing the typical D+5 morphology observed in all vessels analyzed on D+5. For clarity, T75 flasks were used for image capture. [Figures 15A-15B]The results of the analysis of EV particle concentration and size distribution are shown. Figure 15A shows the particle concentration and size distribution of EVs in the clarified conditioned medium before tangential flow filtration (TFF) (*5 (Test 20)) and in the final formulation without trehalose (*7, Sample a (Test 20)) and the final formulation with trehalose (*7, Sample b (Test 20)). Figure 15B shows the particle concentration and size distribution of EVs in the clarified conditioned medium before tangential flow filtration (TFF) (*5 (Test 20)) and in the storage retentate sample without trehalose or histidine (*6, Sample a (Test 20)), the sample with trehalose (*6, Sample b (Test 20)), or the sample with histidine (*6, Sample c (Test 20)). As shown in Figures 15A and 15B, TFF increased particle concentration by approximately 32-fold. [Figures 16A-16D] The results of MACSPlex analysis are shown. Figures 16A and 16B show the results of analysis of small EV-enriched final secretome preparations with and without trehalose for the expression of extracellular vesicle tetraspanins (Figure 16A) that are frequently expressed on the surface of extracellular vesicles (CD9, CD81, and CD63), and for various additional markers that showed little or no expression (Figure 16B). Figures 16C and 16D show the results of analysis of non-sterilized filter-filtered storage retentate samples [see Figure 11B; *6, sample a (Test 20); *6, sample b (Test 20); *6, sample c (Test 20)] for the expression of extracellular vesicle tetraspanins (Figure 16C) that are frequently expressed on the surface of extracellular vesicles (CD9, CD81, and CD63), and for various additional markers that showed little or no expression (Figure 16D). [Figures 17A-17B]Analysis of sample *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. Figure 17A shows the results of the small EV-enriched secretome final preparation with and without trehalose for the expression of cardiac-related markers. Figure 17B shows the results of the non-sterilized-filtered storage retentate samples (with and without trehalose or histidine) for the expression of cardiac-related markers. For all samples shown in Figures 17A and 17B, the investigated markers were found to be present. [Figure 18] Relative scratch wound healing in a HUVEC scratch wound healing assay is shown. Bars are labeled 1–7 from left to right. Small EV-enriched secretome final formulations with (bars 6 and 7) and without (bars 4 and 5) trehalose were tested in a HUVEC scratch wound healing assay. The positive control ("+ve", bar 1) consisted of culturing scratched wells in complete HUVEC cell medium ("Comp") + PBS "treatment", and the negative control ("-ve", bar 2) consisted of culturing scratched wells in basal medium ("Poor") + PBS "treatment". FBS-derived EVs served as the EV control ("EV Ctl", bar 3). 1x treatment is equivalent to secretomes derived from 150,000 cells. Values ​​were subtracted from the baseline (negative control) and normalized to the positive control. [Figure 19]Cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay is shown. Bars are labeled 1–7 from left to right. Final small EV-enriched secretome preparations with (bars 6 and 7) and without (bars 4 and 5) trehalose were tested in the cardiomyocyte survival assay. 1× treatment is equivalent to secretomes derived from 150,000 cells. PBS controls with (bar 2) and without (bar 1) staurosporine served as negative ("-ve") and positive ("+ve") controls, respectively. Mesenchymal stem cell (MSC)-derived EVs served as the EV control ("EV Ctl," bar 3). Plated cells were either stressed with staurosporine for 4 hours ("+") or not stressed with staurosporine ("-") prior to treatment. [Figures 24A-24B] FIG. 24B shows a flow chart illustrating the manufacturing steps (vesicle formation, conditioned medium clarification and TFF, FIG. 24A; final formulation, FIG. 24B) in the second GMP-compliant process described for Examples 12 and 13, i.e., Example 22. The final formulations in this example were manufactured with and without trehalose addition prior to sterile filtration. Different samples that underwent in-process and quality control testing are indicated with an "*" (e.g., *6, *7, etc.). [Figure 25] Figure 1 shows the results of flow cytometry experiments to analyze the cell marker expression profile of CPCs at different times during the vesicle formation process (D+0, D+3, and D+5). iPSCs and cardiomyocytes (CMs) were used as control cells and analyzed separately. Values ​​shown are mean values. [Figure 26] Figure 1 shows the morphology of CPCs during the vesicle formation process, as observed under a light microscope. Cell morphology was analyzed in both T75 and selected CS10 flasks. The image on the left is a representative image showing the typical D+3 morphology observed in all vessels analyzed on D+3. The image on the right is a representative image showing the typical D+5 morphology observed in all vessels analyzed on D+5. For clarity, T75 flasks were used for image capture. [Figures 27A-27B]The results of the analysis of EV particle concentration and size distribution are shown. Figure 27A shows the particle concentration and size distribution of EVs in the conditioned medium before and after clarification in the final formulation (i.e., after TFF) and the final formulation containing trehalose, using nanoparticle tracking analysis. Sample designations are shown in Figures 24A and 24B. Figure 27B shows the particle concentration and size distribution detected by NTA in *6, sample a (Test 22); *7, sample c (Test 22); and *7, sample d (Test 22). [Figures 28A-28B] MACSPlex results are shown for analysis of small EV-enriched secretome final preparations with and without trehalose for expression of extracellular vesicle tetraspanins (Figure 28A), which are often expressed on the surface of extracellular vesicles (CD9, CD81, and CD63), and for various other markers that showed little or no expression (Figure 28B). [Figure 29] MACSPlex results of the final small EV-enriched secretome preparations with and without trehalose for expression of cardiac-related markers are shown. All samples shown in Figure 29 were found to express the markers shown in Figure 29. [Figure 30A-30B]Relative scratch wound healing in the HUVEC scratch wound healing assay is shown. Bars are labeled 1–15 from left to right in 30A and 16–30 from left to right in 30B. The results for sample *7, sample a (Test 22) (bars 4–9), and *7, sample b (Test 22) (bars 10–15) (shown in Figure 24B) are shown in Figure 30A. The results for sample *7, sample c (Test 22) (bars 19–24), and *7, sample d (Test 22) (bars 25–30) (shown in Figure 24B) are shown in Figure 30B. The positive control ("+ve," bars 1 and 16) consisted of culturing scratched wells in complete HUVEC cell culture medium ("Comp") plus PBS "treatment," and the negative control ("-ve," bars 2 and 17) consisted of culturing scratched wells in basal medium (poor) plus PBS "treatment." FBS-derived EVs served as the EV control (EV Ctl, bars 3 and 18). 1× treatment is equivalent to secretomes 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 detailed in Examples 12 and 13, were tested in the scratch wound healing assay as described in Example 17. These four samples originate from the same TFF retentate but differ in the method of final formulation. The four variations involve using fresh retentate and filter-sterilizing it through a Sterivex-GP, 0.22 μm filter (yielding sample a). Fresh retentate was used, supplemented with trehalose, and filter-sterilized through a Sterivex-GP, 0.22 μm filter (to obtain sample b); the retentate was frozen, thawed, and then filter-sterilized through a Sterivex-GP, 0.22 μm filter (to obtain sample c); or the retentate was frozen, thawed, and then filter-sterilized through a Sartopore 2, 0.45 + 0.2 μm filter (to obtain sample d). In Figure 30A, the +ve control result is 100% (first bar on the left). The -ve control is 0% (second bar from the left). The EV Ctl is 29.8% (third bar from the left). Final formulations *7, sample a (Test 22) and *7, sample b (Test 22) showed similar results.Both materials improved scratch wound healing, with dose-response indications ranging from 0.25x to 2.6x. The lowest dose tested was 0.25x, which increased wound healing capacity by at least 17% over the -ve control for both samples. At the 2.6x dose, both samples improved scratch wound healing by more than 25% over the negative control. *7, Sample a (Test 22) improved scratch wound healing by 35.6% at the 2.6x dose. Taken together, the data here show 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 for maintaining the scratch wound healing efficacy of CPC-EV-enriched secretome prepared by the GMP-compliant process for producing small extracellular vesicle-enriched fraction (sEV) formulations as described in Examples 12 and 13. In terms of efficacy in the scratch wound healing assay, all four variations of the final formulation method shown in Figure 24B and described in Example 13 are equally suitable. In Figure 30B, the +ve control result is 100% (first bar from the left). The +ve control is 0% (second bar from the left). The EV Ctl is 25.2% (third bar from the left). Final formulations *7, Sample c (Test 22) and *7, Sample d (Test 22) also showed similar results. Both materials improved scratch wound healing, with dose-response indications from doses ranging from 0.25x to 2.6x. The lowest dose tested was 0.25x, which increased wound healing capacity by at least 17% over the -ve control for both samples. At the 2.6x dose, both samples improved scratch wound healing by over 30% over the negative control. Taken together, the data here show 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 for maintaining the scratch wound healing efficacy of CPC-EV-enriched secretomes prepared by the GMP-compliant process for manufacturing small extracellular vesicle-enriched fraction (sEV) preparations as described in Examples 12 and 13. In terms of efficacy in the scratch wound healing assay, all four variations of the final formulation method shown in Figure 24B and described in Example 13 are equally suitable. [Figure 31A-31B]Cardiomyocyte survival in staurosporine-induced cardiotoxicity assays is shown. Results for sample *7, sample a (Test 22), and *7, sample b (Test 22) (shown in Figure 24B) are shown in Figure 31A, with bars labeled 1 through 9 from left to right. Results for sample *7, sample c (Test 22), and *7, sample d (Test 22) (shown in Figure 24B) are shown in Figure 31B, with bars labeled 1 through 15 from left to right. 1× is equivalent to secretomes derived from 150,000 cells. PBS controls with and without staurosporine served as negative ("-ve," bar 2 in both figures) and positive ("+ve," bar 1 in both figures) controls, respectively. Mesenchymal stem cell (MSC)-derived EVs served as the EV control ("EV Ctl," bar 3 in both figures). Plated cells were either stressed with staurosporine for 4 hours prior to treatment ("+", bars 2-9 in Figure 31A and bars 2-15 in Figure 31B) or not stressed with staurosporine ("-", bar 1 in both figures). Samples a (Test 22); *7, sample b (Test 22); *7, sample c (Test 22); *7, and sample d (Test 22) (shown in Figure 24B), whose preparation is described in detail in Examples 12 and 13, were tested in the cardiomyocyte viability assay described in Example 17. These four samples originate from the same TFF retentate but differ in the method of final formulation. These four variations were: using fresh retentate and filter-sterilizing with a Sterivex-GP, 0.22 μm filter (result *7, sample a (trial 22)); using fresh retentate, supplemented with trehalose, and filter-sterilizing with a Sterivex-GP, 0.22 μm filter (result *7, sample b (trial 22)); freezing the retentate, thawing it, and then filter-sterilizing with a Sterivex-GP, 0.22 μm filter (result *7, sample c (trial 22)); or freezing the retentate, thawing it, and then filter-sterilizing with a Sartopore 2, 0.45 + 0. μm filter (result *7, sample d (trial 22)). In Figure 31A, at the 24-hour time point shown in the figure, the maximal effect seen with *7, sample a (trial 22) was 0.75 x dose (bar 6), which corresponded to an increase (improvement) in cell survival of 10.64% over the -ve control.At the 24 hour time point shown, the greatest effect seen for *7, sample b (Test 22) was 0.5x the dose (bar 8), which corresponded to an 11.85% increase (improvement) in cell viability over the -ve control. In Figure 31B, at the 24 hour time point shown, the greatest effect seen for *7, sample c (Test 22) was 0.75x the dose (bar 6), which corresponded to an 14.82% increase (improvement) in cell viability over the -ve control. At the 24 hour time point shown, the greatest effect seen for *7, sample d (Test 22) was 0.5x the dose (bar 11), which corresponded to an 11.90% increase (improvement) in cell viability over the -ve control. [Figure 34]Figure 1 shows echocardiographic results of mice with induced chronic heart failure after administration of CPC EV ("sEV5.3") or PBS (as a control). Data show absolute changes in left ventricular end-systolic volume (LVESV); left ventricular end-diastolic volume (LVEDV); and ejection fraction (EF). The bottom three graphs show absolute changes for each animal as individual points, with overlaid quantile plots for groups. For illustrative purposes, dotted horizontal lines have been added to each graph to indicate the approximate location of the thresholds used to define severe progressive heart failure for each of the three parameters. For illustrative purposes, dotted borders have been added to the figures to identify animals considered to have severe progressive heart failure in each graph. The actual number of animals with or without severe progressive heart failure is shown in large font on each graph. The left graph shows a threshold of 9.1 μL. Animals above this threshold have severe progressive heart failure. The middle graph shows a threshold of 4 μL. Animals above this threshold have severe progressive heart failure. The graph on the right shows the -5.5% threshold. Animals below this threshold have severely progressive heart failure and a severely reduced EF. In all three graphs, fewer sEV-treated animals had severely progressive heart failure than the PBS group. For absolute changes in LVESV, the sEV5.3 group had significantly fewer animals with severely progressive heart failure than the PBS controls (10 of 11 vs. 5 of 11 for PBS controls, p<0.05). For absolute changes in LVEDV, the sEV5.3 group had significantly fewer animals with severely progressive heart failure than the PBS controls (10 of 11 vs. 5 of 11 for PBS controls, p<0.05). For absolute changes in EVs, the sEV5.3 group had fewer animals with severely progressive heart failure than PBS controls, which approached significance (5 of 11 vs. 1 of 11 for PBS controls, p<0.05). Collectively, these results demonstrate that cardiac therapy candidate 1-extracellular vesicle-enriched secretome (CTC1-EVs) improves heart failure outcomes in animals with chronic heart failure by limiting the progression of that disease. [Figure 35]As shown in Figure 90, the results of lunatic analysis of cellular RNA extracted from *3 (Test 25) are shown. The RNA extracted from *3 (Test 25) is labeled as sample "546" in the figure. Figure 35 also shows the results of lunatic analysis of cellular RNA extracted from *3 (Test 26), as shown in Figure 96. 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. [Figure 36] *3 (Test 25) shows the results of a quality control (QC) test of the cellular RNA extracted from the 546 RNA sample. This RNA is referred to as "546 RNA" in the figure. This analysis was completed to assess the quality of the extracted RNA. [Figure 37] *3 (Study 26) shows the results of a quality control (QC) test of the cellular RNA extracted from the 547 RNA sample. This RNA is referred to as "547 RNA" in the figure. This analysis was completed to assess the quality of the extracted RNA. [Figure 38] *9 (Study 27) shows the results of lunatic analysis of CTC1-EV RNA extracted from the 9th study. This RNA is labeled "45.evrna" in the figure. This analysis was completed to assess the quality of the extracted RNA. [Figure 39] *9 (Trial 27) shows the results of a quality control (QC) test of CTC1-EV RNA extracted from the 9th study. This RNA is labeled "45.evrna" in the figure. This analysis was completed to assess the quality of the extracted RNA. [Figure 40] The results of quality control (QC) tests of cDNA libraries prepared from three different RNA samples are shown below. "Library from 546RNA" is a cDNA library generated from RNA extracted from *3 (Test 25). "Library from 547RNA" is a cDNA library generated from RNA extracted from *3 (Test 26). "Library from 45.evrna" is a cDNA library generated from RNA extracted from *9 (Test 27). These evaluations were performed to assess the quality of the cDNA libraries. [Figure 41]Shown are the results of sequencing read length analysis for small RNA sequencing analysis of CTC1-EV, which in this experiment is *9 (Study 27). [Figure 42] The prevalence (read distribution) of different RNA biotypes in CTC1-EV is shown, which is *9 (study 27) in this experiment. The RNA biotypes shown here were determined by sequence mapping. The results for this sample are identified in the figure as "45RNA." [Figure 43] Shown are the results of an analysis of read distribution for the top 20 miR isomers identified in CTC1-EV, which is *9 in this experiment (Study 27). The results for this sample are identified in the figure as "45RNA." [Figure 44] The top 40 most abundant miRNAs identified in CTC1-EV are shown, which is *9 (Study 27) in this experiment. Data are displayed as a honeycomb representation. Results for this sample are labeled as "45RNA" in this figure. The data used to generate Figure 44 are summarized in Table 9. [Figure 45] A word cloud showing the top localization terms associated with the RNA sequence identified in CTC1-EV, which in this experiment is *9 (Study 27). [Figure 45.1] Scatter plots are shown identifying miRNA signatures in CTC1-EVs compared to extracellular vesicles derived from other cell types included in this study: astrocytes, cardiac fibroblasts, cardiomyocytes, neurons (GABAergic, glutamatergic, dopaminergic, motor neurons, and induced neurons by forward reprogramming), endothelium, hematopoietic progenitor cells, hepatocytes, induced pluripotent stem cells, microglia, macrophages, mesenchymal stem cells, pericytes, and retinal pigment epithelium. The CTC1-EV miR signature was extracted by calculating the 10th percentile per gene of the log2 FPKM values ​​of the CTC1-EV sample replicates and the 90th percentile of all other samples in the study. [Figure 46-47] Cryo-electron micrograph of an extracellular vesicle identified in CTC1-EV, which in this experiment is *9 (study 27). Scale bar = 100 nm. [Figure 48] Cryo-electron micrograph of a large bilayer membrane vesicle approximately 200 nm in diameter (identified in this experiment as CTC1-EV, *9 (Study 27)), containing a second bilayer membrane vesicle of similar diameter and a third, smaller (approximately 50 nm in diameter) bilayer membrane. Scale bar = 100 nm. [Figure 49] Figure 1 shows a 96-well plate map for analysis of the effect of CTC1-EV in a HUVEC plating assay as described in Example 23. The CTC1-EV in this experiment is *5b.uc (Trial 26). This sample is labeled "EV 481" in the figure. A mock EV control is also included (labeled "EV 457" in the figure). [Figure 50]The effect of CTC1-EV in a HUVEC plating assay, as measured using a Tecan for Life Science® plate reader, is shown. Bars are labeled 1 through 7 from left to right. In this experiment, CTC1-EV (Test 26) (results shown in bars 4 and 5) was analyzed in a HUVEC plating assay. In this assay, the number of HUVEC cells in each well is determined by measuring the amount of intracellular ATP in the well, which is a surrogate for cell number. The amount of ATP is determined using the Cell Titer Glo kit as described in Example 23. The readout is luminescence. The higher the luminescence, the more ATP present in the well, which means more cells were present in the well. Therefore, higher luminescence indicates better HUVEC plating. In this assay, the positive control ("+ control," bar 1) is HUVEC cells plated in complete medium as described in Example 23. The negative control ("- control," bar 2) is HUVEC cells plated in poor medium as described in Example 23. For the remaining conditions, HUVEC cells were plated in poor medium supplemented with FBS-EV (bar 3), *5b.uc (Test 26) (bars 4 and 5), or a matched mock EV control ("Mock EV," bars 6 and 7), as described in Example 23. Results were double-normalized, with the negative control set to 0% and the positive control set to 100%. The positive control result is in the first bar on the left (100%). The negative control result is in the second bar from the left (0%). The FBS-EV condition gave a result of 60.37%. *5b.uc (Test 26), when administered at the first dose ("1x"), produced 29.11% of the luminescence of the positive control. *5b.uc (Test 26), when administered at a dose three times higher than the first dose ("3x"), produced 49.37% of the luminescence of the positive control. Matched mock EV controls were also administered at 1x and 3x doses, resulting in luminescence of 9.66% and 17.90% of the positive control. The greater the % luminescence in this assay, the greater the improvement the tested material has over HUVEC cell plating.Both the 1x and 3x doses of CTC1-EV tested here improve HUVEC plating in this assay compared to the negative control and compared to their corresponding mock EV controls, with the improvement in HUVEC seeding in this assay being more than 2-fold greater than that seen from the matched mock EV control. [Figure 51] Figure 1 shows the effect of CTC1-EV (which in this experiment is sample *5b.uc (trial 26)) in a HUVEC plating assay as measured by visual inspection (nuclei of live cells are labeled green, which resembles a light light gray in black and white rendering). This sample is labeled "CTC1-EV*5b.uc (trial 26)" in the figure. In this figure, the mock EV control is labeled "Mock EV." [Figure 52]The effect of CTC1-EV, sample *5b.uc (trial 26) in this experiment, in a HUVEC plating assay as determined by CyQuant nucleic acid staining is shown (bars labeled 1 through 7, from left to right). Sample *5b.uc was analyzed in the HUVEC plating assay 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 intracellularly. The higher the fluorescent 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 1) is HUVEC cells plated in their complete medium as described in Example 23. The negative control ("- control," bar 2) is HUVEC cells plated in poor medium as described in Example 23. For the remaining conditions, HUVEC cells were plated in poor medium supplemented with FBS-EV (bar 3), *5b.uc (bars 4 and 5), or a matched mock EV control ("Mock EV," bars 6 and 7), as described in Example 23. Results were double-normalized, with the negative control set to 0% and the positive control set to 100%. The positive control result is in the first bar on the left (100%). The negative control result is in the second bar from the left (0%). The FBS-EV condition gave a result of 36.34%. *5b.uc (Test 26), when administered at the first dose ("1x"), yielded 15.43% of the positive control. *5b.uc (Test 26), when administered at a dose three times higher than the first dose ("3x"), yielded 36.75% of the positive control. Matched mock EV controls were also administered at 1 and 3 doses, yielding -1.07% and 8.42% of the positive control. Both the 1x and 3x doses of CTC1-EV [*5b.uc (Study 26)] tested here improve HUVEC plating in this assay compared to the negative control and compared to their corresponding mock EV controls.The improvement in HUVEC seeding in this assay by the CTC1-EV tested here [*5b.uc (study 26)] is more than four-fold greater than any improvement seen from the matched mock EV control. [Figure 52.1]The effect of CTC1-EV, sample *5b.uc (Trial 26) in this experiment, in a HUVEC plating assay determined by CyQuant nucleic acid staining is shown (bars labeled 1 through 7, from left to right). Sample *5b.uc (Trial 26) (bars 4 and 5) was analyzed in the HUVEC plating assay described in Example 23. In this assay, the number of HUVEC cells in each well is determined by analyzing microscopic images, where cells are easily identified by CyQuant green staining. The higher 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 1) is HUVEC cells plated in their complete medium as described in Example 23. The negative control ("- control," bar 2) is HUVEC cells plated in poor medium as described in Example 23. For the remaining conditions, HUVEC cells were plated in poor medium supplemented with FBS-EV (bar 3), sample *5b.uc (Test 26) (bars 4 and 5), or a matched mock EV control ("Mock EV," bars 6 and 7), as described in Example 23. Results were double-normalized, with the negative control set to 0% and the positive control set to 100%. The positive control result is in the first bar on the left (100%). The negative control result is in the second bar from the left (0%). The FBS-EV condition gave a result of 54.47%. *5b.uc (Test 26), when administered at the first dose ("1x"), yielded 20.42% of the positive control. *5b.uc (Test 26), when administered at a dose three times higher than the first dose ("3x"), yielded 48.09% of the positive control. Matched mock EV controls were also administered at 1 and 3 doses, yielding -2.13% and 11.71% of the positive control. Both the 1x and 3x doses of CTC1-EV [*5b.uc (Study 26)] tested here improve HUVEC plating in this assay compared to the negative control and compared to their corresponding mock EV controls. The improvement in HUVEC seeding in this assay by CTC1-EV tested here [*5b.uc (Study 26)] is more than four-fold greater than any improvement seen from the matched mock EV control. [Figure 53] Results of analysis of CTC1-EVs in a HUVEC stress assay in which HUVECs were stressed with staurosporine as described in Example 24 are shown. Bars are designated 1 through 6 from left to right. Three different EV types were tested in the HUVEC stress assay. In this assay, HUVEC cells in culture were either unstressed ("Complete"; positive control; bar 1), stressed by culturing in serum-free medium ("Poor"; bar 2), or stressed by culturing in serum-free medium with staurosporine ("Poor + Staurosporine" condition; bars 3 through 6). In the "Poor + Staurosporine" condition, either a vehicle control ("dPBS"; bar 3) or a dose of 5 x 10 particles from one of three different EV-enriched secretome preparations was added to the culture medium. These EV-enriched secretome preparations were isolated from MSC-conditioned medium ("MSC-EV," bar 4), iCell-CPC-conditioned medium ("iCell-CPC-EV," bar 5), or CTC1-conditioned medium ("CTC1-EV," bar 6). In this example, CTC1-EV (bar 6) is *9 (Test 27). The number of HUVEC cells remaining in culture at the end of the assay period was determined. Results for each condition were normalized to the "dPBS" (bar 3) vehicle control condition. The CTC1-EV sample tested here [*9 (Test 27)] improved HUVEC cell survival by 40% in this experiment. [Figure 54]Figure 1 shows the results of analysis of EV-CPCs in a chemotherapy-induced in vitro cardiomyopathy assay, as determined by measuring intracellular ATP concentrations on day 6 (A), day 8 (B), and day 10 (C) in doxorubicin-stressed cardiomyocytes (and non-stressed control cardiomyocytes) as described in Example 25. Results were normalized to the control ("DOX + placebo") on the day of measurement. Results are from five separate experiments, with each sample within each experiment performed in triplicate. Bars represent mean + / - SEM. *p ≤ 0.05 (Kruskal-Wallis and Dunn's multiple comparison test). CM: complete maintenance cardiomyocyte medium; DOX: doxorubicin; EV-CPC: cardiac progenitor cell-derived extracellular vesicles; VM-CPC: CPC virgin medium; ATP: adenosine triphosphate. As can be seen in Figure 54D, the CTC1-EV sample (which in this experiment is *7, sample a (trial 20), and is labeled "CTC1-EV (prod 20)" in the figure) improved (increased) the amount of intracellular ATP per cell in doxorubicin-stressed cardiomyocytes by 40% compared to the stressed control. This result indicates that CTC1-EV [*7, sample a (trial 20)] was able to promote metabolic health of cardiomyocytes in viable cells. The results of the positive control are shown in bar 1 (numbered 1-3 from left to right). The results of the negative control, which were doxorubicin-stressed cells, are shown in bar 2. The results of CTC1-EV treatment of doxorubicin-stressed cells are shown in bar 3. [Figure 55-56] Figure 55 shows the results of an anti-fibrosis assay in which HCF cells were stimulated with TGF-β1, and then the effects of MSC-EVs and CTC1-EVs [*9 (Study 27) in this experiment] on various fibrosis-associated markers were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) as described in Example 26 (bars are indicated as 1-10 in both figures from left to right). The results of MMP2 expression analysis are shown in Figure 55. The results of periostin ("Postn") expression analysis are shown in Figure 56. In this example, CTC1-EVs are *9 (Study 27) (bars 5, 6, 9, and 10 in each figure). [Figure 56.1]1 shows the experimental schedule for the experiment described in Example 27. A timeline is shown showing 5 days when rats received IP injections of doxorubicin (if applicable), 3 days when rats were evaluated by echocardiography, and 3 days when rats received IV injections of placebo (NaCl) or CTC1-EV (if applicable), where CTC1-EV is *7, sample a (trial 20) in this experiment. In this figure, *7, sample a (trial 20) is labeled "GMP-EV." [Figure 57A-57B]Figure 57 shows the effect of CTC1-EV (which is *7 sample a (study 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 (study 20) is labeled "GMP-EV." Figure 57A shows the % change in LV-ESV since D10. Figure 57B shows the % change in LV-EDV since day 10. Results were measured by echocardiography and expressed as percent change (median + / - IQR) from day 10 (post-DOX administration). There were 6 animals that did not receive doxorubicin (sham: n=6). Eleven animals underwent doxorubicin stress followed by NaCl injection ("DOX + placebo"; n = 11), and 12 animals underwent doxorubicin stress followed by *7, sample a (study 20) injection ("DOX + GMP-EV"; n = 12). In this figure, the symbol "*" indicates p < 0.05, Kruskal-Wallis test with Dunn's correction. LV-ESV / LV-EDV: Left ventricular systolic / diastolic function. Cardiac function is related to heart volume. Here, two types of heart volumes are examined: left ventricular end-systolic volume (LVESV or LV-ESV) and left ventricular end-diastolic volume (LVEDV or LV-EDV). These two volumes increase during heart failure. The greater their increase, the more advanced the heart failure. These two volumes are measured by echocardiography (echo). Two volumes are measured for each animal: before doxorubicin injection (or before sham injection in the case of "sham" animals) (baseline echo, Echo #1), then 10 days after the first doxorubicin administration / sham injection (before any CTC1-EV treatment or placebo administration; Echo #2), and finally at the end of the study period, 28 or approximately 29 days after the first doxorubicin injection / sham injection (Echo #3). The CTC1-EV in this experiment was *7, sample a (Study 27). The group of rats administered this substance is referred to as "Dox+GMP-EV" in Figures 57A and 57B. The placebo group received isotonic buffer, NaCl 0.9%, "placebo." This group of animals is referred to as "DOX+Placebo" in Figures 57A and 57B. CTC1-EV or placebo was administered 11, 14, and 16 days after the first doxorubicin injection, as shown in Figure 56.1.The greater the heart volume between Echo #2 and Echo #3, the more heart failure the animal experienced during that time period. "Sham" animals did not have heart failure and did not display markers of a failing heart. Sham animals were not administered doxorubicin or CTC1-EV. For the experiment shown in Figures 57A and 57B, results are expressed as percent change (median + / - IQR) from day 10 after DOX administration (Echo #2) to the end of the study period (Echo #3). There were 6 sham animals, 11 placebo-injected animals, and 12 CTC1-EV-injected animals. (Sham: n = 6; DOX + placebo: n = 11, Dox + GMP-EV: n = 12). For LV-ESV results (shown in Figure 57A), the sham group had an average change in volume of -3.0%, the DOX + placebo group increased LVESV by an average of 28.1%, and Dox + GMP-EV increased LVESV by 12.9%, meaning that their heart failure progressed less than half as much as the placebo group as determined by LV-ESV change, a 2.2-fold improvement in outcome. For LV-EDV results (shown in Figure 57B), the sham group had an average change in volume of -0.1%, the DOX + placebo group increased LVEDV by an average of 19.2%, and Dox + GMP-EV increased LVEDV by 0.7%, meaning that their heart failure progressed less than one-quarter (0.7 / 19.2) as determined by LV-ESV change as the placebo group, a 27-fold improvement in outcome. LV-ESV volume was significantly increased in placebo-injected hearts compared with sham-treated hearts (p = 0.033) but preserved by GMP-EV injection (effect size Hedges' g index = 0.4). Similarly, the percentage of responder rats that did not increase LV-EDV volume by more than 5% from pretreatment values ​​after DOX was 58% (7 of 12) vs. 28% (3 of 11) in Dox + GMP-EV and DOX + placebo hearts, respectively (effect size Hedges' g index = 0.5, OR = 3.7). [Figures 58A-58E]Figure 58A shows the results of an experiment validating the rat model of doxorubicin-induced cardiomyopathy described in Example 27. Figure 58A shows LVEF as percent change (mean + / - SEM) from day 10 (post-DOX administration). Figure 58B shows the final test ratio of diastolic blood pressure to LV-EDV, used as a surrogate marker of ventricular compliance. Figure 58C shows mean blood pressure. Figure 58D shows QT intervals corrected for heart rate. **p≦0.005; (Mann-Whitney test). LVEF: left ventricular ejection fraction (%); QTc: QT interval length (in seconds, "sec") corrected for heart rate (HR); DBP: diastolic blood pressure; LV-EDV: left ventricular end-diastolic volume. Figure 58E shows the results of an experiment further validating the rat model of doxorubicin-induced cardiomyopathy described in Example 27. This figure shows the end-of-study ratio of systolic blood pressure to LV-ESV. This ratio is considered a surrogate marker of ventricular contractility. This ratio is called "end-systolic diastolic volume." LV-EDV: left ventricular end-diastolic volume; SBP: systolic blood pressure. The data show that mean SBP / LV-ESV decreased by 0.46 mmHg / uL (a 34% decrease) in doxorubicin (DOX)-treated animals (the "DOX+placebo" group) from animals that did not receive DOX (the "sham" group). [Figure 58.1] Two experimental designs are shown for experiments used to establish a novel chemotherapy-induced cardiomyopathy model in rats. The first experiment, in which six male rats were injected with doxorubicin, resulted in unacceptable mortality over a 30- or 32-day long-term treatment period. 70% of these male rats died before the end of the study period. The second experiment, which included both male and female rats, is shown. Female rats had a much higher survival rate than males (91% vs. 40%, respectively). d = day; DOXO = doxorubicin injection; Echo = echocardiographic measurement. [Figure 59-60]Figures 59 and 60 show the results of an experiment analyzing the post-thaw viability of CTC1 cells under different conditions, as described in Example 28 (referred to as bars 1 through 6, from left to right in each figure). Three modifications to the process were tested to improve post-thaw CTC1 survival, seeding, and proliferation. Details of this experiment are provided in Example 28. The starting process thawed cells in the same medium used for plating and proliferation (referred to as complete medium A, "CM A," bars 1 and 2 in both Figures 59 and 60). The initiation process used a gentle centrifugation step to pellet the cells after thawing, allowing for removal of the cryopreservation medium (the use of the centrifugation step is referred to as centrifuged, "Centrifuged," bars 1, 3, and 5 in both Figures 59 and 60). The initiation process used thawing medium containing 2 mg / mL human serum albumin. The initiation process used thawing medium without ROC inhibitors. This starting process is referred to as "CM A Centrifuged" in Figures 59 and 60. The number of viable cells placed in each vial during the cryopreservation phase was known. The number of viable cells recovered after the thawing process was recorded. The percentage of cells recovered upon thawing ("% recovered") was calculated by dividing the number of viable cells per vial after the thawing process by the number of viable cells placed in each vial before cryopreservation and multiplying by 100%. The higher the percentage recovered upon thawing, the more successful the thawing process was considered. The % recovered was calculated for the six conditions shown in Figure 59 and the six conditions shown in Figure 60, which varied depending on the thawing medium composition used and whether a centrifugation step was included in the process. The recipes for the various thawing media used are detailed in Example 28. In Figure 59, the results of the start-up process (CM A with a centrifugation step; "CM A Centrifugation") are shown in bar 1. The results of the modified process using the starting thawing medium but omitting the centrifugation step ("No CM A Centrifugation") are shown in bar 2. The results of the modified process using thawing medium containing a higher albumin concentration (20 mg / mL HSA) with and without a centrifugation step ("CM B Centrifugation", "CM B No Centrifugation") are shown in bars 3 and 4, respectively.The results of the modified process using a thawing medium containing a higher albumin concentration (20 mg / mL) and 1 μM H1152 ROC inhibitor, with or without a centrifugation step ("CM C Cent" and "CM C No Cent" respectively), are shown in bars 5 and 6, respectively. Figure 60 shows the results of post-thaw cell viability assays performed under different conditions. In Figure 60, the results of the starting process (CM A with a centrifugation step; "CM A Cent") are shown in a bar graph. The results of the modified process using the starting thawing medium but omitting the centrifugation step ("CM A No Cent") are shown in bar 2. The results of the modified process using a thawing medium containing 1 μM H1152 with and without a centrifugation step ("CM B Cent" and "CM B No Cent") are shown in bars 3 and 4, respectively. Note that this is a different condition from the "CM B" conditions shown in Figure 59. The results of the modified process using a thawing medium containing a higher albumin concentration (20 mg / mL) and 1 μM H1152 ROC inhibitor, with or without a centrifugation step ("CM C Centrifuge" and "No CM Centrifuge," respectively), are shown in bar graphs 5 and 6, respectively. Collectively, the results in Figures 59 and 60 show that when the centrifugation step used to remove cryopreservation medium components is omitted, the % recovery upon thawing increases by an average of 6.45 percentage points over the adapted centrifugation conditions. (6.45 is the average of -0.2, +11.1, +6.6, +6.5, +7.4, and +7.3.) [Figure 61] 1 shows the experimental design for analyzing the post-thaw plating potential of CTC1 cells as described in Example 28. [Figure 62] 1 shows the results of an experiment analyzing cell density (cells / cm 2 ) after thawing and plating CTC1 cells under different conditions as described in Example 28. [Figure 63] 10 shows the results of an experiment to optimize CTC1 cell culture vessels for seeding, as described in Example 29. [Figure 64] 1 shows the experimental design for analyzing the effect of insulin concentration on CTC1 cell yield throughout vesicle formation, as described in Example 30. [Figure 65] 10 shows the results of an experiment analyzing the effect of insulin concentration on CTC1 cell yield throughout vesicle formation, as described in Example 30. [Figure 66] 1 shows the experimental design for analyzing the effect of FGF concentration on CTC1 cells as described in Example 31. [Figure 67] 1 shows the results of an experiment analyzing CTC1 cell numbers after incubation with different FGF concentrations as described in Example 31. [Figure 68] Results of a scratch wound healing experiment using EVs / secretomes from CTC1 cells incubated with different FGF concentrations as described in Example 31 are shown (lines labeled A-H, from highest to lowest at 18 hours). Conditions included in the assay include a positive control (line A); high FGF, MC (labeled "our standard protocol" and line B); intermediate FGF, MC (line C); low FGF, MV (line D); low FGF, MC (line E); intermediate FGF, MV (line F); high FGF, MV (line G); and a negative control (line H). Results at 18 hours are also summarized in Table 22. [Figure 69]

[0033] Figure 23 shows the results of a cardiomyocyte viability assay experiment using EVs / secretomes from CTC1 cells incubated with different FGF concentrations, as described in Example 31. Results at 19 hours are also summarized in Table 23. [Figure 70A-70B]Figure 1 shows the results of monitoring scratch wound healing over time for fresh media samples. The y-axis is % wound confluence, and the x-axis is time from the start of the assay. At the 18-hour time point, UF retentates (lines labeled A-P from top to bottom) were generated from freshly collected and freshly clarified CTC1-conditioned media as described in Example 32 (lines D-H). UF retentates from freshly collected and clarified CPC virgin media controls were prepared as described in Example 32 ("Mock EV control," lines I, K-P). Fresh conditioned media was also subjected to ultracentrifugation as described in Example 32 ("Fresh UC-generated EVs," line B). EV-enriched secretomes were also prepared from FBS by ultracentrifugation ("FBS control," line C). These compositions were tested for their ability to stimulate HUVEC scratch wound healing in a HUVEC scratch wound healing assay as described in Example 32. The positive control was HUVECs maintained in complete medium ("Complete Medium Control," line A). The positive control achieved 33.2% wound confluence at 18 hours. The negative control was HUVECs cultured in serum-free or serum-reduced medium ("Poor Medium Control," line J). To obtain a negative control in this HUVEC scratch wound healing assay, a retentate sample was administered at a 3x dose (the 1x dose was the secretome / EV / secretome fraction produced by 150,000 mother cells). The mock EV control was volume-matched to the corresponding condition. A fresh UC-generated EV sample was administered at 1x. This figure shows the time course results of monitoring scratch wound healing for fresh medium samples. At 18 hours, the fresh UC-generated EV sample (line B) had the strongest effect among all the tested conditions. Fresh UF retentate fractions containing components with molecular weights ranging from 5 kDa to 10 kDa (line H), 10 kDa to -30 kDa (line G), 30 kDa to -50 kDa (line F), 50 kDa to 100 kDa (line D), and 100 kDa to 0.2 µm (line E) all had a greater effect on HUVEC scratch wound healing than mock EV and poor medium controls, indicating that the retentate has a positive effect on endothelial cell migration into the scratch.The strongest effect is caused by components of the conditioned medium with molecular weights between 30 kDa and 0.2 μm. At 15 hours, the 30 kDa-50 kDa retentate (line F) has a 1.4-fold greater wound confluence than the negative control (line J). At 15 hours, the 50 kDa-100 kDa retentate (line D) has a 1.7-fold greater wound confluence than the negative control (line J). At 15 hours, the 100 kDa-2 μm retentate (line D) has a 2.3-fold greater wound confluence than the negative control (line J). The results for the 18-hour time point shown in Figure 70A are shown in Table 24. The results for the 18-hour time point shown in Figure 70B are shown in Table 25. [Figure 70.1] This is an alternative representation of the data presented in Figure 70A. Additionally, results are shown for CTC1-EV secretome compositions prepared by ultracentrifugation of previously frozen CTC1 conditioned medium (labeled "EV181 Frozen UC" in the figure) and its mock EV control (labeled "EV189 Frozen UC MV" in the figure). Results for the 18 hour time point shown in Figure 70.1 are shown in Table 26. [Figure 71]

[0041] Figure 71 shows the results of monitoring scratch wound healing over time for CTC1-EV compositions isolated from fresh or frozen / thawed media samples as described in Example 32. The y-axis is % wound confluence and the x-axis is time from the start of the assay. The results for the 18 hour time point shown in Figure 71 are shown in Table 27. [Figure 72-74] 7 shows histograms of double-normalized data from the cardiomyocyte survival assay described in Example 32. Figure 72, bars labeled 1-7 from left to right. Figure 73, bars labeled 1-14 from left to right. Figure 74, bars labeled 1-14 from left to right. [Figure 77]Results of flow cytometry analysis described in Example 35 are shown. Sample preparation is described in detail in Example 19. Data are expressed as mean fluorescence intensity (MFI) of technical and biological replicates. The MFI of iPSCs is shown by the white bars (first series of bars starting from the left, "iPSC" series). The mean MFI of "CPC D+0" (second series of bars starting from the left) was calculated by averaging the results of *1 (Test 25) and *1 (Test 26). The mean MFI of "CPC D+3" (third series of bars starting from the left) was calculated by averaging the results of *2 (Test 25) and *2 (Test 26). The mean MFI of "CPC D+5" (fourth series of bars starting from the left) was calculated by averaging samples *3 (Test 25) and *3 (Test 26). The mean MFI of CM samples is shown by the black bars ("CM" series, fifth series of bars starting from the left). [Figure 78] Figure 78 shows the results of transcriptome analysis of cells on day +3 ("D+3") and day +5 ("D+5") as described in Example 35. Sample preparation is described in Example 19. From the transcriptome analysis shown in the figure, cells harvested from Example 25 on day +3, Example 25 on day +5, Example 26 on day +3, and Example 26 on day +5 were mRNA-matched to cardiovascular progenitor cells (CPCs). The mRNA profiles of CPCs are similar across the four cell samples. The mRNA profiles of CPCs differ from both the iPSC and CM controls. Heatmaps were generated based on hierarchical clustering analysis using the UPGMA clustering method and correlation distance metrics in TIBCO Spotfire software v11.2.0. The data (log2FPKM) used to generate the heatmap shown in Figure 78 are listed in Table 36. [Figure 79]Figure 1 shows the results of cell morphology analysis of cells on day +3 (labeled "CPC D+3" in the figure) and day +5 (labeled "CPC D+5" in the figure), as described in Example 35. Sample preparation is described in Example 19. Microscopic images shown in the figure indicate that cell morphology is similar between *2 (Test 25) and *2 (Test 26) on day +3. Microscopic images shown in the figure indicate that cell morphology is similar between Test *3 (Test 25) and *3 (Test 26) on day +5. [Figure 80-81] Particle concentrations, mean and mode values ​​are shown 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. Sample preparation is described in Example 19. Particle concentrations increased 67-fold between Sample *5 (Test 25) and Sample *6 (Test 25). Particle concentrations increased 58-fold between Sample *5 (Test 25) and Sample *7 (Test 25). Particle concentrations increased 56-fold between Sample *5 (Test 26) and Sample *7 (Test 26), a factor very similar to the fold change of Test Example 25. There is no significant change in particle concentration between Sample *8 (Test 27) and Sample *9 (Test 27). The final concentration of particles in *9 (Test 27) is 2.8 x 10 particles / mL as measured by Nanosight. Throughout Tests 25, 26, and 27, the mean particle size remained relatively constant, ranging from 119.7 to 169.8 nm. Throughout Tests 25, 26, and 27, the mean modal particle size also remained relatively constant, ranging from 82.4 to 121.5 nm. [Fig. 82-84]Figure 8 shows CTC1-EV surface marker expression assessed as described in Examples 10 and 35. All results are normalized to a 13 μL sample. In these figures, the average of the results for *4 (Test 25) and *4 (Test 26) is indicated as "*4 (Test 25-26)." In these figures, the average of the results for *5 (Test 25) and *5 (Test 26) is indicated as "*5 (Test 25-26)." In these figures, several technical replicates were assessed for *8 (Test 27), and the average is shown. In these figures, a single result obtained for *9 (Test 27) is shown. Figure 82 shows that the MFI of the tetraspanin markers CD9, CD81, and CD63 significantly increases from the medium sample *4 (Test 25-26) to the final formulation *9 (Test 27), by more than 12-fold, 10-fold, and 7-fold for CD9, CD63, and CD81, respectively. The relative MFIs of three standard tetraspanin EV markers (CD9, CD63, and CD81) in *9 (Study 27) are among the top six highest MFI responses of all proteins investigated in this assay. Using a 13 μL sample, CD9 had the sixth-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 in this assay. Collectively, the strong presence of the three tetraspanin markers indicates the presence of extracellular vesicles. Other markers with high MFIs using a 13 μL sample are CD326 (102.3), CD133 / 1 (333.4), and CD29 (47.7). Markers with an MFI greater than 1.5 (= markers expressed in *9 (Study 27)) using a 13 μL sample are shown in Figures 82 and 83. Markers with MFI less than 1.5 (= little or no expression on *9(Test 27)) measured in the final formulation (*9(Test 27)) are shown in Figure 84. [Figure 84.1] *Fragment size results obtained for 9 (Test 27) are shown. Peaks have sizes of 179, 368, 537 and 742 base pairs. [Figure 85]3 shows the results of the HUVEC scratch wound healing assay described in Example 37.1 (lines are labeled A-I from top to bottom based on their position at the 24 hour time point). The complete medium positive control (labeled "Complete (dotted)" on line A in the figure) reached 89.33% wound confluence after 24 hours. The poor medium negative control (labeled "Poor (dashed)" on line I in the figure) reached 13.22% wound confluence after 24 hours. The control EV sample, which is the pellet material obtained after ultracentrifugation of FBS (line D, labeled "Control EV (dotted line)" in the figure), reached 41.29% wound confluence after 24 hours. *9 (Trial 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 secretome collected from 150,000 cells. Results are shown as colored circles connected by solid lines. The 24-hour results shown in Figure 85 are shown in Table 51. [Figure 86] Results of the HUVEC scratch wound healing assay at 18 hours, as described in Example 37.1, are shown (bars 1–9, from left to right). The assay included a complete medium positive control ("+ve" in the figure, bar 1) and a poor medium negative control ("-ve" in the figure, bar 2). The control EV sample (pellet material obtained after FBS ultracentrifugation, labeled "EV Ctl" in the figure, bar 3) reached 31% normalized wound confluence after 18 hours. EV samples (*9 (Test 27), bars 4–9) were tested in this assay at 0.25× (bar 4), 0.5× (bar 5), 0.75× (bar 6), 1× (bar 7), 2× (bar 8), and 2.8× (bar 9). 1× represents 5.79 μL of material, representing secretomes prepared from 150,000 cells. [Figure 87]The cardiomyocyte viability assay described in Example 37.2 (lines labeled A through F from top to bottom based on position at the 24 hour time point) shows the results of an in vitro analysis (Study 27) of the efficacy of *9, the preparation of which is detailed in Example 19. The percent of positive NucLight Red cells normalized to TO are shown at the indicated time points. The 24 hour data points shown in Figure 87 are presented in Table 52. [Figure 88] Results are shown at the 24 hour time point (bars labeled 1-6 from left to right) of an in vitro analysis of the efficacy of *9 in the cardiomyocyte survival assay described in Example 37.2 (Study 27). Results were subtracted from baseline (negative control) and normalized to the positive control. [Figure 89] Figure 3 shows the results of an in vitro analysis of the efficacy of *9 (Test 27) in the scratch wound healing assay (bars designated 1-14 from left to right) described in Example 38. A "complete medium" positive control (bar 1) and an "FBS-EV" control (bar 3) were used as positive controls. The negative control was a "poor medium" control (bar 2). The complete medium (bar 1) control consisted of HUVECs cultured in complete medium [Endothelial Cell Basal Medium (PromoCell; ref. C-22210) supplemented with Endothelial Cell Growth Medium Supplement Pack (PromoCell, ref. C-39210)]. The poor medium control (bar 2) consists of HUVECs cultured in poor medium only [Endothelial Cell Basal Medium (PromoCell; ref: C-22210]. The "FBS-EV" control (bar 3) consists of HUVECs cultured in poor medium supplemented with 5 x 10 particles of FBS-EV (FBS-EV is an EV-enriched secretome produced by ultracentrifugation of fetal bovine serum). *Four separate vials (trial 27) of nine were tested at doses ranging from 1 x 10 to 5 x 10 particles / well as measured by NTA (bars 4–14). The particle dose per well is indicated below the corresponding data bar. [Figure 90] 1 shows the process used to produce Example 25 as described in Example 19. [Figure 91]The results of the HUVEC scratch wound healing assay described in Example 41 (referred to as bars 1 through 12, from left to right) are shown. The positive control (labeled "+ve" in the figure, bar 1) is a HUVEC scratch wound healing assay performed in the presence of complete assay medium (labeled "Complete" in the figure) and treated with a vehicle control, 0.1 μm-filtered PBS (labeled "PBS" in the figure). The negative control (labeled "-ve" in bar 2) is a HUVEC scratch wound healing assay performed in poor assay medium (labeled "Poor" in the figure) and treated with a vehicle control, 0.1 μm-filtered PBS (labeled "PBS" in the figure). The EV control (labeled "EV Ctl" in bar 3) is the pellet collected after FBS ultracentrifugation. Figure 1 shows the results of a HUVEC scratch wound healing assay for 1x (bar 4), 2x (bar 5), and 3x (bar 6) doses of *5a.uc (Study 25), the preparation of which is described in detail in Example 19. Figure 2 shows the results of a HUVEC scratch wound healing assay for 1x (bar 7), 2x (bar 8), and 3x (bar 9) doses of Study Example 26 CTC1-EV (Sample *5b.uc), the preparation of which is described in detail in Example 19. Figure 3 shows the results of a HUVEC scratch wound healing assay for 1x (bar 10), 2x (bar 11), and 3x (bar 12) doses of a mock EV control (labeled "MV Control" in the figure). The results of this assay are doubly normalized so that the "-ve" control (bar 2) is at 0% wound confluence and the "+ve" control (bar 1) is at 100% wound confluence at 18 hours. [Figure 93A-93B] 1 shows the results of a stability study of *9 (Study 27) by NTA (described in Example 43). [Figure 94]Figure 4 shows the results of a HUVEC survival assay at various time points as described in Example 44. The sample is *9 (Test 27), (labeled "Poor medium with staurosporine (0.01 μM) + EV [Test 27 (*9)]" in the figure, and the results are shown in the third bar of each cluster of three bars). Data are shown as fold change relative to the negative control (labeled "Poor medium with staurosporine (0.01 μM)" in the figure, and the second bar in each cluster of three bars). Results for the positive control are also shown as fold change relative to the negative control (labeled "Poor medium without staurosporine" in the figure, and the results are shown in the first bar of each cluster of three bars). [Figure 95] Figure 1 shows the results of the H9c2 cell viability assay described in Example 45. Results of technical replicates were averaged and normalized to the virgin medium 100 kDa test condition. [Figure 96] 1 shows the process used to produce Example 26 as described in Example 19. [Figure 97] 1 shows the process used to produce Example 27 as described in Example 19. [Figure 98A] Figure 1 shows the correlation found between particle number measured by NTA (106 particles) and CD9 MFI measured by the MACSPlex Exosome Kit®. [Figure 98B] 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 probing for CD9 (Miltenyi Reference: 130-108-813)) versus input volume for a range of 1 and 120 µL of input is shown in *9 (Test 27). [Figure 99]Figure 99B shows a representative cluster from *9 (Study 27) visualized by ONi, CD81 / CD63 / CD9_TP. CD9, CD81, and CD63 signals are shown individually as an overlay, confirming the presence of each of the three markers in a single cluster. Figure 99B shows the relative abundance of each cluster subtype in *9 (Study 27) detected by ONi super-resolution microscopy (shown as % below each bar). Absolute counts are graphed in the figure and shown above each bar. [Figure 100A] The results of GO (Gene Ontology) enrichment analysis for biological processes were analyzed using String Prot. [Figure 100B] Some of the molecular components identified in CTC1-EV are shown. Image created with BioRender.com. [Figure 100C] Some of the protein components identified in CTC1-EVs and the biological processes they participate in are shown in Figure 1. The various processes shown highlight the potential for CTC1-EVs to have multiple beneficial effects on multiple cell types, resulting in improved physiological outcomes. [Figure 100D] Some of the key biological effects of CTC1-EV treatment described herein are summarized below. Importantly, data from mice, rats, human cells, and human patients confirm that CTC1-EVs can confer molecular, cellular, and physiological effects on target cells and tissues that are beneficial to stressed human cells and mammals with ventricular dysfunction, including humans with heart failure. [Figure 101] Schematic diagram of clinical trial design. DETAILED DESCRIPTION OF THE INVENTION

[0171] It 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 this 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.

[0172] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although other methods and materials similar or equivalent to those described herein can be useful in the present invention, the preferred materials and methods are described herein.

[0173] As used herein, the terms "subject," "individual," or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, including, but not limited to, humans and other primates, including non-human primates such as rhesus monkeys, chimpanzees, and other monkey and ape species; livestock, e.g., cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including chickens, turkeys, and other poultry, poultry such as ducks, geese, and wild and game birds. The term does not denote a particular age or sex. Thus, the term includes adults, juveniles, and newborns, as well as males and females. In some embodiments, cells (e.g., stem cells, including pluripotent stem cells, progenitor cells, or tissue-specific cells) are derived from a subject. In some embodiments, the subject is a non-human subject.

[0174] As used herein, "differentiation" refers to the process by which an unspecialized cell (e.g., a pluripotent or other stem cell) or a multipotent or hypopotent cell acquires specialized structural and / or functional features characteristic of, for example, a more mature or fully mature cell. "Transdifferentiation" is the process of transforming one differentiated cell type into another differentiated cell type or specific fate.

[0175] As used herein, "embryoid body" refers to a three-dimensional aggregate of pluripotent stem cells. These cells can differentiate into cells of three germ layers: endoderm, mesoderm, and ectoderm. The three-dimensional structure, including the establishment of complex cell adhesion and paracrine signaling within the embryoid body microenvironment, allows for differentiation and morphogenesis.

[0176] As used herein, "stem cells" refer to cells that have the ability to self-renew, i.e., to undergo multiple cell division cycles while maintaining their non-terminally differentiated state. Stem cells can be totipotent, pluripotent, multipotent, oligodendrocyte, or unipotent. Stem cells can be, for example, embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.

[0177] As used herein, "pluripotent stem cells" (PSCs) refer to cells that have the ability to endlessly regenerate themselves and differentiate into any other cell type of an adult organism. Generally, pluripotent stem cells are stem cells capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; they can differentiate into cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types); and they express one or more markers characteristic of PSCs. Examples of such markers expressed by PSCs, such as embryonic stem cells (ESCs) and iPSCs, include Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and REX1.

[0178] As used herein, "induced pluripotent stem cells" (iPSCs) refer to a type of pluripotent stem cell 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, skin fibroblasts, bone marrow-derived mesenchymal cells, cardiomyocytes, keratinocytes, liver cells, stomach cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Further 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.

[0179] iPSCs can be generated by reprogramming somatic cells, by expressing or inducing the expression of one or a combination of factors (referred to herein as reprogramming factors) in the somatic cells. iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In some examples, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, OCT4 (OCT3 / 4), SOX2, c-MYC, and KLF4, NANOG, and LIN28. In some examples, somatic cells can be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors to reprogram somatic cells into pluripotent stem cells. Cells can 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 can also be engineered to introduce reprogramming factors or activate endogenous programming genes, for example, using CRISPR-Cas9-based techniques.

[0180] As used herein, "embryonic stem cells" are embryonic cells derived from embryonic tissue, preferably the inner cell mass of a blastocyst or morula, optionally serially passaged as a cell line. 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, for example, from a zygote, blastomere, or blastocyst-stage mammalian embryo produced by sperm-egg fusion, nuclear transfer, or parthenogenesis. Human ESCs include, but are not limited to, MAO1, MAO9, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of a blastocyst-stage embryo and 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 asexual reproductive means, including fertilization or somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone cannot develop into fetuses or adult animals when implanted in utero because they do not have the potential to contribute to all extraembryonic tissues (e.g., placenta in vivo or trophoblast cells in vitro).

[0181] As used herein, the term "progenitor cells" refers to the descendants of stem cells that can further differentiate into one or more specialized cells but cannot divide and regenerate indefinitely. That is, unlike stem cells (which have unlimited potential for self-renewal), progenitor cells have only limited potential for self-renewal. Progenitor cells can be multipotent, hypopotent, or unipotent and are typically classified according to the type of specialized cell they can differentiate into. For example, "cardiomyocyte progenitor cells" are progenitor cells derived from stem cells that have the potential to differentiate into cardiomyocytes. Similarly, "cardiac progenitor cells" can differentiate into multiple specialized cells that constitute cardiac tissue, including, for example, cardiomyocytes, smooth muscle cells, and endothelial cells. Furthermore, "cardiovascular progenitor cells" have the potential to differentiate into cells of the cardiac and vascular lineages, for example.

[0182] As used herein, "expanding" or "growing" can refer to the process by which the number of cells in a cell culture is increased by cell division.

[0183] "Pluripotent" means that a cell is capable, through its progeny, of giving rise to several different cell types found in the adult animal.

[0184] "Pluripotent" means that a cell can, through its progeny, give rise to all cell types that make up an adult animal, including germ cells. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.

[0185] As used herein, the term "autologous cells" refers to donor cells that are genetically identical to the recipient.

[0186] As used herein, the term "allogeneic cells" refers to cells derived from different, genetically non-identical individuals of the same species.

[0187] As used herein, the term "totipotent" can refer to a cell that gives rise to a live, born animal. The term "totipotent" can also refer to a cell that gives rise to all cells within a particular animal. A totipotent cell, when utilized in a procedure to develop an embryo from one or more nuclear transfer steps, can give rise to all of the cells of the animal.

[0188] As used herein, the term "extracellular vesicles" refers collectively to biological nanoparticles derived from cells, examples of which include, but are not limited to, exosomes, ectosomes, exovesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, membrane vesicles, open vesicles, membrane particles, open microvesicles, oncosomes, exomers, and apoptotic bodies.

[0189] Extracellular vesicles can be classified, for example, according to size. For example, as used herein, the term "small extracellular vesicles" refers to extracellular vesicles having a diameter of approximately 50-200 nm. In contrast, extracellular vesicles having a diameter of more than approximately 200 nm but less than 400 nm may be referred to as "medium extracellular vesicles," and extracellular vesicles having a diameter of more than approximately 400 nm may be referred to as "large extracellular vesicles." As used herein, the term "small extracellular vesicle fraction" ("sEVs") refers to a portion, extract, or fraction of a secretome or conditioned medium that has been enriched and / or concentrated for small extracellular vesicles having a diameter of approximately 50-200 nm. Such enrichment and / or concentration can be achieved 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, achieved, or possible.

[0190] As used herein, the term "exosomes" refers to extracellular vesicles that are released from cells upon fusion of multivesicular bodies (MVBs) (intermediate endocytic compartments) with the plasma membrane.

[0191] "Exosome-like vesicles," which share a common origin with exosomes, are typically described as having size and sedimentation properties that distinguish them from exosomes, particularly as lacking lipid raft microdomains. As used herein, "ectosomes" are typically microvesicles derived from neutrophils or monocytes.

[0192] As used herein, "microparticles" are typically about 50-1000 nm in diameter and are derived from the plasma membrane. "Extracellular membrane structures" also include membrane structures from other cellular sources, including linear or folded membrane fragments from necrotic death, as well as secreted lysosomes and nanotubes.

[0193] As used herein, an "apoptotic bleb or body" is typically about 1-5 μm in diameter and is released as a bleb of cells undergoing apoptosis, i.e., diseased, unwanted and / or abnormal cells.

[0194] Within this class of extracellular vesicles, a key component is the "exosome" itself, which can range in diameter from about 40-50 nm to about 200 nm and are membranous vesicles of endocytic origin, i.e., vesicles surrounded by a phospholipid bilayer, resulting from exocytic fusion or "exocytosis" of multivesicular bodies (MVBs). In some cases, exosomes can range in diameter from about 40-50 nm up to about 200 nm, e.g., 60 nm to 180 nm.

[0195] As used herein, the terms "secretome" and "secretome composition" refer interchangeably to one or more molecules and / or biological factors secreted by cells into the extracellular space (e.g., into culture medium). Secretomes or secretome compositions can include, but are not limited to, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space (e.g., into culture medium). Secretomes or secretome compositions can be left unpurified or further processed (e.g., components of a secretome or secretome composition can be present in a culture medium, such as a conditioned medium. Alternatively, components of a secretome or secretome composition can be purified, isolated, and / or enriched from a culture medium or an extract, part, or fraction thereof). Secretomes or secretome compositions can further include one or more substances not secreted by cells, including medium components (e.g., medium, additives, nutrients, etc.). Alternatively, the secretome or secretome composition does not include media components (eg, media, additives, nutrients, etc.).

[0196] As used herein, the term "conditioned medium" refers to a culture medium (or an extract, part, or fraction thereof) in which one or more cells of interest have been cultured. Preferably, the conditioned medium is separated from the cultured cells before use and / or further processing. Cultivation of cells in the culture medium may result in the secretion and / or accumulation of one or more molecules and / or biological factors (which may include, but are not limited to, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space). A medium containing one or more molecules and / or biological factors is a conditioned medium. Examples of methods for preparing conditioned medium are described, for example, in U.S. Pat. No. 6,372,494, the entire contents of which are incorporated herein by reference.

[0197] As used herein, the term "cell culture" refers to cells grown under controlled conditions outside the cells' natural environment. For example, cells can be grown entirely outside their natural environment (in vitro) or removed from their natural environment and cultured (ex vivo). During cell culture, cells can survive in a non-replicating state or can replicate and increase in number, depending, for example, on the particular culture medium, culture conditions, and cell type. The in vitro environment can be any medium known in the art suitable for maintaining cells in vitro, such as, for example, an appropriate liquid medium or agar.

[0198] As used herein, the term "cell line" can refer to a culture of cells that can be passaged at least one time without termination.

[0199] As used herein, the term "suspension" can refer to a cell culture condition in which the cells are not attached to a solid support. Cells grown in suspension can be agitated while growing using equipment well known to those of skill in the art.

[0200] As used herein, the term "monolayer" can refer to cells that adhere to a solid support while growing under appropriate culture conditions. A small portion of cells growing in a monolayer under appropriate growth conditions may adhere to cells in the monolayer but not to the solid support.

[0201] The terms "plated" or "plating" as used herein with respect to cells can refer to establishing a cell culture in vitro. For example, cells can be diluted in cell culture medium and then added to a cell culture plate, dish, or flask. Cell culture plates are commonly known to those skilled in the art. Cells can be seeded at various concentrations and / or cell densities.

[0202] The term "cell plating" can also be extended 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 includes (1) releasing cells from a solid support or substrate and dissociating the cells, and (2) diluting the cells with a medium suitable for further cell growth. Cell passaging can also refer to removing a portion of the liquid medium containing the cultured cells and adding the liquid medium back to the original culture vessel to dilute the cells and allow for further cell growth. Additionally, cells can also be added to a new culture vessel supplemented with a medium suitable for further cell growth.

[0203] As used herein, the terms "culture medium," "growth medium," or "culture medium" are used interchangeably and refer to a composition intended to support the growth and survival of an organism. Culture media are often in liquid form, although other physical forms may be used, such as, for example, a solid, semi-solid, gel, suspension, etc.

[0204] As used herein, the term "serum-free" in the context of a culture or growth medium refers to a culture or growth medium that is free of serum. Serum typically refers to the liquid component of clotted blood after clotting factors (e.g., fibrinogen and prothrombin) have been removed by clot formation. Serum, such as fetal bovine serum, is routinely used in the art as a component of cell culture media because the various proteins and growth factors therein are particularly useful for cell survival, growth, and division.

[0205] As used herein, the term "basal medium" refers to a synthetic, unsupplemented medium that may contain a buffer, one or more carbon sources, amino acids, and salts. Depending on the application, the basal medium may be supplemented with growth factors and supplements, including, but not limited to, additional buffers, amino acids, antibiotics, proteins, and growth factors useful for promoting the growth of specific cell types (e.g., fibroblast growth factor basic (bFGF), also known as fibroblast growth factor 2 (FGF-2)), or for maintaining or altering the differentiation state.

[0206] 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 that occurs in nature. For example, the typical form of a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA, or genome exists in and can be isolated from a natural source. The wild-type form, appearance, phenotype, or strain serves as the original parent prior to intentional modification. Thus, mutants, variants, engineered forms, recombinant forms, and modified forms are not wild-type.

[0207] As used herein, the term "isolated" refers to material that has been removed from its original environment and has therefore been altered "by the hand of man" from its natural state.

[0208] As used herein, the term "enriched" means selectively concentrating or increasing the amount of one or more components in a composition relative to one or more other components. For example, enrichment can include reducing or decreasing (e.g., removing or eliminating) the amount of undesired material and / or specifically selecting or isolating a desired material from a composition.

[0209] The terms "engineered," "genetically engineered," "genetically modified," "recombinant," "modified," "non-naturally occurring," and "non-naturally occurring" refer to the deliberate human manipulation of the genome of an organism or cell. This term encompasses methods of genome modification, including genome editing, as defined herein, as well as techniques that alter gene expression or inactivation, enzymatic engineering, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, codon optimization, and the like. Methods of genetic engineering are known in the art.

[0210] 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 contain one or more nucleic acid sequences. Nucleotides can be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and can be of any length. Polynucleotides can perform any function and can have a variety of secondary and tertiary structures. These terms encompass natural nucleotides and known analogs of nucleotides modified at the base, sugar, and / or phosphate moieties. Analogs of a particular nucleotide have the same base-pairing specificity (e.g., an analog of an A base pair with a T). A polynucleotide can contain one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. The nucleotide structure can be modified before or after the polymer is assembled. After polymerization, the polynucleotide can be further modified, for example, by conjugation with a labeling moiety or a target-binding moiety. Nucleotide sequences can incorporate non-nucleotide components. The term encompasses nucleic acids that have similar binding properties to a reference polynucleotide (e.g., DNA or RNA), including synthetic, naturally occurring, and / or non-naturally occurring modified backbone residues or linkages. 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 acids (LNA™) (Exiqon, Inc., Woburn, MA), nucleosides, glycol nucleic acids, bridged nucleic acids, and morpholino structures. Peptide-nucleic acids (PNAs) are synthetic homologs of nucleic acids in which the polynucleotide phosphate-sugar backbone is replaced with a flexible pseudo-peptide polymer. Nucleobases are linked to the polymer. PNAs have the ability to hybridize with high affinity and specificity to complementary sequences of RNA and DNA.Polynucleotide sequences are presented herein in conventional 5' to 3' orientation unless otherwise indicated.

[0211] 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 an algorithm with 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, etc.) available through the World Wide Web, including, but not limited to, sites such as GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk). Sequence identity between two polynucleotide or two polypeptide sequences is generally calculated using standard default parameters for the various methods or computer programs. A high degree of sequence identity between two polynucleotides or two polypeptides is often about 90% to 100% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 90% or more identity, about 91% or more identity, about 92% or more identity, about 93% or more identity, about 94% or more identity, about 95% or more identity, about 96% or more identity, about 97% or more identity, about 98% or more identity, or about 99% or more identity over the length of the reference polynucleotide or polypeptide or query sequence. Sequence identity can also be calculated for regions of overlap of two sequences where only a portion of the two sequences can be aligned.

[0212] A moderate sequence identity between two polynucleotides or two polypeptides is often between about 80% identity and about 90% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 80% or more identity, about 81% or more identity, about 82% or more identity, about 83% or more identity, about 84% or more identity, about 85% or more identity, about 86% or more identity, about 87% or more identity, about 88% or more identity, or about 89% or more identity, but less than 90%, over the length of the reference polynucleotide or polypeptide or query sequence.

[0213] 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% or more identity, about 60% or more identity, about 70% or more identity, but less than 75% identity over the length of the reference polynucleotide or polypeptide or query sequence.

[0214] 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, etc.). Such non-covalent interactions are also referred to as "associating" or "interacting" (e.g., when a first macromolecule interacts with a second macromolecule, the first macromolecule binds non-covalently to the second macromolecule). Some portions of the binding interaction may be sequence-specific (the terms "sequence-specific binding," "sequence-specific binding," "site-specific binding," and "site-specific binding" are used interchangeably herein). Binding interactions can be characterized by the dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. Increased binding affinity is correlated with a lower Kd.

[0215] As used herein, "gene" refers to a polynucleotide sequence comprising exons and associated regulatory sequences. A gene may further comprise introns and / or untranslated regions (UTRs).

[0216] As used herein, "expression" refers to the transcription of a polynucleotide from a DNA template, resulting in, for example, messenger RNA (mRNA) or other RNA transcripts (e.g., non-coding RNAs, such as structural or scaffolding RNAs). The term also refers to the process by which the transcribed mRNA is translated into peptides, polypeptides, or proteins. The transcript and the encoded polypeptide are sometimes collectively referred to as "gene product." Expression can include splicing of the mRNA in a eukaryotic cell if the polynucleotide is derived from genomic DNA.

[0217] A "coding sequence," or a sequence "encoding" 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.

[0218] As used herein, a "different" or "altered" level of, for example, a characteristic or characteristic is measurably different, preferably a statistically significant difference (e.g., not due to the standard error of the assay). In some embodiments, for example, the difference compared to a control or reference sample can be, for example, more than 10% difference, more than 20% difference, more than 30% difference, more than 40% difference, more than 50% difference, more than 60% difference, more than 70% difference, more than 80% difference, more than 90% difference, more than 2-fold difference; more than 5-fold difference; more than 10-fold difference; more than 20-fold difference; more than 50-fold difference; more than 75-fold difference; more than 100-fold difference; more than 250-fold difference; more than 500-fold difference; more than 750-fold difference; or, for example, more than 1,000-fold difference.

[0219] As used herein, the term "between" includes the end values ​​of a given range (eg, a length of about 1 to about 50 nucleotides includes 1 nucleotide to 50 nucleotides).

[0220] As used herein, the term "amino acid" refers to amino acid analogs, modified amino acids, peptidomimetics, natural and synthetic (unnatural) amino acids, including glycine and D or L optical isomers.

[0221] As used herein, the terms "peptide," "polypeptide," and "protein" are interchangeable and refer to a polymer of amino acids. A polypeptide can be of any length. It can be branched or linear, interrupted by non-amino acids, and can contain modified amino acids. The term also refers to amino acid polymers that have been modified, for example, by acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and / or conjugation (e.g., with a labeling moiety or ligand). Polypeptide sequences are presented herein in the conventional N-terminal to C-terminal orientation unless otherwise specified. Polypeptides and polynucleotides can be produced using routine techniques in the field of molecular biology.

[0222] As used herein, a "moiety" refers to a portion of a molecule. A moiety can be a functional group or can represent a portion of a molecule that has multiple functional groups (e.g., sharing a common structural aspect). 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 includes some common chemical behavior. A "moiety" is often used as a structural description.

[0223] The term "effective amount" or "therapeutically effective amount" of a composition or agent, such as a therapeutic composition provided herein, refers to an amount of the composition or agent sufficient to provide a desired response, such response depending on the particular disease and associated conditions at issue.

[0224] As used herein, "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for the insertion. For example, transformation can be by direct uptake, transfection, infection, etc. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, an episome, or alternatively, may be integrated into the host genome.

[0225] As used herein, the terms "hypoxia" or "hypoxia" refer to a condition in which oxygen (O2) concentrations are below atmospheric O2 concentrations (typically 20-21%). In some embodiments, hypoxia refers to a condition having an O2 concentration that is 0%-19%, 2%-18%, 3%-17%, 4%-16%, 5%-15%, 5%-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%.

[0226] As used herein, the term "normoxia" refers to normal atmospheric oxygen concentrations, typically about 20%-21% O2.

[0227] Generation of progenitor cells from stem cells The present disclosure relates, in part, to methods for generating secretomes containing extracellular vesicles (EVs) from progenitor cells. In certain embodiments herein, progenitor cells can be isolated from a subject or tissue and used in the methods of the present disclosure. In other embodiments, progenitor cells can be generated from pluripotent stem cells, such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs).

[0228] Generation of iPSC cells iPSC cells can be derived from somatic cells, including, for example, human somatic cells. Somatic cells can be derived from humans or non-human animals, including, for example, humans and other primates, including non-human primates such as rhesus monkeys, chimpanzees, and other monkey and ape species; livestock, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; domestic fowl, such as chickens, turkeys, other poultry, ducks, geese, and birds, including wild birds and game birds.

[0229] In some embodiments, the somatic cells are selected from keratinized epithelial cells, mucosal epithelial cells, exocrine epithelial cells, endocrine cells, hepatocytes, epithelial cells, endothelial cells, fibroblasts, muscle cells, cells of the blood and immune systems, cells of the nervous system including neurons and glial cells, pigment cells, and progenitor cells including hematopoietic stem cells. Somatic cells may be fully differentiated (specialized) or not. For example, undifferentiated progenitor cells that are not PSCs can be used, including somatic stem cells and terminally differentiated mature cells. Somatic cells can be derived from animals of any age, including adult and fetal cells.

[0230] The somatic cells may be of mammalian origin. For example, when secretomes (or extracellular vesicles) from progenitor cells thereof are used for in vivo administration, allogeneic or autologous stem cells can be used. In some embodiments, the iPSCs are MHC- / HLA-mismatched to the subject. In some embodiments, the iPSCs are MHC- / HLA-matched to the subject. In embodiments, for example, when iPSCs are used to produce PSC-derived progenitor cells (to obtain secretomes or extracellular vesicles for therapeutic use in the subject), the somatic cells can be obtained from the subject to be treated or another subject with the same or substantially the same HLA type as the subject. The somatic cells can be cultured before nuclear reprogramming, or can be reprogrammed without culture, for example, after isolation.

[0231] Viral vectors can be used to introduce reprogramming factors into somatic cells, including vectors derived from viruses such as SV40, adenovirus, vaccinia virus, adeno-associated virus, herpesviruses including HSV and EBV, Sindbis virus, alphavirus, human herpesvirus vectors (HHVs) such as HHV-6 and HHV-7, and retroviruses. Lentiviruses include, but are not limited to, human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), ovine visna virus (VISNA), and caprine arthritis-encephalitis virus (CAEV). Lentiviral vectors can infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and in vitro. Viral vectors can be targeted to specific cell types by conjugation of viral proteins, such as envelope proteins, to binding agents such as antibodies or specific ligands (e.g., to target receptors or proteins on or within specific cell types).

[0232] In some embodiments, viral vectors, such as lentiviral vectors, can integrate into the genome of a host cell. The genetic material thus transferred is then transcribed and possibly translated into proteins within the host cell. In other embodiments, viral vectors are used that do not integrate into the genome of the host cell.

[0233] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Episomal gene delivery systems can be, for example, plasmids, Epstein-Barr virus (EBV)-based episomal vectors, yeast-based vectors, adenovirus-based vectors, Simian virus 40 (SV40)-based episomal vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0234] Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells, see, for example, U.S. Patent Application Publication No. 2009 / 0246875; U.S. Patent Application Publication No. 2010 / 0210014; published U.S. Patent Application Publication No. 2012 / 0276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; and U.S. Patent No. 8,268,620, all of which are incorporated herein by reference.

[0235] Generally, reprogramming factors that can be used to generate induced pluripotent stem cells, alone, in combination, or as fusions with transactivation domains, include, but are not limited to, one or more of the following genes: Oct4 (Oct3 / 4, Pou5f1), Sox (e.g., Sox1, Sox2, Sox3, Sox18, or Sox15), 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 these gene and protein sequences, 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: N M_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. Similar sequences are also contemplated, including those with 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.

[0236] Exemplary reprogramming factors for the production of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18. Klf4 can be replaced 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 papillomavirus (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, Bmi1;(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 can be replaced by 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, Bmil; (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 can be replaced with Esrrg).

[0237] iPSCs typically exhibit 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) can also be used to identify fully reprogrammed human cells. Furthermore, at the functional level, PSCs, such as ESCs and iPSCs, demonstrate the ability to differentiate into lineages from all three embryonic germ layers and form teratomas in vivo (e.g., in SCID mice).

[0238] Differentiation of PSCs to generate progenitor cells The present disclosure further contemplates differentiating PSCs, including ESCs and iPSCs, into progenitor cells, which can then be used to produce the secretomes (and extracellular vesicles) of the present disclosure.

[0239] Progenitor cells of the present disclosure include, for example, hematopoietic progenitor cells, bone marrow 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, periosteal cells, endothelial progenitor cells, blast cells, borderline caop cells, and mesenchymal stem cells. Methods for differentiating pluripotent stem cells into progenitor cells and culturing and maintaining progenitor cells are described, for example, in U.S. Patent Application Publication No. 17 / 931,669, entitled "Methods for the Production of Committed Cardiac Progenitor Cells," which is incorporated herein by reference in its entirety.

[0240] Secretome / extracellular vesicle production The present disclosure encompasses culturing progenitor cells for secretome / extracellular vesicle production under GMP-compliant and / or GMP-compliant conditions, e.g., to produce a GMP-compliant and / or GMP-compliant product. The present disclosure also encompasses culturing progenitor cells for secretome / extracellular vesicle production under non-GMP-compliant and / or non-GMP-compliant conditions, e.g., to produce a non-GMP-compliant and / or non-GMP-compliant product.

[0241] In the methods of producing secretomes or extracellular vesicles of the present disclosure, progenitor cells are typically subjected to two or more culture steps in serum-free culture medium.

[0242] In the first culture step, one or more progenitor cells are cultured in a first serum-free culture medium containing a basal medium, human serum albumin, and one or more growth factors. The first serum-free culture medium is then replaced with a second serum-free culture medium containing the basal medium but not containing human serum albumin or one or more growth factors. In the second culture step, one or more progenitor cells are then cultured in the second serum-free culture medium. Following the second culture step, the second serum-free culture medium is collected to obtain a conditioned medium containing the secretome of one or more progenitor cells.

[0243] The one or more progenitor cells can be, for example, recently isolated or differentiated progenitor cells (e.g., from stem cells). Alternatively, in some embodiments, previously refrigerated, frozen, and / or cryopreserved progenitor cells can be used in the culture methods of the present disclosure. In some embodiments, the progenitor cells are thawed from a cryopreserved state (e.g., at -80°C or below) prior to use. In some such embodiments, the cells are thawed in a thawing medium. In some embodiments, the thawing medium can include a liquid medium (e.g., α-MEM, STEMdiff™ Cardiomyocyte Support Medium (StemCell, Reference: 05027)) containing one or more supplements. In some embodiments, the supplements in the thawing medium can be one or more of a carbon source (e.g., glucose), albumin, B-27, insulin, FGF-2, FGF, and an antibiotic (e.g., gentamicin). In some embodiments, the cells can be thawed in a thawing device, such as a water bath or an anhydrous thawing system (e.g., ThawSTAR™ Automated Thawing System, Biolife Solutions®). The cells can be thawed, for example, in a tube or bottle (e.g., plastic, glass) or a bag (e.g., an ethyl vinyl acetate (EVA) bag) such as a 500-1000 mL capacity bag (e.g., Corning, References: 91-200-41, 91-200-42).

[0244] The one or more growth factors may be selected based on, for example, the type of progenitor cell. In some embodiments, the one or more growth factors are selected from adrenomedullin, angiopoietin, autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), macrophage colony-stimulating 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 B1, ephrin B2, ephrin B3, erythropoietin, and the like. 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 (FGF-F22), fibroblast growth factor 23 (FGF-23), fetal bovine growth factor (FBS), glial cell line-derived neurotrophic factor (GDN) F), neurons, persephin, artemin, growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatocyte-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),The inhibitor may be selected from 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).

[0245] The amount of growth factor can be adjusted depending on the desired culture conditions and / or needs. In some embodiments, one or more growth factors can each independently be present in an amount of 0.001 μg / mL to 1000 μg / mL, 0.01 μg / mL to 100 μg / mL, 0.1 μg / mL to 10 μg / mL, 0.05 μg / mL to 5 μg / mL, 0.5 μg / mL to 2.5 μg / mL, or 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.

[0246] 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.

[0247] The basal culture medium can be any basal medium appropriate for the cell type to be cultured, including, for example, Dulbecco'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.

[0248] Additional supplements may be added to the basal medium to provide cells with trace elements for optimal growth and proliferation. Such supplements include, for example, insulin, transferrin, sodium selenium, Hank's balanced salt solution, Earle's salt solution, antioxidant supplement, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, and 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.

[0249] Optionally, hormones can also be used in cell culture, including, but not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine. β-Mercaptoethanol can also be supplemented to the cell culture medium.

[0250] Depending on the cell type, lipids and lipid carriers can also be used to supplement the cell culture medium, which may 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 conjugated to albumin, and unconjugated and albumin-conjugated oleic acid, among others.

[0251] In certain embodiments, albumin, such as human serum albumin, is present in the first serum-free culture medium. Albumin, including human serum albumin, can be, for example, isolated, synthetic, recombinant, and / or modified. The amount of albumin can be adjusted depending on the desired culture conditions and / or needs. In some embodiments, albumin can be present in an amount of 0.1 μg / mL to 50 mg / mL, 1 μg / mL to 25 mg / mL, 10 μg / mL to 20 mg / mL, 100 μg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, 1 mg / mL to 3 mg / mL, or about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL.

[0252] In some embodiments, the serum-free medium 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; triiodo-I-thyronine; amino acids; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.

[0253] The basal medium can also be supplemented with one or more carbon sources. The one or more carbon sources can be selected from carbon sources such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose. In some embodiments, a carbon source such as glucose can 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.

[0254] A lock inhibitor may also be included in the culture medium, such as, for example, the lock inhibitor H1152.

[0255] The first and second culture steps may be carried out for different periods of time, for example, the first and second culture steps may be carried out independently for 6 to 96 hours, 12 to 72 hours, 36 to 60 hours, 42 to 56 hours, or 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.

[0256] In some embodiments, the first culturing step is carried out for 42 to 56 hours, for example, about 48 hours, and in some embodiments, the second culturing step is carried out for 42 to 56 hours, for example, about 48 hours.

[0257] In some embodiments, the first culturing step is carried out for 42 to 96 hours, for example, about 72 hours, and in some embodiments, the second culturing step is carried out for 42 to 56 hours, for example, about 48 hours.

[0258] In some embodiments, all or part of the first and / or second culturing step is performed under hypoxic conditions. In some embodiments, all or part of the second culturing step is performed under hypoxic conditions. In some embodiments, the last 6 to 72 hours, the last 10 to 48 hours, or the last 12 to 36 hours of the second culturing step are performed under hypoxic conditions. In some embodiments, the hypoxic conditions are O2 concentrations of 0% to 15%, 0% to 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%.

[0259] In some embodiments, all or part of the first and / or second culturing step is performed under normoxic conditions. In some embodiments, all or 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 are performed under normoxic conditions. In some embodiments, normoxic conditions are an O2 concentration of 20%-21%.

[0260] In some embodiments, all or part of the first and / or second culturing step is carried out in the presence of insulin. In some embodiments, all or part of the first culturing step is carried out 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 part of the second culturing step is carried out 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.

[0261] 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 (e.g., removing the first culture medium and then adding the second culture medium).

[0262] The first and / or second culturing step can be carried out in suspension or attached to a solid support. The culture can be a two-dimensional or three-dimensional cell culture.

[0263] For example, in some embodiments, the culture vessel used for culturing can be, for example, a flask, a tissue culture flask (e.g., T25, T75), a HyperFlask (e.g., CellBind surface HYPERFlask®; Corning, Reference: 10024) or a HyperStack (e.g., 12 or 36 chamber, HYPERStacks®; Corning, References: 10012, 10036, 10013, 10037), a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, CellSTACK® Chambers (e.g., 1ST, 2ST, 5ST, 10ST; Corning, References: 3268, 3269, 3313, 3319), a culture bag, a roller bottle, a bioreactor, a stirred culture vessel, a spinner flask, a microcarrier, or a vertical wheel bioreactor. The one or more progenitor cells may be cultured in a volume of, for example, 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, 5 L, 10 L, 50 L, 100 L, 1000 L, 5000 L, or 10,000 L.

[0264] In embodiments in which the culture comprises a two-dimensional cell culture, for example, on the surface of the culture vessel, the culture surface (to which the cells are intended to adhere) can be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, types I, II, and 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, Corning® Cell-Tak™ Cell and Tissue Adhesive, Corning® PuraMatrix® peptide hydrogel, and / or vitronectin.

[0265] In some embodiments, when the cells are cultured as adherent cultures, e.g., when the cells adhere to a solid support, the cells are cultured at a density of 25,000 to 250,000 cells / cm. 2 ,50,000~200,000 cells / cm 2 ;75,000~175,000 cells / cm 2 or 100,000 to 150,000 cells / cm 2 The seeds can be seeded in an amount of

[0266] In some embodiments, when the cells are cultured as an adherent culture, e.g., when the cells adhere to a solid support, the cells can be seeded onto the solid support under gravity. In other embodiments, the cells can be seeded onto the solid support under centrifugation.

[0267] In some embodiments, after the second culturing step, the second serum-free culture medium used in the second culturing step is collected to obtain a conditioned medium containing the secretome of one or more progenitor cells.

[0268] 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.

[0269] In some embodiments, the recovered conditioned medium can be pre-cleaned or clarified to remove particulates above a certain size. For example, the recovered conditioned medium can be pre-cleaned or clarified by one or more centrifugation and / or filtration techniques. In some embodiments, in-line filters can be used with gradually decreasing pore sizes to minimize clogging and loss of material. In some embodiments, such as when TFF is performed, pore sizes as small as 0.2 μm can be used to avoid clogging / high pressure during the TFF stage.

[0270] In some embodiments, the recovered conditioned medium is further processed to obtain specific extracts or fractions of the recovered conditioned medium. For example, the recovered conditioned medium can be further processed to separate a small extracellular vesicle-enriched fraction (sEVs) therefrom. The sEV fraction can 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, density gradient ultracentrifugation, tangential flow filtration, size exclusion chromatography, ion exchange chromatography, affinity capture, polymer-based precipitation, or organic solvent precipitation.

[0271] In some embodiments, the conditioned medium is subjected to clarification by one or more filtration steps. In some embodiments, the one or more filtration steps utilize a membrane filter having a specific pore size. In some embodiments, a filter having a pore size of 0.1 μm to 500 μm, or 0.2 μm to 200 μm, or 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 or less is used.

[0272] In some embodiments, 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, clarification comprises 4 filtration steps. In some embodiments, the successive filtration steps utilize filters with increasingly smaller pores.

[0273] In some embodiments thereof, the first filtration step involves the use of an approximately 200 μm filter (e.g., a 200 μm drip chamber filter; specific gravity blood set, BD careFusion, Reference: VH-22-EGA). The second filtration step involves the use of an approximately 15 μm filter (e.g., DIDACTIC, Reference: PER1FL25). The third filtration step involves the use of an approximately 0.2 μm filter, optionally including a prefilter, such as an approximately 1.2 μm prefilter (e.g., Sartoguard PES XLG MidiCaps, pore size: 1.2 μm + 0.2 μm, Sartorius, Reference: 5475307F7--OO--A). The fourth filtration step involves the use of an approximately 0.22 μm filter (e.g., a vacuum filter / storage bottle system, 0.22 μm pores, 33.2 cm), as shown in Example 5 and FIG. 11A. 2 This involves the use of a PES membrane (Corning, ref: 431097).

[0274] In another embodiment, as shown in Example 12 and FIG. 24A, the first filtration step involves the use of an approximately 5 μm filter (e.g., Sartopure PP3 MidiCaps, pore size: 5 μm, Sartorius, Reference: 5055342P9--OO--A), the second filtration step involves the use of a prefilter, e.g., an approximately 0.2 μm filter, optionally including an approximately 1.2 μm prefilter (e.g., Sartopure 2 MidiCaps, pore size: 0.45 μm+0.2 μm, Sartorius, Reference: 5475307F9--OO--A), and the third filtration step involves the use of a prefilter, e.g., an approximately 0.45 μm prefilter (e.g., Sartopure 2 MidiCaps, pore size: 0.45 μm+0.2 μm, Sartorius, Reference: 5475307F9--OO--A). This involves the use of a filter of approximately 0.2 μm, optionally containing MidiCaps, pore size: 0.45 μm + 0.2 μm, Sartorius, reference: 5445307H8--OO--A).

[0275] In some embodiments, the conditioned medium may be subjected to clarification by one or more centrifugation steps, hi some embodiments, the conditioned medium may be subjected to clarification by a combination of centrifugation and filtration steps.

[0276] In some embodiments, one or more additives are added to the conditioned medium, such as before and / or after clarification. In some embodiments, an additive that reduces aggregation is added. In some embodiments, the additive is one or more selected from trehalose, histidine (e.g., L-histidine), arginine (e.g., L-arginine), citrate-dextrose solution, Dnase (e.g., Dnase I), ferric citrate, or an anti-clumping agent (Gibco / Life technologies, Reference: 01-0057; Lonza, Reference: BE02-058E).

[0277] In some embodiments, conditioned medium or sEVs can be subjected to isolation, concentration, and / or enrichment using tangential flow filtration (TFF). In some embodiments, conditioned medium or sEVs are subjected to TFF after clarification using one or more clarification steps (e.g., 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, for example, for concentration (e.g., concentrating small extracellular vesicles from conditioned medium); diafiltration; and concentration and diafiltration. Diafiltration is a type of ultrafiltration process in which the retentate (the fraction that does not pass through the membrane) is diluted with a buffer and re-ultrafiltered to reduce the concentration of soluble permeate components and further increase the concentration of retentate components.

[0278] In some embodiments, TFF is used to enrich, concentrate, and diafilter conditioned medium or sEVs (e.g., for EV secretome concentration and diafiltration). In some embodiments, TFF is first used to concentrate conditioned medium or sEVs, followed by diafiltration. In some embodiments, the TFF process may include an additional step of concentration after diafiltration. In some embodiments, TFF is used for diafiltration but not concentration. In some embodiments, TFF is used for concentration but not diafiltration.

[0279] In some embodiments, the TFF membrane has a cutoff of 10 kDa or less, 20 kDa or less, 30 kDa or less, 40 kDa or less, 50 kDa or less, 60 kDa or less, 70 kDa or less, 80 kDa or less, 90 kDa or less, 100 kDa or less, or 150 kDa or less. In some embodiments, the TFF membrane has a cutoff of about 10 kDa, about 30 kDa, about 100 kDa, or about 500 kDa. In some embodiments, the TFF membrane has a cutoff of 30 kDa or about 30 kDa.

[0280] 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.

[0281] In some embodiments, TFF pressures 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 can be used. In some embodiments, TFF pressures of 3.5 bar or less are used to address filter clogging and slow filtration rates (e.g., when using large-scale (>5 L) media processing of spent media using low cutoffs such as 30 kDa).

[0282] In some embodiments, the conditioned medium or sEVs subjected to TFF can be further purified, isolated, and / or enriched (after TFF) using one or more purification, isolation, and / or enrichment techniques. For example, the product obtained from TFF can be subjected to a chromatography step, such as an ion exchange chromatography step or a steric exclusion chromatography step, to further purify the small extracellular vesicles. In some embodiments, the conditioned medium subjected to TFF, with or without further purification, isolation, and / or enrichment, can be further concentrated, such as by ultracentrifugation.

[0283] Any of the above processing techniques can be performed, for example, on collected conditioned medium (or previously processed extracts or fractions thereof), which are fresh or previously frozen and / or refrigerated.

[0284] In some embodiments, the secretome, extracellular vesicles, and sEV-containing compositions produced by the methods herein may contain 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, Dnase (e.g., Dnase I), ferric citrate, or an anti-aggregating agent (Gibco / Life Technologies, Reference: 01-0057; Lonza, Reference: BE02-058E). In some embodiments, trehalose is added. In some embodiments, trehalose or L-histidine is added.

[0285] 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 one or more of exosomes, microparticles, and extracellular vesicles. The sEV fraction may also contain secreted proteins (envelope and / or non-envelope). The extracellular vesicles in the conditioned medium or sEV fraction of the present disclosure may contain one or more components selected from, for example, 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 synthase), nucleic acids (e.g., RNA, miRNA), heat shock proteins (e.g., HSP70 and HSP90), exosome biogenesis proteins (ALIX, Tsg101), LT, prostaglandins, and S100 proteins.

[0286] In some embodiments, the presence of the desired extracellular vesicle type in the fraction can be determined, for example, by nanoparticle tracking analysis (to determine the size of particles in the fraction) and / or by confirming the presence of one or more markers associated with the desired extracellular vesicle type. For example, fractions of the collected conditioned medium can be analyzed for the presence of the desired extracellular vesicle type by detecting the presence of one or more markers, such as CD9, CD63, and / or CD81, in the fraction.

[0287] In some embodiments, the sEV preparation or composition is positive for CD9, CD63, and CD81 (canonical EV markers) and positive for the cardiac-related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142. In some embodiments, the sEV preparation or composition comprises a lower amount of one or more markers selected from the group consisting of CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD40, CD11c, CD86, CD31, CD20, CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69 compared to the amount of CD9, CD63, and / or CD81 in the sEV preparation or composition. In some embodiments, the sEV preparation or composition contains an undetectable amount (e.g., by MACSPlex assay, immunoassay, etc.) of or is negative for one or more markers selected from the group consisting of CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69.

[0288] In some embodiments, the sEV preparation or composition is at least one of the following: an sEV preparation or composition enriched in extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm; an sEV preparation or composition enriched in extracellular vesicles having a diameter of about 50-150 nm or 50-150 nm; an sEV preparation or composition that is substantially free or free of whole cells; and an sEV preparation or composition that is substantially free of one or more culture medium components (e.g., phenol red).

[0289] In some embodiments, such as, for example, some GMP-compliant processes, test panels are performed to analyze and / or determine one or more characteristics of the process, its products, intermediate products, or the like.

[0290] For example, during the vesicle formation stage (e.g., including thawing, plating, culturing, and / or harvesting steps), one or more characteristics of the cells can be examined (e.g., including: number of viable cells, cell viability; cell morphology; cell identity; cell karyotype; and / or cell transcriptome).

[0291] Additionally or alternatively, one or more properties of the secretome and / or extracellular vesicle-containing fraction, extract, or composition can be analyzed using one or more tests (e.g., including particle concentration and / or size distribution; protein concentration; protein profile concentration; RNA profile; potency; marker identification; host cell protein assessment; residual DNA quantification and / or characterization; sterility; mycoplasma; endotoxin; appearance; pH; osmolality; 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, the EV composition, formulation, fraction, or secretome, etc., can be analyzed by electron microscopy.

[0292] In some embodiments, RNA can be extracted from EVs to analyze the RNA transcriptome of an EV composition, preparation, fraction, or secretome, etc. In some embodiments, microRNAs are analyzed, for example, by generating a cDNA library from the extracted RNA. This includes sequencing all or a portion of the library. In some embodiments, the sequence analysis includes sorting the sequenced RNA into different biotypes. In some embodiments, an EV composition, preparation, fraction, secretome, etc. contains all or a portion of the miRNAs listed in Figure 43 or Table 9. In some embodiments, an EV composition, preparation, fraction, secretome, etc. contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of the miRNAs listed in Figure 43 or Table 9. In some embodiments, an EV composition, preparation, fraction, secretome, etc. contains all or a portion of the miRNAs listed in Table 80. In some embodiments, the 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 listed in Table 80. In some embodiments, the EV composition, formulation, fraction, etc., includes at least one of miR-302, miR-16, miR-126, and miR-93.

[0293] In some embodiments, proteomic analysis of the EV composition, preparation, fraction, secretome, etc. can be performed. In some embodiments, proteins can be isolated from the EV composition, preparation, fraction, secretome, etc. and analyzed by mass spectrometry, e.g., nanoLC-MS / MS and HPLC-MS / MS analysis. In some embodiments, the EV composition, preparation, fraction, 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 of the proteins listed in Table 81.

[0294] Furthermore, one or more of the above properties can be assessed in the conditioned medium before clarification; in the conditioned medium after clarification; in the isolated and / or enriched secretome / extracellular vesicles; and / or in the final formulation.

[0295] In some embodiments, the final formulation may be tested after formulation, immediately after manufacture and / or after 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 18 months, and several years.

[0296] An exemplary process / product testing panel is shown in Table 49. This exemplary process adds to the description above. This panel was developed to characterize and ensure the reproducibility of our process, which further led to the development of CTC1-EV.

[0297] [Table 1] JPEG2025536321000003.jpg255161JPEG2025536321000004.jpg255161

[0298] Therapeutic Compositions and Uses The present disclosure contemplates the production 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.

[0299] Tissues that can be treated according to the methods of the present disclosure include, but are not limited to, cardiac tissue, brain or other neural tissue, skeletal muscle tissue, lung tissue, arterial tissue, capillary tissue, kidney tissue, liver tissue, gastrointestinal tissue, epithelial tissue, connective tissue, urinary tract tissue, etc. The tissues to be treated may be damaged or fully or partially non-functional due to, for example, injury, age-related degeneration, acute or chronic disease, cancer, or infection. Such tissues may be treated, for example, by intravenous administration of a secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition.

[0300] In some embodiments, compositions of the present disclosure can be used to treat diseases such as myocardial infarction, stroke, heart failure, and critical limb ischemia. In some embodiments, compositions of the present disclosure can be used to treat heart failure having one or more of the following characteristics: 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 can 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 chemotherapy-induced heart failure. In some embodiments, compositions of the present disclosure can be used to treat diseases such as congestive heart failure, heart disease, ischemic heart disease, valvular heart disease, connective tissue disease, viral or bacterial infection, myopathy, dystrophinopathy, liver disease, kidney disease, sickle cell disease, diabetes, eye disease, and neurological disease. In some embodiments, the compositions of the present disclosure can be used to treat chemotherapy-induced cardiomyopathy (e.g., caused by anthracycline administration). It will be appreciated that the appropriate progenitor cell type can be selected depending on the disease being treated or the tissue being targeted.

[0301] 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 vesicles, and / or sEV-containing composition produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells.

[0302] Additionally, secretome-, extracellular vesicles-, and / or sEV-containing compositions produced from appropriate progenitor cell types can also be used to improve tissue function or performance. For example, improved angiogenesis or cardiac function can be achieved by delivering secretome-, extracellular vesicles-, and / or sEV-containing compositions produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells to a subject in need thereof.

[0303] In some embodiments, administration includes administration at the same tissue or organ site as the target tissue. In some embodiments, administration includes administration at a different tissue or organ site than the target tissue. Such administration may include, for example, intravenous administration.

[0304] The secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition may contain or be administered with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, such compositions may also contain one or more pharmaceutically acceptable concentrations of salt, buffer, preservative, or other therapeutic agent. Some examples of substances that can function 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; buffers such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic, compatible substances used in pharmaceutical formulations. For example, in some embodiments, the secretome-containing composition, extracellular vesicle-containing composition, 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, which is incorporated by reference in its entirety.

[0305] The secretome-containing compositions, extracellular vesicle-containing compositions, and / or sEV-containing compositions of the present disclosure can be administered in effective amounts, e.g., therapeutically effective amounts, depending on the intended purpose. The effective amount depends on various factors, including the material selected for administration, whether the administration is a single dose or multiple doses, and individual patient parameters, including age, physical condition, size, weight, and stage of disease. These factors are well known to those skilled in the art.

[0306] Any suitable route of administration may be used, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intrahepatic, intracapsular, intrathecal, intracapsular, intraperitoneal, intranasal, intramyocardial, intracoronary, aerosol, suppository, epicardial patch, oral administration, or via perfusion. For example, therapeutic compositions for parenteral administration may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; and for intranasal formulations, the formulation may be in the form of a powder, nasal spray, or aerosol. For example, in some embodiments, a subject with a cardiac disease, such as acute myocardial infarction or heart failure, may 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, and the composition is administered intravenously.

[0307] In some embodiments, a single dose of the secretome-, extracellular vesicle-, and / or sEV-containing composition can be administered. In other embodiments, multiple doses, one or more times per day, week, or month, are administered to a subject. In some embodiments, single or repeated administrations of the secretome-, extracellular vesicle-, and / or sEV-containing composition can be administered, including two, three, four, five, or more administrations. In some embodiments, the secretome-, extracellular vesicle-, and / or sEV-containing composition can be administered continuously. Repeated or continuous administrations can occur over the course 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, or 1-7 days), or several weeks (e.g., 1-2, 1-3, or 1-4 weeks) or months, depending on the nature and / or severity of the condition being treated. When administration is repeated but not consecutively, the time between administrations can be several hours (e.g., 4, 6, or 12 hours), several days (e.g., 1, 2, 3, 4, 5, or 6 days), or several weeks (e.g., 1, 2, 3, or 4 weeks). The time between administrations can be the same or different. For example, if symptoms worsen or do not improve, the secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition can be administered more frequently. Conversely, if symptoms stabilize or diminish, the secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition can be administered less frequently.

[0308] In some embodiments, the secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition is administered intravenously in several doses, e.g., three doses, spaced apart over several days, weeks or about several months, weeks or about several months, or two weeks or about two weeks. In some embodiments, the composition may be diluted, formulated, and / or administered with a carrier, diluent, or suitable material (e.g., saline).

[0309] Assays for determining the activity, functionality and / or potency of secretomes and extracellular vesicles The present disclosure also encompasses methods for analyzing the activity, functionality and / or efficacy of conditioned medium or secretome-containing compositions, extracellular vesicle-containing compositions and / or sEV-containing compositions.

[0310] The activity, functionality, and / or efficacy of conditioned medium; or secretome-containing compositions, extracellular vesicle-containing compositions, and / or sEV-containing compositions can be assessed by a variety of techniques, depending, for example, on the type of progenitor cells used to produce the conditioned medium or composition and the desired use of the conditioned medium or composition.

[0311] For example, the activity, functionality, and / or efficacy of conditioned medium, or secretome-, extracellular vesicle-, and / or sEV-containing compositions can be assessed by administering the conditioned medium, secretome-, extracellular vesicle-, and / or sEV-containing compositions to target cells in vitro, ex vivo, or in vivo. One or more properties of the target cells or secretome-, extracellular vesicle-, and / or sEV-containing compositions, such as cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology, can then be analyzed to determine, for example, the activity, functionality, and / or efficacy of the conditioned medium.

[0312] In some embodiments, assays known in the art can be used to determine the activity, functionality, and / or efficacy of the conditioned medium or secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition.

[0313] For example, in the case of conditioned medium; or secretome-containing compositions, extracellular vesicle-containing compositions, and / or sEV-containing compositions obtained from cardiovascular progenitor cells or cardiomyocyte progenitor cells, the activity, functionality, and / or efficacy can be measured using known cardiomyocyte viability assays, such as those described in El Harane et al. (Eur. Heart J., 2018, 39(20):1835-1847).

[0314] Specifically, serum-starved cardiomyoblasts (e.g., H9c2 cells) can be contacted with conditioned medium or a secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition, and cell viability can then be measured. In some embodiments of this assay, serum is removed from the cells before administering the conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition. In other embodiments, serum is removed from the cells after administering the conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition. In some embodiments, the cells are serum-deprived before or after administering the conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition.

[0315] In other embodiments, the angiogenic activity of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition can be measured, for example, using a HUVEC scratch wound healing assay. In the HUVEC scratch wound healing assay, HUVEC cells are cultured on a culture surface, and then the cultured cell layer(s) are scratched. The angiogenic activity of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition can then be determined by the ability of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition to cause wound closure under serum-free conditions.

[0316] In some embodiments, the activity, functionality, and / or efficacy of conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions; can be analyzed using a HUVEC (human umbilical vein endothelial cell) plating assay. In some embodiments, HUVEC cells are cultured in basal medium in the presence of conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing compositions to analyze the effect of conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions on HUVEC viability. In some embodiments, conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions improve cell seeding, survival, and / or proliferation of HUVEC cells.

[0317] Cell viability (in cell viability assays) can be measured, for example, using DNA labeling dyes or nuclear staining dyes. The dyes can be used in conjunction with live cell imaging. Cell viability can also be measured by microscopy, for example, fluorescence microscopy, using such DNA labeling dyes or nuclear staining dyes. Cell viability can also be measured, for example, by analyzing ATP content in a HUVEC plating assay.

[0318] In some embodiments, the conditioned medium, or secretome-, extracellular vesicle-, and / or sEV-containing composition, can be analyzed in an anti-fibrosis assay to determine the effect of the conditioned medium, or secretome-, extracellular vesicle-, and / or sEV-containing composition in treating or reducing fibrosis. In some embodiments, cells are stimulated to induce a fibrotic state. In some embodiments, the cells induced to a fibrotic state are fibroblasts, e.g., cardiac fibroblasts. In some embodiments, the cells are stimulated with at least one stimulatory agent to induce a fibrotic state. In some embodiments, the stimulatory agent is 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, one or more markers of fibrosis are analyzed by quantifying the amount of transcripts encoding the markers of fibrosis. In some embodiments, the amount of transcripts is quantified by quantitative reverse transcription polymerase chain reaction. In some embodiments, transcript abundance is quantified using arrays or next generation sequencing. In some embodiments, expression of at least one of MMP2 and periostin is analyzed.

[0319] The activity, functionality, and / or efficacy of the conditioned medium, or secretome-, extracellular vesicle-, and / or sEV-containing composition, can also be determined with reference to one or more control samples. For example, the control cells can be one or more of: serum-starved control cells that are not administered conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition; control cells that are not serum-starved; or serum-starved control cells that are administered mock-conditioned medium or mock secretome-, extracellular vesicle-, and / or sEV-containing composition.

[0320] In some methods of the present disclosure, at least one activity, functionality, and / or efficacy of a conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition can be assessed by a method comprising administering the conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition to target cells cultured under at least one stress-inducing condition and analyzing at least one characteristic of the cells. The one or more characteristics of the target cells that can be analyzed can be selected from, for example, 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 characteristic measured is cell adhesion, cell number, cell proliferation, and / or cell morphology, and the cell adhesion, cell number, cell proliferation, and / or cell morphology are determined by measuring electrical impedance across the surface of a culture vessel in culture.

[0321] In the first method, target cells are cultured in a pre-conditioning medium under at least one stress-inducing condition, followed by administration of 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 secretome-, extracellular vesicle-, and / or sEV-containing composition, and at least one characteristic of the cultured cells is measured one or more times during the culture. In some embodiments, the at least one characteristic is measured multiple times during the culture in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition (e.g., 5 minutes to 10 hours apart, 10 minutes to 4 hours apart, or 30 minutes to 2 hours apart).

[0322] In some embodiments of this first method, culturing in the presence of the conditioned medium or secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition is performed in the presence of at least one stress-inducing condition. In other embodiments of this first method, culturing in the presence of the conditioned medium or secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition is performed in the absence of at least one stress-inducing condition.

[0323] In some embodiments of this first method, the pre-treatment medium is removed from the cells prior to 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 in which 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), culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition is performed in the absence of the at least one stress-inducing condition.

[0324] In other embodiments of this first method, the pre-treatment medium is not removed from the cells prior to 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 in which 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), culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and / or sEV-containing composition is performed in the presence of at least one stress-inducing condition.

[0325] In a second method, target cells are cultured in a pre-conditioning medium, followed by administration of conditioned medium or a secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition (optionally followed by culturing the target cells in the presence of the conditioned medium or a secretome-containing composition, an extracellular vesicle-containing composition, and / or an 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 culture under the at least one stress-inducing condition (which also occurs in the presence of the conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition). In some embodiments, the at least one property is measured multiple times during culture under the at least one stress-inducing condition (and in the presence of the conditioned medium or secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition), e.g., 5 minutes to 10 hours apart, 10 minutes to 4 hours apart, or 30 minutes to 2 hours apart.

[0326] In some embodiments of this second method, the target cells are cultured in the presence of conditioned medium or a secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition before being cultured under at least one stress-inducing condition. In other embodiments of this second method, the target cells are not cultured in the presence of conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition before being cultured under at least one stress-inducing condition. In some embodiments of this second method, the conditioned medium or a secretome-containing composition, an extracellular vesicle-, and / or sEV-containing composition is removed from the target cells before being cultured under at least one stress-inducing condition.

[0327] In some embodiments of the first and second methods, the stress-inducing condition is culture in the presence of a cell stress agent. In some embodiments of the second method, the cell stress agent is co-administered to the target cells with the conditioned medium or the secretome-containing composition, the extracellular vesicle-containing composition, and / or the sEV-containing composition.

[0328] In some embodiments of the first and second methods above, the cell stress agent is one or more apoptosis-inducing agents.

[0329] The one or more apoptosis inducers may be selected from, for example, doxorubicin, staurosporine, etoposide, camptothecin, paclitaxel, vinblastine, gambogic acid, daunorubicin, tyrphostin, thapsigargin, okadaic acid, mifepristone, colchicine, ionomycin, 24(S)-hydroxycholesterol, cytochalasin D, brefeldin A, raptinal, carboplatin, C2 ceramide, actinomycin D, rosiglitazone, kaempferol, berberine chloride, bioimif, betulinic acid, tamoxifen, emberine, phytosphingosine, mitomycin C, birinapant, anisomycin, genistein, cycloheximide, and the like.

[0330] In some embodiments, the apoptosis inducer is indolocarbazole. In some embodiments, the apoptosis inducer is indolo(2,3-a)pyrrole(3,4-c)carbazole. In some embodiments, the apoptosis inducer is staurosporine or a derivative thereof. In other embodiments, the apoptosis inducer is doxorubicin or a derivative thereof.

[0331] In some embodiments of the first and second methods described above, the stress-inducing conditions include culturing in the presence of a chemotherapeutic agent, and the conditioned medium or secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition is analyzed in a chemotherapy-induced cardiomyopathy assay. In some embodiments, the chemotherapeutic agent is an anthracycline. In some embodiments, the anthracycline is one or more of aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments, the chemotherapeutic agent is or includes doxorubicin.

[0332] In some embodiments, the chemotherapy-induced cardiomyopathy assay includes treating cells, such as cardiomyocytes, with a cardiomyopathy-inducing chemotherapeutic agent and then culturing the treated cells in the presence of conditioned medium or a secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition. In some embodiments, the induction of chemotherapy-induced cardiomyopathy and / or the treatment or reduction of chemotherapy-induced cardiomyopathy is measured by analyzing ATP content. In some embodiments, the induction of chemotherapy-induced cardiomyopathy and / or the treatment or reduction of chemotherapy-induced cardiomyopathy is measured by analyzing mitochondrial function, for example, using the Seahorse method (e.g., Seahorse Mito Stress Test (Seahorse XFp Cell Mito Stress Test Kit, Agilent)).

[0333] In some embodiments of the first and second methods, the at least one characteristic measured is viability of the cultured cells. Viability can be measured, for example, using a DNA-labeling dye or a nuclear-staining dye. In some embodiments, the DNA-labeling dye or the nuclear-staining dye is a fluorescent dye, such as a far-red fluorescent dye.

[0334] In some embodiments, conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing compositions can be analyzed in an animal model of chemotherapy-induced cardiomyopathy. In some embodiments, the animal model is a rat model of chemotherapy-induced cardiomyopathy. In some embodiments, the chemotherapeutic agent is an anthracycline. In some embodiments, the anthracycline is one or more of aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments, the chemotherapeutic agent is or includes doxorubicin. In some embodiments, induction, treatment, or reduction of chemotherapy-induced cardiomyopathy can be measured by one or more of echocardiography (e.g., to determine LVESV and LVEDV); electrocardiography; blood pressure (systolic, diastolic, etc.) measurement; functional status assessed by the NYHA score; quality of life; measurement of LVEF and LV volumes; maximal oxygen consumption during exercise; immune response by detection of donor cell-specific antibodies after each infusion; and assays of pro-inflammatory and anti-inflammatory cytokines. In some embodiments, the conditioned medium; or secretome, extracellular vesicles and / or sEV-containing compositions may have the ability to counteract energy stress (metabolic / energy-related pathologies) induced by chemotherapy (e.g., mitochondrial damage, insufficient energy production).

[0335] In some embodiments of the first and second methods, one or more of the culturing of target cells in (a) pre-conditioning medium; (b) conditioned medium or 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, serum may be removed from target cells before administering conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition. In other embodiments, serum may be removed from target cells after administering conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition. In some embodiments, cells are serum-deprived before or after administering conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition.

[0336] In embodiments of the first and second methods, the target cells can be cultured in the pretreatment medium for different periods of time. For example, the target cells can be cultured in the pretreatment medium for 30 minutes to 10 hours, 1 hour to 5 hours, or for more than 1, 2, 3, 4, or 5 hours, less than 1, 2, 3, 4, or 5 hours, or for about 1, 2, 3, 4, or 5 hours.

[0337] In embodiments of the first and second methods, the target cells are cultured with the conditioned medium or the secretome, extracellular vesicles 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.

[0338] In some embodiments of the first and second methods, the target cells are cultured in vitro before culturing in the pretreatment medium. For example, the target cells may be cultured in vitro for 1 to 21 days, 3 to 17 days, 5 to 14 days, or less than 20 days, 18 days, 16 days, 14 days, 12 days, 10 days, 8 days, 6 days, 4 days, or 2 days before culturing in the pretreatment medium. In certain embodiments in which the target cells are cultured in vitro before culturing in the pretreatment medium, the target cells are fed fresh culture medium before culturing in the pretreatment medium. For example, the target cells may be fed fresh culture medium for 6 to 72 hours, 8 to 60 hours, 10 to 48 hours, or 12 to 36 hours before culturing in the pretreatment medium.

[0339] In embodiments of the first and second methods, the culture of target cells can be two-dimensional or three-dimensional cell culture. For example, in some embodiments, the culture vessel used for the culture can be, for example, a flask, a tissue culture flask (HyperFlask), a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, CellSTACK® Chambers, a culture bag, a roller bottle, a bioreactor, a stirred culture vessel, a spinner flask, a microcarrier, or a vertical wheel bioreactor.

[0340] In embodiments where the culture comprises a two-dimensional cell culture, for example, on the surface of the culture vessel, the culture surface (to which the cells are intended to adhere) can be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, types I, II, and IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly-D and poly-L-lysine, matrigel, thrombospondin, and / or vitronectin.

[0341] In the first and second method embodiments, the at least one property may also be analyzed with reference to one or more control samples.

[0342] For example, the first and second methods may further include culturing positive control cells in parallel, where the positive control cells are not administered the conditioned medium or the secretome-containing composition, the extracellular vesicle-containing composition, and / or the sEV-containing composition, and are not cultured under at least one stress-inducing condition. Thus, in embodiments where the stress-inducing condition is the presence of an apoptosis-inducing agent, the positive control cells are not administered the apoptosis-inducing agent.

[0343] The first and second methods may include culturing negative control cells in parallel, where the negative control cells are not administered conditioned medium or a secretome-containing composition, an extracellular vesicle-containing composition, and / or an sEV-containing composition. In some embodiments, the negative control cells include negative control cells that are subjected to the same process as the target cells, except that they are not administered a secretome.

[0344] In certain embodiments, the negative control cells comprise negative control cells cultured in a pre-treatment medium under at least one stress-inducing condition, and the at least one property measured in the target cells can then be measured in the negative control cells either during or after they are cultured in the pre-treatment medium under at least one stress-inducing condition.

[0345] In some embodiments, negative control cells include negative control cells to which mock conditioned medium or mock secretome-, extracellular vesicle-, and / or sEV-containing compositions have been added, in certain embodiments of which the mock conditioned medium or mock secretome-, extracellular vesicle-, and / or sEV-containing compositions are produced by excluding cells from the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing compositions, e.g., processes of the present disclosure.

[0346] The use of such a negative control allows for the evaluation of the activity, functionality, and / or efficacy of the conditioned medium or secretome, extracellular vesicles, and / or sEV-containing composition. For example, if at least one measured property is the viability of cultured cells, the conditioned medium or secretome, extracellular vesicles, and / or sEV-containing composition can be determined to be active, functional, or efficacious (and / or exhibit a therapeutic effect) if the viability of the target cells is greater than the viability of the negative control cells.

[0347] Alternatively, for example, if at least one property measured is cell adhesion, cell proliferation and / or cell number, and cell adhesion, cell proliferation and / or cell number are determined by measuring electrical impedance across the surface of the culture vessel in the culture, the conditioned medium or secretome, extracellular vesicles and / or sEV-containing composition can be determined to be active, functional, efficacious (and / or exhibit a therapeutic effect) if the electrical impedance across the surface of the culture vessel in the culture is higher than the electrical impedance across the surface of the culture vessel in a culture of negative control cells.

[0348] Any one or more samples and / or any one or more positive and / or negative controls may be performed in replicates, e.g., in duplicate, triplicate, etc. In some embodiments where cell viability is measured and replicate cultures are performed, the number of positive control cells in the replicate cultures may be averaged to generate an average maximum cell number (the number of target cells in each replicate culture may be normalized to the average maximum cell number to calculate cell viability).

[0349] To more accurately compare the activity, functionality, and / or potency of different conditioned medium or secretome-containing compositions, extracellular vesicle-containing compositions, and / or sEV-containing compositions, it may be beneficial to determine the amount of conditioned medium or secretome-containing composition, extracellular vesicle-containing composition, and / or sEV-containing composition added to target cells. This can be determined, for example, based on one or more of the amount of secretory cells that produced the secretome, the protein content of the secretome, the RNA content of the secretome, the exosome content of the secretome, and the particle number.

[0350] Methods of Treatment Using the Compositions of the Present Disclosure The present disclosure further contemplates the use of the compositions of the present disclosure for the treatment or prevention of various diseases and conditions in a subject in need thereof. Methods of treatment or prevention contemplated herein include, for example, the 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.

[0351] 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.

[0352] In some embodiments, the cardiomyopathy is chemotherapy-induced cardiomyopathy. In some embodiments, the chemotherapy-induced cardiomyopathy is anthracycline-induced cardiomyopathy. In some embodiments, the anthracycline is doxorubicin.

[0353] In some embodiments, the methods of the present disclosure improve cardiac function, health, endurance, or recovery in a subject.

[0354] In some embodiments, the disclosed methods improve angiogenesis, e.g., by improving the migration and / or wound healing capacity of stressed endothelial cells, improve cardiomyocyte viability, improve endothelial cell viability, health, and function, reduce fibrosis in cardiac fibroblasts, improve stressed cardiomyocyte viability, improve stressed endothelial cell viability, survival, and proliferation, and improve cell migration and / or wound healing capacity in a subject. In some embodiments, the cardiac fibroblasts whose fibrosis is reduced by the disclosed methods are stimulated cardiac fibroblasts, such as cardiac fibroblasts stimulated with TGF-β1. In some embodiments, the methods reduce expression of the pro-fibrotic marker POSTN in TGF-β1-stimulated cardiac fibroblasts below levels prior to stimulation with TGF-β1.

[0355] In some embodiments, the disclosed methods improve or maintain left ventricular end-systolic volume (LVESV), hi some embodiments, the methods maintain LVESV within 20%, 15%, 10%, 5%, or 2% of the pre-treatment LVESV.

[0356] In some embodiments, the methods of the disclosure do not induce an allogeneic inflammatory response, do not induce 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 CD107-expressing NK cells in a subject.

[0357] In some embodiments, compositions of the present disclosure are administered to a subject by interventional cardiology methods such as intravenous infusion, direct cardiac injection, intra-arterial or catheter-based administration.

[0358] The dose, route of administration, frequency of administration and duration of treatment with the compositions of the present invention can be determined by considering the disease or condition for which the treatment is being administered, the severity and duration of the disease or condition, the medical history and general health of the subject being treated, the tolerability of the composition, adverse effects and other factors.

[0359] The compositions of the present disclosure may be administered in doses containing secretomes derived from, for example, 100-10 million cells per kg of the subject's body weight, 500-5 million cells per kg of the subject's body weight, 1-3 million cells per kg of the subject's body weight, 1-2 million cells per kg of the subject's body weight, or 1 million cells per kg of the subject's body weight. The dose may be administered all at once, for example, with each intravenous infusion, 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 a mixture thereof.

[0360] The compositions of the present disclosure may be administered at a dose of, for example, 1 x 10 per kg of subject body weight. 9 ~60×10 9 particles, 10 x 10 per kg of subject's body weight 9 ~60×10 9 particles or 10 x 10 per kg of subject body weight 9 ~40×10 9 particles, 20 x 10 per kg of subject's body weight 9 ~40×10 9particles, 20 x 10 per kg of subject's body weight 9 particles or 10 x 10 per kg of subject body weight 9 The dose may be administered in a dose containing particles. The number of particles may be measured, for example, by nanoparticle tracking analysis (NTA). The dose may be administered all at once, for example, with each intravenous infusion, direct cardiac injection, or intra-arterial administration, or over several administrations.

[0361] The compositions of the present disclosure may be administered at a dose of, for example, 20×10 per kg of subject body weight. 9 ~200×10 9 particles, 30 x 10 per kg of subject's body weight 9 ~100×10 9 particles, 60 x 10 per kg of subject's body weight 9 particles, 50 x 10 per kg of subject's body weight 9 particles, 40 x 10 per kg of subject's body weight 9 particles, 30 x 10 per kg of subject's body weight 9 particles, 20 x 10 per kg of subject's body weight 9 particles or 10 x 10 per kg of subject body weight 9 The cumulative daily dose may be administered in a cumulative daily dose containing particles. The number of particles can be measured by NTA. The cumulative daily dose may be administered at once, for example, as a single intravenous infusion, direct cardiac injection, or intra-arterial administration, or over several administrations.

[0362] 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.

[0363] The treatment period can 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.

[0364] During the course of treatment, the compositions of the present disclosure may be administered, for example, every day, every other day, every day to every 30 days, every 7 to 21 days, every 21 days, every 14 days, or every 7 days.

[0365] Testing products in 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 allows for testing the effects of products on the physiology of tissues, organs, and organisms. Novel animal models of heart failure are described herein. While the term "heart failure" is typically used in human subjects, its use herein extends to animal models. Two different heart failure models are described. One is a murine postischemic chronic heart failure model induced by surgical means (permanent occlusion of the left ventricular coronary artery), and the other is a nonischemic, chemotherapy-induced cardiomyopathy accompanied by left ventricular dysfunction and other signs of heart failure. Both models involve left ventricular dysfunction.

[0366] In the chemotherapy-induced cardiomyopathy (CCM) model described herein, left ventricular dysfunction is induced in rats by administration of doxorubicin, a drug belonging to the anthracycline class of anticancer therapy and one of the most widely used antineoplastic agents due to its broad spectrum of activity. Anthracyclines are chemotherapeutic agents known to induce heart failure in some patients. The model described herein effectively reproduces many physiological features of chemotherapy-induced cardiomyopathy (CCM) in humans, including progressively larger left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV), reduced ejection fraction (LVEF), decreased systolic elastance, and slower LV depolarization (increased QTc on EKG). In human patients, these features are characteristic of reduced cardiac function and are associated with poor prognosis. The model described herein is useful for determining the effects of extracellular vesicles (EV)-containing compositions on cardiac physiology. The beneficial effects of EV-containing compositions in this non-ischemic model are expected to predict the beneficial effects of EV-containing compositions on cardiac function in human subjects with non-ischemic heart failure, including subjects with chemotherapy-induced heart failure.

[0367] The beneficial effects of EV-containing compositions in post-ischemic models of chronic heart failure are expected to predict the beneficial effects of EV-containing compositions on cardiac function in human subpopulations with post-ischemic heart failure, such as patients with pre-existing myocardial insufficiency.

[0368] Using the proposed rat model of CCM, we demonstrate herein that the final CTC1-EV formulation has a favorable effect on the physiology of the failing heart. We demonstrated that animals treated with the EV-containing composition experienced reduced progression of left ventricular dysfunction compared to controls, as indicated by less expansion of cardiac volume over the study period. In contrast, controls continued to deteriorate over the study period, as indicated by increased systolic and diastolic cardiac volumes.

[0369] In addition to testing product efficacy in animal models, it is useful to test products in in vitro models of human disease using human cells. In vitro models are key to understanding the specific effects a product has on a particular cell type by allowing for direct and specific analysis of that cell type. The most useful in vitro cell models involve human cells, as they best match the intended use of a product designed to treat human subjects. If necessary, in vitro models can be limited to a single cell type, allowing the biological effects of EV-containing compositions on that specific cell type to be examined. Furthermore, coculture and mixed cell models are also useful for exploring the interactions of different cell types. Herein, we describe the effects of EV-containing compositions on four human cell types in monoculture and one mixed peripheral blood mononuclear cell (PBMC) model. 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. The inventors found a positive biological effect of the EV-containing composition on the lack of allogeneic activation of human cardiomyocytes, human endothelial cells, human cardiac fibroblasts and human NK cells or human PBMCs.

[0370] Cardiomyocytes are stressed in heart failure and may undergo programmed cell death (apoptosis). Preserving the health and survival of cardiomyocytes has beneficial effects on failing hearts or hearts with ventricular dysfunction. The inventors have shown that EV-containing compositions promote the survival of human cardiomyocytes under apoptosis-inducing stress. EV-containing compositions are expected to promote the health and survival of cardiomyocytes in human subjects with heart failure. EV-containing compositions are expected to improve cardiomyocyte-related function in heart failure in human subjects.

[0371] Endothelial cells are essential components of the cardiac vascular and lymphatic tissues. Lack of adequate circulation and drainage to and from cardiac tissue contributes to the decline of cardiac function in failing hearts. Promoting endothelial cell survival, proliferation, and migration under stress can promote beneficial cardiac tissue remodeling or reduce negative cardiac tissue remodeling. Together, this helps improve or preserve cardiac function. The inventors have demonstrated that EV-containing compositions support human endothelial cell survival in vitro under two different forms of stress: in vitro human endothelial cell proliferation and in vitro human endothelial cell migration when these cells are under stress. EV-containing compositions are expected to promote endothelial cell survival, proliferation, and migration in human subjects with heart failure, where vascular health and wound healing capacity are impaired. Increased angiogenesis in failing cardiac tissue supports the maintenance of cardiac tissue health. EV-containing compositions are expected to improve endothelial cell-related functions in human subjects with heart failure.

[0372] Fibrosis increases in heart failure. Fibrosis contributes to negative remodeling of the failing heart. Reducing fibrosis supports better cardiac function in failing hearts. The inventors have shown that an EV-containing composition reduces signs of fibrosis in human cardiac fibroblasts stressed to conditions with increased fibrosis. It is expected that the EV-containing composition will reduce fibrosis in human subjects with heart failure and reduce the adverse effects of fibrosis on cardiac function.

[0373] Heart failure leads to a pro-inflammatory state in humans, contributing to the deterioration of cardiac function. The inventors have demonstrated in vitro and in vivo that the EV-containing composition of the present invention does not stimulate allogeneic NK degranulation. The inventors have also demonstrated that the EV-containing composition does not induce allogeneic PBMC activation in vitro. The EV-containing composition of the present invention does not promote NK degranulation in human subjects with heart failure, nor does it induce PBMC activation in human subjects with heart failure. The EV-containing composition of the present invention is immunologically neutral or anti-inflammatory when administered to human patients with heart failure.

[0374] In vivo preclinical safety testing was conducted on the EV-containing composition (see Clinical Trial NCT05774509, incorporated herein by reference in its entirety). Using GLP mouse and GLP rat models, the preclinical safety profile of the EV-containing composition of the present invention was demonstrated. The selected immunocompetent animal model, even xenografts, showed no signs of acute immune reaction after repeated administration. Both mouse and rat models showed no toxicity of the EV-containing composition when administered at high doses. Furthermore, a GLP immunocompromised mouse model did not demonstrate tumorigenicity of the EV-containing composition. Demonstration in animal models that are more complex than in vitro models is important because they have functional immune and circulatory systems. The animal model test results demonstrated that the EV-containing composition of the present invention has a favorable safety profile, which needs to be established before testing in human subjects.

[0375] The EV-containing compositions of the present invention are non-toxic and non-tumorigenic when administered to human subjects.

[0376] Another aspect of the EV-containing compositions of the present invention is their combination of multiple parallel beneficial biological effects that are expected to affect the biology of multiple cell types in a beneficial way in patients in need of treatment, combined with a positive safety profile. The EV-containing compositions of the present invention are complex mixtures of biological molecules that enable the above-mentioned simultaneous protective, therapeutic, or regenerative properties that cooperate to support the health, survival, and function of multiple cell types, contributing to the physiological effects observed in animal models of chemotherapy-induced cardiomyopathy (CCM), as shown herein as therapeutic effects when used to treat humans.

[0377] In addition to the novel and unexpected biological and therapeutic effects of the disclosed EV-containing compositions of the present invention, a manufacturing process has been designed, optimized, and tested at a Phase 1 clinical manufacturing scale using GMP-compatible methods, materials, and reagents to produce EV-containing compositions for use in human subjects. The use of multi-layer cell stacks, an in-line clarification process, TFF (tangential flow filtration), the ability to work from frozen cells if needed, the ability to freeze the TFF retentate for future pooling if needed, and the lack of a centrifugation step make the process defined herein scalable to Phase 2 and Phase 3 manufacturing and commercial manufacturing scales.

[0378] Furthermore, the inventors have disclosed a novel and innovative inventive comprehensive panel of in-process quality control tests and release tests for quality control that ensure the reproducibility, stability, safety, and efficacy of EV-enriched secretomes or EVs and compositions containing them as therapeutics. The panel features described herein can be applied to the quality control of other EV-enriched therapeutics to ensure process control, reproducibility of EV-containing compositions, safety of EV-containing compositions, efficacy of EV-containing compositions, and stability of EV-containing compositions to enable their use in human subjects. [Example]

[0379] Non-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, etc.), but some experimental error and deviation 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 inventors regard as various embodiments of the present invention. Not all of the following steps described in each example are required, and the order of the steps in each example does not have to be as presented.

[0380] Example 1 Generation of cardiovascular progenitor cells from iPSCs Human iPS cells (iPSCs) were expanded and differentiated into cardiovascular progenitor cells (CPCs) by suspension culture in PBS-mini vessels (PBS MINI 0.5L Bioreactor Disposable Vessels; PBS Biotech, Reference: 1A-0.5-D-001) using the process shown in Figure 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 vessel, the cell aggregates were allowed to settle by gravity, the supernatant was removed, and the aggregates were resuspended in 3–5 mL of room-temperature TrypLE Select (Invitrogen, Reference: 12563029) and incubated at 37°C for 3–10 minutes. The digestion was stopped using two volumes of RPMI-B27 quench medium (RPMI 1640 medium (Gibco reference: 118875-085) supplemented with B-27 XenoFree, CTS grade 50x (Gibco reference: A14867-01, fc = 1x), filter-sterilized using a 0.2 µm filter (ThermoScientific, reference 567-0020)). The cell suspension was then centrifuged at 300 x g for 5 min, and the resulting supernatant was discarded. The remaining cell pellet was carefully loosened, and the cells were resuspended in 5–10 mL of MEM α medium base (MEM α, GlutaMAX™, without nucleosides, Gibco, reference 32561-.37). A 500 µL sample of these resuspended cells was counted using a ViCell XR cell viability analyzer (Beckman Coulter) according to the manufacturer's instructions. Viable cells per mL were recorded. As shown in Figure 2, two different differentiation runs were performed, resulting in similar yields of CPCs per input iPSC.

[0381] To confirm that the obtained cells were indeed CPCs, we analyzed RNA expression by the obtained cells. Specifically, 1–2 million cells were removed from the cell sample and lysed in RLT Plus buffer (Qiagen 1030963) for RNA extraction. RNA was extracted from the lysate using the Qiasymphony RNA Kit (Qiagen, Ref: 931636) according to the manufacturer's instructions. The mRNA levels of 48 custom-selected genes were assessed using the qPCR Fluidigm platform. Unsupervised hierarchical clustering was performed on the raw data using the "SINGuLAR Analysis Toolset" package in R v3.1.1 (Figure 3A). Unsupervised hierarchical clustering was also performed on gene z-scores using JMP software v17 (method = ward, not standardized) (Figure 3B). RNA expression by the obtained cells was compared with that by iPSC and cardiomyocyte control cells to confirm that gene expression by the obtained cells was consistent with their identification as CPCs (Figure 3A, Figure 3B, and Table 1). Table 1 shows the Ct data required to generate Figures 3A and 3B.

[0382] To dissociate CPC aggregates into single cells, 300–800 mL of CPC aggregate suspension was collected from differentiation suspension cultures and allowed to settle for approximately 5 minutes in a 500 mL conical tube. The spent medium was then removed, and the cell aggregates were washed with DPBS- / -. The washed cell aggregates were then resuspended in room temperature TrypLE (approximately 25 mL of TrypLE for 100 mL of original aggregate suspension volume) and allowed to dissociate for 10 minutes at 37 °C. Dissociation of the cell aggregates was quenched with an equal volume of RPMI-B27 quench medium, and the dissociated cells were spun at 400 × g for 5 minutes. The resulting cell pellet was resuspended in RPMI-B27 quench medium and then strained (Falcon 100 μm cell strainer, Corning Ref: 352360) into a conical tube and counted using a ViCell XR cell viability analyzer (Beckman Coulter).

[0383] A subset of these cells was respun at 300 × g for 5 min and cultured in alpha-MEM complete medium (MEM alpha medium base (MEM alpha, GlutaMAX™, without nucleosides, Gibco, ref. 32561-.37); gentamicin (Gibco, ref. 15750060, final concentration (fc) = 0.025 mg / mL); glucose supplement (Gibco, ref. A2494001, ratio of 1:200); flexibumin (25% w / vol human serum albumin, Baxter reference: NDC0944-0493-02 code 2G0012, fc HSA = 2 mg / mL); B27 (minus insulin) (50x, Gibco, ref. A1895601, fc = 1x); human FGF-2 premium grade (Miltenyi The cells were resuspended for fresh CPC-plating follicle-forming culture in a medium containing 0.2 μm (Biotec, Reference: A12873-01, fc=1 μg / mL); filter sterilized using a 0.2 μm filter (ThermoScientific, Reference 567-0020); medium was used the same day. The freshly collected single cells were counted again using a ViCell XR cell viability analyzer (Beckman Coulter) and plated (see Example 2 below). The remainder of the single-cell suspension was spun at 400 × g for 5 minutes, and the cells were resuspended at 25 million cells / mL in cryopreservation medium (CryoStor CS-10, BioLife Solution Reference: 210102), frozen at -80 °C, and then stored in liquid nitrogen for later use in thawed CPC-plating follicle-forming cultures.

[0384] The Ct data used to generate Figures 3A and 3B are shown in Table 1.

[0385] [Table 2] JPEG2025536321000006.jpg255162

[0386] Example 2 Vesicle-forming culture of cardiovascular progenitor cells CPCs were cultured in the vesiculation process as fresh aggregates in suspension culture, as fresh single cells plated in HyperFlask, or as thawed single cells plated in HyperFlask after cryopreservation and subsequent maintenance at -80°C or below until use. Specifically, the CPCs produced in Example 1 were used for suspension vesiculation and adherent vesiculation in HyperFlask, as described below.

[0387] For suspension vesicle culture, the volume of the aggregates in the PBS-mini vessels at the end of the CPC differentiation process was recorded (300–400 mL per vessel; "day + 0" volume). The cell aggregates underwent a 100% medium change according to the following steps: (1) The cell aggregates were transferred from the PBS-mini vessels to a conical tube and allowed to settle for approximately 15 minutes. (2) The PBS-mini vessels were rinsed three times with MEM alpha medium base (MEM α, GlutaMAX™, nucleoside-free, Gibco, reference 32561-.37). (3) The spent medium was removed from the settled cell aggregates. (4) The cell aggregates were washed three times with the appropriate volume of MEM alpha medium base. (5) The washed cell aggregates were reseeded into their original (washed) PBS-mini vessels in α-MEM complete medium (as described above) at their day 0 volume to maintain cell density.

[0388] The seeded cell aggregates were then cultured in suspension (37 °C, 5% CO2 in atmospheric oxygen) with agitation at 40 rpm for 2 days (until "day +2"). On day +2, after rinsing three times with MEM alpha medium base, the cell aggregates underwent a 100% medium change. For this day +2 medium change, the cell aggregates were reseeded in their original PBS-mini vessels in the same volume as their day +0 volume in alpha-MEM poor medium (MEM alpha, GlutaMAX™, nucleoside-free, Gibco, ref. 32561-.37) supplemented with gentamicin (Gibco, ref. 15750060, final concentration (fc) = 0.025 mg / mL) and glucose supplement (Gibco, ref. A2494001, 1:200 ratio), filter sterilized using a 0.2 μm filter (ThermoScientific, ref. 567-0020)). The cell aggregates were then cultured in suspension (37°C, 5% CO2 in atmospheric oxygen) with agitation at 40 rpm for an additional 2 days until the end of the vesicle formation period ("day +4").

[0389] For HyperFlask adherent culture, fresh single-cell CPCs were plated onto vitronectin-coated HyperFlasks in alpha-MEM complete medium ("day +0") at 100,000 cells / cm. 2 Cryopreserved CPCs were then thawed at 37°C for 3 minutes, transferred to an empty conical tube, and resuspended (dropwise) in alpha-MEM complete medium. The thawed cell suspension was centrifuged, and the cell pellet was resuspended in alpha-MEM complete medium. The thawed CPCs were plated at 100,000 cells / cm in vitronectin-coated hyperflasks in alpha-MEM complete medium ("day +0"). 2 The seeded cells, both fresh and thawed CPCs, were then cultured for two days (37°C, 5% CO2 in atmospheric oxygen) (until "Day +2"). On Day +2, the spent medium was removed and the flasks were rinsed three times with 50-100 mL of pre-warmed MEM alpha medium base. The culture vessels were then filled with alpha-MEM poor medium according to the manufacturer's instructions and incubated for an additional two days (37°C, 5% CO2 in atmospheric oxygen) until the end of the vesicle formation period ("Day +4").

[0390] On days +2 and +4, cells in suspension cultures were counted as described above in Example 1. On day +4, cells in adherent cultures were harvested by: 1) rinsing the cells with DPBS; 2) incubating the cells with 100 mL of prewarmed 0.05% trypsin-EDTA (Gibco, 15400-054, diluted in DPBS) for 2-3 minutes at room temperature; 3) quenching the harvest with 100 mL of aMEM + glutamax supplemented with B27 (minus insulin) (fc1x); 4) harvesting the bulk cell suspension into a 500 mL conical centrifuge tube; and 5) rinsing the harvested flask with basal aMEM medium to harvest any remaining cells and adding this rinse to the bulk cell suspension. The concentration of cells in suspension was determined using a ViCell Automated Cell Counter, and a 1 cm sample from the harvested vessel was collected. 2 Cells per well were back-calculated.

[0391] In addition to CPC adherent and suspension vesicle-forming cultures, virgin medium controls were also performed for adherent and suspension cultures.

[0392] For the suspension vesicle-forming culture virgin medium control, a new 0.5 L PBS-mini vessel was filled with 400 mL of alpha-MEM complete medium ("Day +0") and incubated for 2 days (37 °C, 5% CO2 in atmospheric oxygen) with agitation at 40 rpm. After 2 days ("Day +2"), the spent culture medium was removed and the vessel was thoroughly rinsed (3 times with 50-100 mL of pre-warmed MEM alpha medium base). The PBS-mini vessel was then filled with 400 mL of alpha-MEM poor medium and incubated for another 2 days (37 °C, 5% CO2 in atmospheric oxygen) until "Day +4".

[0393] For the adherent vesicle formation culture virgin medium control, vitronectin-coated HyperFlasks were filled with alpha-MEM complete medium and incubated for two days (37°C, 5% CO2 in atmospheric oxygen). After these two days ("Day +2"), the spent culture medium was removed and the vessel was rinsed thoroughly (three times with 50-100 mL each of pre-warmed MEM alpha medium base). The HyperFlasks were then filled with alpha-MEM poor medium and incubated for two more days (37°C, 5% CO2 in atmospheric oxygen) until "Day +4."

[0394] On day +4, medium from the suspension and adherent cell cultures (conditioned medium, MC) and day +4 medium from the virgin control container (virgin medium, MV) were collected and pre-clarified by sequential centrifugation (400 x g for 10 minutes at 4°C, then 2000 x g for 30 minutes at 4°C). The pre-clarified medium was then aliquoted into conical tubes and frozen at -80°C. Figure 4 shows the process flow diagram for the generation of conditioned medium and virgin medium control.

[0395] Example 3 Preparation of small extracellular vesicle-enriched fraction (sEVs) To validate the vesicle formation process, samples of conditioned and control media were subjected to ultracentrifugation to generate sEV and MV preparations for molecular characterization and in vitro functional analysis. Two biological replicates of each sample type were prepared. Figure 5 shows the process flow diagram for the isolation of sEV or mock (virgin media) control samples.

[0396] MC and MV were thawed for 1–4 h at room temperature or overnight at 4°C. After thawing, MC and MV were ultracentrifuged at 100,000 × g for 16 h at 4°C (wX+ Ultra Series centrifuge, ThermoScientific; rotor: F50L-8x39; acceleration: 9; deceleration: 9), and the resulting supernatant was removed. The bottom of each tube was rinsed twice with 100 μL of 0.1 μm-filtered DPBS− / − (0.1 μm PES filter unit, ThermoFisher 565-0010) without disturbing the pellet. Each pellet was then resuspended in 0.1 μm-filtered DPBS− / − by gently stirring the solvent with a sterile glass stir bar. sEV preparations were collected, and the tubes were rinsed with 0.1 μm-filtered DPBS− / − for maximum product recovery (to the total resuspension + rinse target volume, as calculated based on the number of secreting cells giving rise to the conditioned medium). Calculated using the following formula: 1.4 x 10 6 45 μL was targeted per secretory cell on day +4.

[0397] Target sEV resuspension volume = (total viable cells on day + 4 ÷ total volume conditioned medium on day + 4) × centrifuged MC volume × (45 µL ÷ 1.4 x 10 6 living cells).

[0398] The target resuspension volume of the MV control was matched to the relevant MC target resuspension volume. For MC and MV produced in PBS minivessels, sEV preparations were filtered through a 0.65 μm filter (Ultrafree 0.65 μm DV Durapore, Millipore reference: UFC30DV05) to remove large particulates. sEV and MV control preparations were aliquoted and frozen at -80°C.

[0399] sEV and MV control preparations were further analyzed as described below.

[0400] First, particle concentrations and size distributions in sEV and MV control preparations were determined by nanoparticle tracking analysis (NTA; NanoSight). Nanoparticle tracking analysis confirmed the presence of exosome- and microparticle-sized particles in sEV prepared from CPC-conditioned medium, but not in the MV control. Figure 6 shows representative size distribution curves from two sEV and two control MV samples. Observable particle sizes ranged from approximately <30 nm to 300 nm, with peaks generally between 50 and 150 nm, corresponding to the size of exosomes or small microparticles.

[0401] Second, we also analyzed the presence of the exosome-associated vesicle surface marker CD63 using a PS Capture Exosome ELISA Kit (Wako Chemicals, Reference: 293-77601) with an anti-CD63 antibody (Wako Chemicals, Reference: 292-79251) as the primary antibody and an HRP-conjugated anti-mouse IgG antibody (Wako Chemicals, Reference: 299-79261) as the secondary antibody. The input volume was set so that 400 ng of protein from the sEV and MV control preparations was added to each well. This anti-CD63 ELISA evaluation confirmed the presence of the exosome-associated CD63 surface antigen in each of the sEV samples but not in any of the MV controls (Figure 7). The CD63 signal was higher in the aggregated samples than in the plated samples, but 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 reference: 23235).

[0402] Example 4 In vitro analysis of sEV functionality To 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-starved H9c2 cells; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.

[0403] For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed for Incucyte by Essen BioSciences) was used according to the manufacturer's instructions. Briefly, HUVEC cells were grown using HUVEC complete medium:endothelial cell basal medium (PromoCell, ref. C-22210) supplemented with an endothelial cell growth medium supplement pack (PromoCell, ref. C-39210). After growth, cells were aliquoted at 1-2 x 10 per aliquot in CS10 (Cryostore, ref. 210102). 6 Cells (enough for half to a full 96-well plate) were cryopreserved. Two days before the assay, an aliquot of HUVECs was thawed and seeded at 10,000 cells / well onto ImageLock 96-well plates (EssenBio, Ref: 4379) and grown in HUVEC complete medium for 2 days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. According to the manufacturer's instructions, wells were scratched using a Wound Maker (EssenBio, Ref: 4493). Cells were then rinsed with endothelial cell basal medium and cultured overnight (either in HUVEC complete medium alone as a positive control; endothelial cell basal medium alone as a negative control; or in endothelial cell basal medium supplemented with sEV or MV preparations). Plates were imaged every 3 h for a total of 18 h using an Incucyte with a Scratch Wound Healing Module. Wound closure was determined using the manufacturer's software, and values ​​were subtracted from the baseline (negative control) and normalized to the positive control. Figure 8 shows that the sEV preparation, but not the control MV preparation, promoted wound healing, demonstrating the functionality of the sEV preparation.

[0404] A cardiomyocyte viability assay using serum-starved H9c2 cells was performed essentially as described by El Harane et al. (Eur. Heart J., 2018;39:1835-1847). In this assay, H9c2 cardiomyocytes are proliferative when the culture medium is serum-rich (e.g., cultured in H9c2 complete medium), but cease proliferation and lose viability when serum-free (e.g., cultured in H9c2 poor medium). The ability of sEV and MV preparations to promote H9c2 cardiomyocyte viability was determined by supplementing H9c2 poor medium with increasing concentrations of sEV and MV control preparations. Figure 9 shows that the sEV preparation, but not the control MV preparation, improved H9c2 cardiomyocyte viability in the absence of serum, demonstrating the functionality of the sEV preparation.

[0405] For cardiomyocyte viability assays using staurosporine-treated human cardiomyocytes, iCell Cardiomyocytes were plated at 50,000 cells / well in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., Ref. M1001) on fibronectin-coated 96-well plates. 2Cells were seeded with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: CMC-100-012-001) and cultured for 4 hours. The medium was then replaced with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: M1003), and the cells were cultured for up to 7 days, with complete medium changes every 2–3 days. After a minimum of 4 days, cells were exposed to iCMM containing NucSpot Live 650 dye (Biotium, Reference: 40082) (which served as a viable cell control) or iCMM with NucSpot Live 650 dye and staurosporine (Abcam, Reference: ab146588) at a final well concentration of 2 μM (which also served as an apoptotic cell control). Dye, PBS, and DMSO concentrations, as well as final well volumes, were equivalent in all wells. Cells were cultured in these preincubation media for 4 hours. After this incubation, the pre-incubation medium was removed and the wells were rinsed with iCMM. The cells were then fed with iCMM containing NucSpot Live 650 dye and PBS, or iCMM containing NucSpot Live 650 dye supplemented with increasing concentrations of sEV or MV control preparations while maintaining the final volume of PBS. The wells were imaged every 24 hours in an Incucyte and nuclei counts were determined. Figure 10 shows that the sEV preparation, but not the control MV preparation, improved cardiomyocyte survival, demonstrating the functionality of the sEV preparation. The results shown in Figure 10 are detailed in Table 2.

[0406] [Table 3]

[0407] Example 5 An exemplary Good Manufacturing Practice (GMP)-compliant process for producing an extracellular vesicle-enriched fraction (sEV) preparation of CTC1-EV A first exemplary GMP-compatible process for manufacturing sEV-containing formulations was developed. The manufacturing process included four major steps: vesicle formation, conditioned medium clarification, enrichment and concentration of small EV-enriched secretomes, and production of the final sEV formulation. A flow diagram outlining the GMP-compatible process implemented is shown in Figures 11A and 11B.

[0408] vesicle formation For the vesicle formation step, cardiovascular progenitor cells (CPCs) that had been cryopreserved and stored under vapor-phase liquid nitrogen (or in a -150°C freezer) were first thawed in EVA bags (Corning) containing thawing medium (MEM alpha, GlutaMAX™ supplement, no nucleosides; Gibco / Life Technologies; ref. 32561-029), glucose (30%) supplement (Macopharma, ref. CARELIDE, to a total final glucose concentration of 2 mg / mL; Ydralbum® (LFB), final concentration 20 mg / mL; B-27™ supplement (50x, Life Technologies, ref. 17504001, final concentration 1x); and Rock inhibitor H1152 (Sigma, ref. 555550, final concentration 0.392 μg / mL) for 2 min at 37°C. 18 mL of thawing medium was used per mL of CPCs.

[0409] After thawing, CPCs were sterilized using vitronectin (Life Tech, ref: VTN-N; recombinant human protein, truncated (ref: A31804); 5 μg / mL, 0.22 μm filter (syringe filter 0.2 μm polyethersulfone (PES) membrane) coated culture flasks (8 × 10ST CellStack culture chambers, tissue culture (TC) treated (Corning, ref: 3271); also 2 × TC treated vitronectin coated T75 flasks) at 0.2 mL / cm. 2Complete medium (MEM alpha, GlutaMAX™ Supplement, no nucleosides; Gibco / Life Technologies; ref: 32561-029; glucose (30%) supplement (Macopharma, ref: CARELIDE, to a final total glucose concentration of 2 mg / mL; Ydralbum® (LFB; 200 g / L); B-27™ supplement (50x, Life Tech, ref: 17504001 or 17504044, final concentration 1x); gentamicin (Panpharma, final concentration 25 μg / mL); and human FGF-2 premium grade (Miltenyi Biotec, ref: A12873-01, final concentration 1 μg / mL)) was used to grow cells at approximately 100,000 cells / cm. 2 The cells were seeded at a seeding density of 1000 μg / ml. Seeding was performed without prior centrifugation of the cell suspension. The seeded CPCs were then cultured in complete medium at 37°C in the presence of 5% CO2 and atmospheric oxygen for 3 days.

[0410] Immediately prior to seeding ("D+0"), the 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), their identity (see Figure 12 and Example 7) by flow cytometry using a MACSQuant 10 flow cytometer, and their transcriptome (see Figure 13 and Example 8) was analyzed.

[0411] After 3 days of culture ("D+3"), cells were harvested from one of the cultured T75 flasks. 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), their identity (see Figure 12 and Example 7) by flow cytometry using a MACSQuant 10 flow cytometer, and their transcriptome (see Figure 13 and Example 8) was analyzed. Spent medium from the 10ST CellStack culture chamber was also tested for sterility and the presence of mycoplasma and endotoxin.

[0412] For the remaining flasks (8 x 10ST CellStack culture chambers; 1 x T75), cells were visualized by microscopy to determine their morphology (see Figure 14), washed twice with wash medium (MEM alpha (Macopharma, ref: BC0110021), glucose (30%) supplement (Macopharma, ref: CARELIDE, to a final total glucose concentration of 2 mg / mL), and then cultured in starvation medium (poor medium) (MEM alpha (1000 mL Macopharma, ref: BC0110021), glucose (30%) supplement (Macopharma, ref: CARELIDE, to a final total glucose concentration of 2 mg / mL)) at 37°C in the presence of 5% CO for 2 days. After this 2-day incubation ("D+5"), the culture medium (conditioned medium) was collected, and cells were harvested from the 10ST CellStack culture chambers and the remaining T75 flasks.

[0413] Similar to the cells on D+3, the cells on D+5 were again visualized by microscopy to determine their morphology (see Figure 14), and the cells harvested on D+5 were further analyzed to determine the number and percentage of viable cells (see Table 5, column 4, *3 (Study 20)), their identity by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 12 and Example 7), and their transcriptome (see Figure 13 and Example 8) was analyzed. The collected conditioned media was tested for sterility and the presence of mycoplasma and endotoxin before further processing.

[0414] Clarification of conditioned medium The conditioned medium was clarified by a series of four filtration steps. First, filtration was performed using a 200 μm drip chamber filter (specific gravity blood set, BD careFusion, reference: VH-22-EGA). The filtrate was then filtered through a syringe using a 15 μm filter (DIDACTIC, reference: PER1FL25). The filtrate was then filtered through Sartoguard PES XLG MidiCaps (pore size (prefilter + filter): 1.2 μm + 0.2 μm, size 7 (0.065 μm)). 2 The resulting filtrate was then filtered using a vacuum filter / storage bottle system (0.22 μm, pore size 33.2 cm). 2 Further filtration was performed using a PES membrane (Corning, ref: 431097).

[0415] Enrichment and concentration After clarification of the conditioned medium, the conditioned medium was subjected to enrichment and concentration of small EV secretome.

[0416] First, the clarified conditioned medium was subjected to tangential flow filtration (TFF) using a TFF Allegro™ CM150 (PALL / Sartorius). For the TFF manifold, a sterile disposable flow-through manual valve P&F (PALL / Sartorius, reference: 744-69N) was used with a 5 L retentate assembly (sterile, single-use; PALL / Sartorius reference: 744-69L). The TFF cassette contained a sterile single-use regenerated cellulose filter (30 kDa cutoff; 0.14 m 2 Sartorius, Reference: Opta Filter Assembly + 3D51445901MFFSG) was used. For collection of the retentate (i.e., what is retained in the TFF), a benchtop TFF 1 L Bag was used (PALL / Sartorius, Reference: 7442-0303P).

[0417] The TFF device was first washed with 10 L of HO and 1 L of 1x PBS (sterilized by filtration using a 0.2 μm filter) before operation. Next, the clarified conditioned medium was administered to the TFF device, and the retentate was concentrated (to 500 mL; pressure less than 3 bar). After this initial concentration step, the retentate was subjected to diafiltration (6 diafiltration volumes; using 1x DPBS, sterilized by filtration using a 0.2 μM filter). After diafiltration, the retentate was further concentrated to obtain a total volume of at least 100 mL. The parameters of the TFF process were as follows: feed manifold pressure (PT01) - 0.86 to 2.1 bar; retentate manifold pressure (PT02) - 0.11 to 0.14 bar; retentate manifold flow rate (FT01) - 0.03 to 0.32 L / min; transmembrane pressure (TMP01) - 0.4 to 1.1 bar; and quatroflow pump (P01) - 18 to 23%.

[0418] Example 6 Formulations / Compositions After enrichment and concentration by TFF, the retentate was processed as shown in Figure 11B. Briefly, the retentate alone, the retentate containing 25 mM trehalose, and the retentate containing 5 g / L L-histidine were each stored in glass vials (2 mL, bromobutyl cap; Adelphi, Reference: VCDIN2RDLS1) and stored at -80 °C. Quality control tests were performed on these samples (different stages at which quality control tests were performed are indicated with an "*", e.g., *6, *7, etc.). In addition, final sEV formulations were also prepared by filter-sterilizing the retentate (with or without 25 mM trehalose) using a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, Reference: SVGPL10RC). After the sterilization step, the final formulations (with or without the addition of 25 mM trehalose) were bottled in glass vials (2 mL, bromobutyl cap; Adelphi, Reference: VCDIN2RDLS1). Furthermore, any pharmaceutically suitable carrier may be utilized. The final formulation was stored at -80°C for future use or testing.

[0419] Thus, the final preparations were in PBS (with or without trehalose) and were positive for CD9, CD63, and CD81 (canonical EV markers) as detected by MACSPlex, and for the cardiac-associated markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142 (as shown in Figures 16A, 16C, 17A, and 17B).

[0420] Example 7 Characterization of CPC identity during vesicle formation in a GMP-compliant process To assess the identity of cells during the vesicle formation process in Example 5, D+0 CPCs and cells harvested on D+3 and D+5 were analyzed by flow cytometry. iPSCs and cardiomyocyte (CM) cells were included as controls. As shown in Figure 12, flow cytometry analysis performed using a MACSQuant 10 flow cytometer with iPSC, CPC, and cardiac markers demonstrated that CPCs became more mature over the 5-day vesicle formation period. Specifically, CPCs maintained little or no NANOG or SOX2 protein expression and showed a continued increase in CD56, cTNT, and aMHC protein expression (however, they did not reach CD56, cTNT, and aMHC expression levels similar to those of cardiomyocytes, indicating that they remained precursors throughout the process). iPSC and CM control cells were analyzed separately, and average values ​​are shown in Figure 12 for comparison purposes.

[0421] Example 8 Transcriptome analysis of CPC during vesicle formation in a GMP-compliant process To evaluate the transcriptome of cells during the vesicle formation process in Example 5, RNA was extracted from CPCs on D+0 and from cells harvested on D+3 and D+5 of the vesicle formation process. RNA was also extracted from iPSCs (pluripotent cell control) and iPSC-derived cardiomyocytes (differentiated cardiomyocyte control). Total RNA was sequenced on an Illumina NovaSeq 6000 platform, and differential gene expression was determined with normalized data.

[0422] The heat maps shown in Figures 13 and 13B were generated based on hierarchical clustering analysis using the UPGMA clustering method and the correlation distance metric in TIBCO Spotfire software v11.2.0. The genes included in the panel are expressed at different differentiation stages (from iPSCs to beating cardiomyocytes) as well as related off-target cells. Therefore, the gene expression analysis results shown in Figures 13 and 13B confirmed that the cells retained cardiovascular progenitor characteristics throughout the vesicle formation process. The data used to generate the heat maps in Figures 13 and 13B are listed in Table 3.

[0423] [Table 4] JPEG2025536321000009.jpg255162JPEG2025536321000010.jpg255162

[0424] Example 9 Analysis of EV particle concentration and EV particle size distribution in a GMP-compliant process To assess the particle concentration and size distribution of the EVs generated in Example 5, the clarified conditioned medium (before TFF) and the final formulation (with and without trehalose) were analyzed by nanoparticle tracking analysis (NTA; NanoSight). Figure 15A shows a representative size distribution curve for each sample. The overall size distribution, mean, and mode were similar between samples. Peaks were generally observed between approximately 50 and 150 nm, corresponding to the size of exosomes or small microparticles. The TFF process resulted in an approximately 32-fold increase in particle concentration. Similar experiments were also performed on the non-sterilized retentate samples (with and without trehalose or histidine) shown in Figure 11B (samples a-c). The results of these experiments are shown in Figure 15B.

[0425] Example 10 Analysis of EV markers in CTC1-EVs produced by a GMP-compliant process To assess the presence of EV markers in the clarified conditioned medium (pre-TFF) and final formulations (with and without trehalose) of Example 5, the MACSPlex Exosome Kit Human (Miltenyi Reference: 130-108-813) was used to identify and quantify the presence of EV markers. As shown in Figure 16A, analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81, and CD63) in both the conditioned medium (pre-TFF) and the final formulations (with and without trehalose). Additionally, as shown in Figure 16B, MACSPlex analysis also revealed various markers that were found to be either present in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD40, CD11c, CD86, CD31, and CD20) or virtually absent (CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69) in the conditioned medium (pre-TFF) and / or in the final formulation (with and without trehalose). Similar experiments were also performed on the storage retentate samples (with and without trehalose or histidine) shown in Figure 11B that were not sterile filtered ("*6", Samples a-c, Experiment 20). The results of these experiments are shown in Figures 16C and 16D.

[0426] Furthermore, as shown in Figure 17A, additional cardiac-related markers were also observed in the conditioned medium (pre-TFF) and the final formulation (with and without trehalose). Similar experiments were performed to confirm the presence of these additional cardiac-related markers in the storage retentate samples shown in Figure 11B (with and without trehalose or histidine) that were not filter-sterilized ("*6", Samples a-c, Test 20). The results of these experiments are shown in Figure 17B.

[0427] Example 11 In vitro analysis of the potency of CTC1-EVs produced by a GMP-compliant process Two in vitro assays were used to analyze the functionality and potency of the final formulation produced by the GMP-compliant process of Example 5: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.

[0428] For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed for Incucyte by Essen BioSciences) was used according to the manufacturer's instructions. Briefly, HUVEC cells were grown using HUVEC complete medium:endothelial cell basal medium (PromoCell, ref. C-22210) supplemented with an endothelial cell growth medium supplement pack (PromoCell, ref. C-39210). After growth, cells were aliquoted at 1-2 x 10 per aliquot in CS10 (Cryostore, ref. 210102). 6 Cells (enough to fill a half-to-full 96-well plate) were cryopreserved. Two days before the assay, an aliquot of HUVECs was thawed and seeded at 10,000 cells / well onto ImageLock 96-well plates (EssenBio, Ref: 4379) and grown in HUVEC complete medium for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. According to the manufacturer's instructions, wells were scratched using a Wound Maker (EssenBio, Ref: 4493). Cells were then rinsed with endothelial cell basal medium and cultured overnight (either in HUVEC complete medium and PBS as a positive control; in endothelial cell basal medium and PBS as a negative control; or in endothelial cell basal medium supplemented with sEV preparation in PBS). Plates were imaged 21 hours after treatment using an Incucyte equipped with a scratch wound healing module. Wound closure was determined using the manufacturer's software, and values ​​were subtracted from the baseline (negative control) and normalized to the positive control. Figure 18 shows that the final formulations with and without trehalose (samples b and a, respectively) promoted wound healing.

[0429] For cardiomyocyte viability assays using staurosporine-treated human cardiomyocytes, iCell Cardiomyocytes were plated at 50,000 cells / well in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., Ref. M1001) on fibronectin-coated 96-well plates. 2Cells were seeded with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: CMC-100-012-001) and cultured for 4 hours. The medium was then replaced with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: M1003), and the cells were cultured for up to 7 days, with complete medium changes every 2–3 days. After a minimum of 4 days, cells were exposed to iCMM containing NucSpot Live 650 dye (Biotium, Reference: 40082) (which served as a viable cell control) or iCMM with NucSpot Live 650 dye and staurosporine (Abcam, Reference: ab146588) at a final well concentration of 2 μM (which also served as an apoptotic cell control). Dye, PBS, and DMSO concentrations, as well as final well volumes, were equivalent in all wells. Cells were cultured in these preincubation media for 4 hours. After this incubation, the pre-incubation medium was removed and the wells were rinsed with iCMM. Cells were then fed with iCMM containing NucSpot Live 650 dye and PBS, or iCMM containing NucSpot Live 650 dye supplemented with increasing concentrations of sEV preparations (samples a and b) while maintaining the final volume of PBS. After 24 hours, wells were imaged in an Incucyte and nuclei counted. Figure 19 shows that the final formulations, with and without trehalose, promoted cardiomyocyte survival.

[0430] The test panel used for the process / product of Example 5 and performed, for example, in Examples 6-11, is shown in Table 4. The results are therefore shown in Table 5. Additionally, Table 6 shows the enrichment of the retentate and final formulations produced in Example 6 compared to the conditioned medium after clarification.

[0431] [Table 5] JPEG2025536321000012.jpg255162

[0432] [Table 6]

[0433] [Table 7]

[0434] Example 12 A second exemplary Good Manufacturing Practice (GMP)-adapted process for producing a small extracellular vesicle-enriched fraction (sEV) preparation of CTC1-EV A second exemplary GMP-compatible process for producing sEV-containing formulations was developed. The manufacturing process included four major steps: vesicle formation, conditioned medium clarification, enrichment and concentration of small EV-enriched secretomes, and production of the final sEV formulation. A flow diagram outlining the GMP-compatible process that was implemented is shown in Figures 24A and 24B.

[0435] vesicle formation For the vesicle formation step, cardiovascular progenitor cells (CPCs) that had been cryopreserved and stored under vapor-phase liquid nitrogen (or in a -150 °C freezer) were first thawed in thawing medium (MEM α (1000 mL Macopharma, ref. BC0110021); glucose (30%) supplement (Macopharma, ref. CARELIDE to a final total glucose concentration of 2 mg / mL); Ydralbum® (LFB), final concentration 20 mg / mL; B-27™ supplement (50x, LifeTech, ref. 17504001, final concentration 1x); and Rock inhibitor H1152 (Sigma, ref. 555550, final concentration 0.392 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter) in an EVA bag (Corning) at 37 °C for 2.5 min. 18 mL of thawing medium was used per mL of CPCs.

[0436] After thawing, 0.2mL / cm 2Complete medium (MEM α (1000 mL Macopharma, ref: BC0110021); glucose (30%) supplement (Macopharma, ref: CARELIDE, to a final total glucose concentration of 2 mg / mL; Ydralbum® (LFB; 200 g / L); B-27™ supplement (50x, Life Tech, ref: 17504001 or 17504044, final concentration 1x); gentamicin (Panpharma, final concentration 25 μg / mL); and human FGF-2 premium grade (Miltenyi Biotec, ref: A12873-01, final concentration 1 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter)) was used to culture the cells in vitronectin (Life Tech). 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 × 10ST CellStack culture chambers, tissue culture (TC) treated (Corning, ref: 3271); 2 × similarly TC-treated vitronectin-coated T75 flasks) at approximately 100,000 cells / cm. 2 CPCs were seeded at a seeding density of 1000 x g / ml. Seeding was performed without prior centrifugation of the cell suspension. The seeded CPCs were then cultured in complete medium at 37°C in the presence of 5% CO2 and atmospheric oxygen for 3 days.

[0437] Immediately prior to seeding ("D+0"), cells were analyzed to determine the number and percentage of viable cells using a NucleoCounter NC-200 (Chemometec) with DAPI / AO staining (Ph.Eur.2.7.29) (see column 1 of Table 7 ("D+0 cells")) and their identity by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 25 and Example 14).

[0438] After 3 days of culture ("D+3"), cells were harvested from one of the cultured T75 flasks. These harvested cells were analyzed to determine the number and percentage of viable cells using a NucleoCounter NC-200 (Chemometec) with DAPI / AO staining (Ph.Eur.2.7.29) (see column 2 of Table 7 ("D+3 Material")) and their identity by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 25 and Example 14). Spent medium from the 10ST CellStack culture chamber was also tested for sterility and the presence of mycoplasma and endotoxin.

[0439] For the remaining flasks (12 x 10ST CellStack culture chambers; and 1 x T75), cells were visualized by microscopy to determine their morphology (see Figure 26), washed twice with wash medium (MEM alpha (1000 mL Macopharma, ref: BC0110021); glucose (30%) supplement (Macopharma, ref: CARELIDE, to a final total glucose concentration of 2 mg / mL), and then cultured for 2 days in starvation medium (poor medium) (MEM alpha (1000 mL Macopharma, ref: BC0110021); glucose (30%) supplement (Macopharma, ref: CARELIDE, to a final total glucose concentration of 2 mg / mL) at 37°C in the presence of 5% CO and atmospheric oxygen. After this 2-day incubation ("D+5"), the culture medium (conditioned medium) was collected, and cells were harvested from the 10ST CellStack culture chambers and the remaining T75 flasks.

[0440] Similar to the D+3 cells, the D+5 cells were again visualized by microscopy to determine their morphology (see Figure 26), and the cells harvested on D+5 were further analyzed to determine the number and percentage of viable cells (see column 3 ("D+5 cells") of Table 7) and their identity by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 25 and Example 14). The collected conditioned media was tested for sterility and the presence of mycoplasma and endotoxin before further processing.

[0441] [Table 8]

[0442] Clarification of conditioned medium The conditioned medium was clarified by a series of three filtration steps. First, filtration was carried out using a Sartopure PP3 MidiCaps 5 μm PES filter (Sartorius, Reference: 5055342P9--OO--A (Sartorius)). The resulting filtrate was then filtered using a Sartoguard PES MidiCaps filter (pore size (prefilter + filter): 1.2 μm + 0.2 μm; Sartorius, Reference: 5475307F9--OO--A). The resulting filtrate was then filtered using a Sartopure2 MidiCaps filter (pore size (prefilter + filter): 0.45 μm + 0.2 μm; Sartorius, Reference: 5445307H8--OO--A).

[0443] Enrichment and concentration After clarification of the conditioned medium, the conditioned medium was subjected to enrichment and concentration of small EV secretome.

[0444] First, the clarified conditioned medium was subjected to tangential flow filtration (TFF) using a TFF Allegro™ CM150 (PALL / Sartorius). For the TFF manifold, a sterile disposable flow-through manual valve P&F (PALL / Sartorius, reference: 744-69N) was used with a 10 L retentate assembly (sterile, single-use; PALL / Sartorius reference: 744-69M). The TFF cassette contained a sterile single-use regenerated cellulose filter (30 kDa cutoff; 0.14 m 2 Sartorius, Reference: Opta Filter Assembly + 3D51445901MFFSG) was used. For collection of the retentate (i.e., what is retained in the TFF), a benchtop TFF 1 L Bag was used (PALL / Sartorius, Reference: 7442-0303P).

[0445] The TFF device was first washed with 10 L of HO and 2 L of 1x PBS before operation. Next, the clarified conditioned medium was dosed into the TFF device, followed by concentration of the retentate (to 500 mL; pressure ≤ 3 bar). After this initial concentration step, the retentate was subjected to diafiltration (6 diafiltration volumes; using 1x DPBS). After diafiltration, the retentate was further concentrated to obtain a total volume of at least 100 mL. The TFF process parameters were as follows: feed manifold pressure (PT01)—0.94–2.1 bar; retentate manifold pressure (PT02)—0.12–0.13 bar; retentate manifold flow rate (FT01)—0.012–0.58 L / min; transmembrane pressure (TMP01)—0.53–1.11 bar; and quatroflow pump (P01)—14–20%.

[0446] Example 13 Formulations / Compositions After enrichment and concentration by TFF, the resulting retentate was then filter-sterilized using a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, ref. SVGPL10RC) to prepare the final sEV preparation. In some experiments, 25 mM trehalose was added before this sterilization step to avoid aggregation. After the sterilization step, the final preparation (with or without the addition of 25 mM trehalose) was bottled in glass vials (2 mL, bromobutyl cap; Adelphi, ref. VCDIN2RDLS1). The final product preparation 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, and then sterile filtered using either a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, ref: SVGPL10RC) or a Sartopure2 filter (pore size (prefilter+filter): 0.45 μm+0.2 μm; Sartorius, standard: 5441307H4--OO--B) to produce the final formulation, as shown in FIG. 24B.

[0447] Thus, the final preparations were in PBS (with or without trehalose) and were positive for CD9, CD63 and CD81 (canonical EV markers) as detected by MACSPlex, and for the cardiac-associated markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29 and CD142 (as shown in Figures 28A and 29).

[0448] Example 14 Characterization of CPC identity during vesicle formation in a GMP-compliant process To assess the identity of cells during the vesicle formation process in Example 12, D+0 CPCs and cells harvested on D+3 and D+5 were analyzed by flow cytometry. iPSCs and cardiomyocyte (CM) cells were included as controls. As shown in Figure 25, flow cytometry analysis performed using a MACSQuant 10 flow cytometer with iPSC, CPC, and cardiac markers demonstrated that CPCs became more mature over the 5-day vesicle formation period. Specifically, CPCs maintained little or no Nanog or SOX2 protein expression and showed a continued increase in CD56, cTNT, and aMHC protein expression (however, they did not reach CD56, cTNT, and aMHC expression levels similar to those of cardiomyocytes, indicating that they remained precursors throughout the process). iPSC and CM control cells were analyzed separately, and average values ​​are shown in Figure 25 for comparison purposes.

[0449] Example 15 Analysis of EV particle concentration and EV particle size distribution in CTC1-EV in a GMP-compliant process To evaluate the particle concentration and size distribution of the EVs produced in Examples 12 and 13, the conditioned medium before (*4 (Test 22)) and after (*5 (Test 22)) clarification, as well as the final formulations (with and without trehalose, Samples b and a, respectively) were analyzed by nanoparticle tracking analysis (NTA; Nanocytometer). Figure 27A shows a representative size distribution curve for each sample. The overall size distribution, mean, and mode were similar between samples. Peaks were generally observed between 50 and 150 nm, corresponding to the size of exosomes or small microparticles. The TFF process resulted in an approximately 32-fold increase in particle concentration. Similar experiments were also performed on previously frozen retentate and final formulation samples (filtered with STerivex-GP or Sartopore 2) shown in Figure 24B (*6, Sample a (Test 22); *7, Sample c (Test 22); and *7, Sample d (Test 22)). The results of these experiments are shown in Figure 27B. The TFF process resulted in approximately a 20-fold higher concentration of particles, even though particles were lost during the final sterile filtration (especially for the final formulation made from thawed retentate).

[0450] Example 16 Analysis of EV markers in the final CTC1-EV formulation manufactured by a GMP-compliant process To assess the presence of EV markers in the clarified conditioned medium (pre-TFF) and final formulations (with and without trehalose) of Example 12, the MACSPlex Exosome Kit Human (Miltenyi Reference: 130-108-813) was used to identify and quantify the presence of EV markers. As shown in Figure 28A, analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81, and CD63) in both the conditioned medium (pre-TFF) and the final formulations (with and without trehalose). Additionally, as shown in Figure 28B, MACSPlex analysis also revealed a variety of markers that were found to be either present in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD40, CD11c, CD86, CD31, and CD20) or virtually absent (CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69) in the conditioned medium (pre-TFF) and / or in the final formulation (with and without trehalose).

[0451] Furthermore, as shown in Figure 29, additional cardiac-related markers were also observed in the conditioned medium (pre-TFF) and the final formulations (with and without trehalose).

[0452] Example 17 In vitro analysis of the potency of CTC1-EV final formulations produced by a GMP-compliant process Two in vitro assays were used to analyze the functionality and efficacy of the final formulation produced by the GMP-compliant process of Example 12: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.

[0453] For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed for Incucyte by Essen BioSciences) was used according to the manufacturer's instructions. Briefly, HUVEC cells were grown using HUVEC complete medium:endothelial cell basal medium (PromoCell, ref. C-22210) supplemented with an endothelial cell growth medium supplement pack (PromoCell, ref. C-39210). After growth, cells were aliquoted at 1-2 x 10 per aliquot in CS10 (Cryostore, ref. 210102). 6 Cells (enough for a half-to-full 96-well plate) were cryopreserved. Two days before the assay, an aliquot of HUVECs was thawed and seeded at 10,000 cells / well onto ImageLock 96-well plates (EssenBio, Ref: 4379) and grown in HUVEC complete medium for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. According to the manufacturer's instructions, wells were scratched using a Wound Maker (EssenBio, Ref: 4493). Cells were then rinsed with endothelial cell basal medium and cultured overnight (either in HUVEC complete medium alone as a positive control; in endothelial cell basal medium with PBS alone as a negative control; or in endothelial cell basal medium supplemented with sEVs in PBS). Plates were imaged 18 hours after treatment using an Incucyte with a scratch wound healing module. Wound closure was determined using the manufacturer's software, and values ​​were subtracted from baseline (negative control) and normalized to the positive control. Figure 30A shows that the final formulations with and without trehalose (*7, samples b and a, respectively, (Test 22)) promoted wound healing. Figure 30B shows that the pre-frozen final formulations without trehalose (*7, samples c and d, (Test 22)) promoted wound healing.

[0454] For cardiomyocyte viability assays using staurosporine-treated human cardiomyocytes, iCell Cardiomyocytes were plated at 50,000 cells / well in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., Ref. M1001) on fibronectin-coated 96-well plates. 2 Cells were seeded with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: CMC-100-012-001) and cultured for 4 hours. The medium was then replaced with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: M1003), and the cells were cultured for up to 7 days, with complete medium changes every 2–3 days. After a minimum of 4 days, cells were exposed to iCMM containing NucSpot Live 650 dye (Biotium, Reference: 40082) (which served as a viable cell control) or iCMM with NucSpot Live 650 dye and staurosporine (Abcam, Reference: ab146588) at a final well concentration of 2 μM (which also served as an apoptotic cell control). Dye, PBS, and DMSO concentrations, as well as final well volumes, were equivalent in all wells. Cells were cultured in these preincubation media for 4 hours. After this incubation, the pre-incubation medium was removed, and the wells were rinsed with iCMM. Cells were then fed with iCMM containing NucSpot Live 650 dye and PBS, or iCMM containing NucSpot Live 650 dye supplemented with increasing concentrations of sEV preparations while maintaining the final volume of PBS. After 24 hours, wells were imaged in an Incucyte and nuclei counted. Figure 31A shows that the final formulations with and without trehalose (*7, samples b and a, respectively (Test 22)) promoted cardiomyocyte survival. Figure 31B shows that the pre-frozen final formulations without trehalose (*7, samples c and d, (Test 22)) promoted cardiomyocyte survival.

[0455] The test panel used for the process / product of Example 12 and performed, for example, in Examples 13-17, is shown in Table 4. The results are therefore shown in Table 7. Additionally, Table 8 shows the enrichment (calculated by the increase in particles per unit protein) for the retentate and final formulations produced in Example 12 compared to the conditioned medium after clarification.

[0456] [Table 9]

[0457] Example 18 Analysis of the effects of cardiovascular progenitor cell (CPC) EVs on cardiac function in a mouse heart failure model To analyze the in vivo functionality and efficacy of sEV preparations produced according to the methods described herein, a mouse model was used to determine the effect of the sEV preparations on cardiac function (in mice with induced heart failure).

[0458] 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 herein by reference in its entirety). Briefly, a total of 42 mice underwent surgical occlusion of the left coronary artery to induce chronic heart failure (CHF). Three weeks after occlusion, 22 of the mice were treated with either PBS vehicle control (60 μL, n=11) or sEVs (60 μL, n=11), delivered via percutaneous injection under echocardiographic guidance into the peri-infarct myocardium (as described in Kervadec et al.). The administered sEVs were produced according to the "sEV 5.3" scheme shown in Figure 2 (whereby sEVs were prepared by ultracentrifugation from clarified "MC5"), and the resulting EVs were resuspended in half the typical PBS volume (producing a 2x concentrated sEV preparation containing secretomes from 6.22E+04 cells per μL of sEV preparation).

[0459] Four weeks after occlusion, cardiac function was assessed by echocardiography. The results are shown in Figure 34. Among CHF mice, significantly fewer sEV-treated mice (compared to PBS-treated mice) had severely progressive LVESV hypertrophy (here defined as an increase in left ventricular end-systolic volume (LVESV) of 9.1 μL or greater; p<0.05). Among CHF mice, significantly fewer sEV-treated mice (compared to PBS-treated mice) had severely progressive LVEDV hypertrophy (here defined as an increase in left ventricular end-diastolic volume (LVEDV) of 4 μL or greater; p<0.05). Although not significant, there was also a strong trend for fewer sEV-treated CHF mice (compared to PBS-treated mice) to have a severely progressive loss of ejection fraction (EF) (here defined as a loss of EF of 5.5% or greater; p<0.056). Furthermore, although not statistically significant, the mean ejection fraction in the PBS group was 2.5-fold worse than in 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.

[0460] Example 19 Manufacturing of small extracellular vesicle-enriched fraction (sEV) clinical candidate (CTC1-EV) formulation; CTC1-EV final formulation Three CPC sEV clinical candidate formulations (Examples 25, 26, and 27 herein) were generated for further analysis. Examples 25 and 26 were made from CPC essentially as described in Example 12 herein, except that the TFF was prepared in a total volume of 15 L; 0.28 m 2 A TFF cassette with a filter of size (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 bar was used.

[0461] Figure 90 shows the process used to generate Example 25. In this example, FCDI CTC1 cardiovascular progenitor cells were thawed and seeded onto vitronectin-coated flasks. This cell population is designated "Clin001." After thawing and prior to plating, a sample of the cells was taken ("*1 (Test 25)"). Twelve 10-layer cell stacks (CS10) and two T-75 flasks were seeded with FCDI CTC1 cells at a density of 100,000 cells / cm² in complete medium. They were cultured for three days at 37°C and 5% CO2 in a humidified incubator. After three days of growth, cells were taken from one of the T-75 flasks for in-process characterization. This sample is designated "*2 (Test 25)." After three days of growth, spent medium was removed from all remaining vessels, and "poor medium" was returned to the vessels ("change to poor medium") three days after plating ("D+3"). The cells were maintained in this poor medium for an additional two days at 37 degrees Celsius in a humidified incubator containing 5% CO2. This is referred to as vesiculation medium. Five days after seeding the cells, spent medium was collected from the CS10 flasks. This medium is referred to as "*4 (Test 25)." An aliquot of this material was used for in-process testing. Five days after seeding 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 collected for in-process testing. The cells collected 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 was then clarified.

[0462] To clarify the spent medium, the medium was filtered three times, first using a Sartopure® PP3 filter with a 5 μm filter size, then using a Sartoguard PES filter with a nominal filter size of 0.2 μm, and finally using a Sartopore® 2 filter with a filter size of (0.45 + 0.2 μm). The filtrate obtained after these three filtrations is the "clarified conditioned medium" or "conditioned medium" for short. For this Example 25, 15 liters of conditioned medium were produced. This conditioned medium is referred to as "*5 (Test 25)." An aliquot of this material was used for in-process testing. A 30 mL aliquot of this material was subjected to ultracentrifugation to generate EV-enriched secretome for further testing. This material is referred to as "*5a.uc (Test 25)."

[0463] The clarified conditioned medium was then processed by tangential flow filtration ("TFF") using a regenerated cellulose filter with a 30 kilodalton cutoff. The surface area of ​​the filter used in Example 25 was 0.28 square meters. The TFF process involved first concentrating the retentate and then diafiltering the retentate using 6 volumes of DPBS. The diafiltered retentate was then concentrated again. An aliquot of the resulting retentate (referred to as "*6 (Test 25)") was analyzed by in-process control. In Example 25, the final retentate was concentrated 50-fold. The remaining retentate was stored in a 1 L bag at 4°C overnight and then placed in a -80°C freezer until needed for Example 27.

[0464] Figure 96 shows the process used to generate Example 26. In this example, FCDI CTC1 cardiovascular progenitor cells were thawed and seeded onto vitronectin-coated flasks. This cell population is designated "Clin002." After thawing and prior to plating, a sample of the cells was taken ("*1 (Test 26)"). Twelve 10-layer cell stacks (CS10) and two T-75 flasks were seeded with FCDI CTC1 cells at a density of 100,000 cells / cm² in complete medium. They were cultured for three days at 37°C and 5% CO2 in a humidified incubator. After three days of growth, cells were taken from one of the T-75 flasks for in-process characterization. This sample is designated "*2 (Test 26)." After three days of growth, spent medium was removed from all remaining vessels, and "poor medium" was returned to the vessels ("change to poor medium") on the third day after plating ("D+3"). The cells were maintained in this poor medium for an additional two days at 37 degrees Celsius in a humidified incubator containing 5% CO2. This is referred to as vesiculation medium. Five days after seeding the cells, spent medium was collected from the CS10 flasks. This medium is referred to as "*4 (Test 26)." An aliquot of this material was used for in-process testing. Five days after seeding 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 collected for in-process testing. The cells collected 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 was then clarified.

[0465] To clarify the spent medium, the medium was filtered three times, first using a Sartopure® PP3 filter with a 5 μm filter size, then using a Sartoguard PES filter with a nominal filter size of 0.2 μm, and finally using a Sartopore® 2 filter with a filter size of (0.45 + 0.2 μm). The filtrate obtained after these three filtrations is the "clarified conditioned medium" or "conditioned medium" for short. For this Example 26, 15 liters of conditioned medium were produced. This conditioned medium is referred to as "*5 (Test 26)." An aliquot of this material was used for in-process testing. A 30 mL aliquot of this material was subjected to ultracentrifugation to generate EV-enriched secretome for further testing. This material is referred to as "*5b.uc (Test 26)."

[0466] The clarified conditioned medium was then processed by tangential flow filtration ("TFF") using a regenerated cellulose filter with a 30 kilodalton cutoff. The surface area of ​​the filter used in Example 26 was 0.28 square meters. The TFF process involved first concentrating the retentate, then diafiltering the retentate using 6 volumes of DPBS. The diafiltered retentate was then concentrated again. An aliquot of the resulting retentate (referred to as "*6 (Test 26)") was analyzed by in-process control.

[0467] The final retentate was concentrated 46-fold for Example 26. The remaining retentate was stored in 1 L bags overnight at 4° C. until needed for Example 27.

[0468] Figure 97 shows the process used to produce Example 27. To produce Example 27, the frozen retentate obtained at the end of Example 25 was thawed overnight at 4°C. This material is designated "Thawed Retentate Test 25." A sample of the thawed retentate of Example 25 was collected for in-process testing. This material is designated "*7 (Test 25)." The retentate obtained at the end of Example 26 was removed from the oven where it had been stored overnight. This material is designated "Retentate Fresh Test 26." A sample of Retentate Fresh Test 26 was taken for in-process testing. This material is designated "*7 (Test 26)." The thawed retentate of Test 25 and Retentate Fresh Test 26 were pooled (combined) together (designated the "pool"). A sample of the pool was taken for in-process testing. This material is designated "*8 (Test 27)." The pool was sterile filtered using Sartopore® 2, Sterile Capsules (pore size (prefilter + filter): 0.45 μm + 0.2 μm; Sartorius, Reference: 5441307H4--OO--B) to obtain the final formulation (without trehalose). The sterilized material was vialed into glass cryotubes and stored at -80°C until further use. The material in the glass cryotubes is referred to as "CTC1-EV Final Formulation". It is also referred to as "*9 (Test 27)".

[0469] Example 20 Transcriptome analysis of CTC1-EV final preparations To assess the RNA transcriptome of the final CTC1-EV formulation, RNA was extracted (during the vesicle formation phase) from the CPCs used to generate Examples 25 and 26 on 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 *9 (Test 27), the preparation of which is described in detail in Example 19.

[0470] For RNA extraction from CPCs (Examples 25 and 26), cell lysates (1 million cells per 450 μL of RLT buffer (Qiagen, USA)) were obtained on D+3 and D+5 from the CPCs used to generate Examples 25 and 26. Small RNAs were enriched from the CPCs using the mirVana RNA Isolation Kit (Thermofisher, Reference AM1561) according to the manufacturer's protocol, and the resulting RNA was eluted in 100 μL of nuclease-free water (Teknova, Reference W3330). Two μL of this RNA preparation was then used to assess RNA concentration using Lunatic (Unchained Labs). The results of Lunatic analysis of cellular RNA extracted from Examples 25 and 26 (D+5), showing RNA concentration, are ...

Claims

1. 1. A method for generating a secretome, comprising: (a) culturing one or more progenitor cells in a first serum-free culture medium, the first serum-free culture medium comprising a basal medium, human serum albumin, and one or more growth factors; (b) removing the first serum-free culture medium from the one or more progenitor cells; (c) culturing the one or more progenitor cells in a second serum-free culture medium, the second serum-free culture medium comprising a basal medium but not including human serum albumin or growth factors; (d) recovering the second serum-free culture medium after the culturing of step (c), thereby obtaining a conditioned medium comprising the secretome of the one or more progenitor cells. A method comprising:

2. The method of claim 1, wherein one of the one or more growth factors is fibroblast growth factor 2 (FGF-2).

3. 3. The method of claim 1 or 2, wherein the first and second serum-free media are supplemented with a carbohydrate source.

4. The method of claim 3 , wherein the carbohydrate source is glucose.

5. The method of any one of claims 1 to 4, wherein the first and second serum-free media are supplemented with an antibiotic.

6. 6. The method of claim 5, wherein the antibiotic is gentamicin.

7. The first serum-free medium may contain 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; triiodo-I-thyronine; amino acids; Sodium pyruvate; Lipoic acid; 7. The method of claim 1, further comprising one or more selected from the group consisting of vitamin B12; a nucleoside; and ascorbic acid.

8. The method according to any one of claims 1 to 7, wherein the basal medium is a minimum essential medium (MEM).

9. The method of claim 8, wherein the MEM is α-MEM.

10. 10. The method of any one of claims 1 to 9, wherein the culturing in step (a) is for 6 to 96 hours.

11. 11. The method of claim 10, wherein the culturing in step (a) is for 12 to 96 hours.

12. 12. The method of claim 11, wherein the culturing in step (a) is for 36 to 84 hours.

13. 13. The method of claim 12, wherein the culturing in step (a) is for about 72 hours.

14. 14. The method of any one of claims 1 to 13, wherein the culturing in step (c) is for 6 to 96 hours.

15. 15. The method of claim 14, wherein the culturing in step (c) is for 12 to 72 hours.

16. 16. The method of claim 15, wherein the culturing in step (c) is for 36 to 60 hours.

17. 17. The method of claim 16, wherein the culturing in step (c) is for about 48 hours.

18. 15. The method of claim 14, wherein the final 12 to 36 hours of the culturing in step (c) is carried out under hypoxic conditions.

19. 19. The method of claim 18, wherein the culture conditions include culturing in an atmosphere having 1 to 21% oxygen.

20. 20. The method of any one of claims 1 to 19, wherein after step (b) but before step (c), the one or more progenitor cells are washed.

21. 21. The method of any one of claims 1 to 20, wherein the 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.

22. 22. The method of any one of claims 1 to 21, wherein the one or more progenitor cells are obtained from induced pluripotent stem cells (iPSCs).

23. 23. The method of any one of claims 1 to 4 or 7 to 22, wherein the first and second serum-free media do not contain antibiotics.

24. 24. The method of any one of claims 1 to 23, wherein the culture in one or more of steps (a) and (c) is a two-dimensional cell culture.

25. 25. The method of claim 24, wherein the two-dimensional cell culture comprises culturing the one or more progenitor cells on a surface of a culture vessel.

26. 26. The method of claim 25, wherein the culture vessel surface is coated with a substance to promote cell adhesion.

27. 27. The method of claim 26, wherein the substance for promoting cell adhesion is vitronectin or fibronectin.

28. 24. The method of any one of claims 1 to 23, wherein the culture in one or more of steps (a) and (c) is a three-dimensional cell culture.

29. 30. The method of claim 28, 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.

30. 30. The method of any one of claims 1 to 29, further comprising pre-clarifying the medium recovered in step (d) by centrifugation, filtration, or a combination of centrifugation and filtration.

31. 31. The method of any one of claims 1 to 30, optionally further comprising freezing the medium collected in step (d).

32. The method according to any one of claims 1 to 31, wherein the one or more progenitor cells cultured in step (a) have been previously frozen.

33. 33. The method of any one of claims 1 to 32, further comprising concentrating and / or enriching small extracellular vesicles-enriched fraction (sEVs) from the medium collected in step (d).

34. 34. The method of claim 33, wherein the sEVs are concentrated and / or enriched from the harvested medium by at least one process selected from the group consisting of ultracentrifugation, filtration, ultrafiltration, tangential flow filtration, size exclusion chromatography, and affinity capture.

35. The enrichment is based on the following characteristics: (a) CD63 ＋ , CD81 ＋ and / or CD9 － 34. The method of claim 33, wherein the method enriches extracellular vesicles having one or more of the following: (a) a diameter of 50 to 200 nm; (b) a diameter of 50 to 200 nm; (c) being positive for one or more of CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142; and / or (d) being negative for one or more of CD19, CD4, CD209, HLA-ABC, CD62P, CD42a, and CD69.

36. 34. The method of claim 33, wherein the sEVs comprise one or more of exosomes, microparticles, extracellular vesicles, and secreted proteins.

37. A secretome-containing composition obtainable by the method according to any one of claims 1 to 32.

38. 37. An sEV-containing composition obtainable by the method according to any one of claims 33 to 36.

39. A method for preparing a therapeutic composition suitable for administration to a patient, the method comprising preparing a secretome-containing composition according to the method of any one of claims 1 to 32.

40. 40. The method of claim 39, further comprising purifying, concentrating, isolating and / or enriching the secretome-containing composition by one or more purification, concentration, isolation and / or enrichment steps.

41. 40. The method of claim 39, further comprising adding a pharmaceutically acceptable excipient or carrier to the secretome-containing composition.

42. A method for producing a therapeutic composition suitable for administration to a patient, the method comprising producing an sEV-containing composition according to the method of any one of claims 33 to 36.

43. 43. The method of claim 42, further comprising purifying, concentrating, isolating and / or enriching the sEV-containing composition by one or more purification, concentration, isolation and / or enrichment steps.

44. 43. The method of claim 42, further comprising adding a pharmaceutically acceptable excipient or carrier to the sEV-containing composition.

45. 38. A therapeutic composition comprising the secretome-containing composition of claim 37 and a pharmaceutically acceptable excipient or carrier.

46. A therapeutic composition comprising the sEV-containing composition of claim 38 and a pharmaceutically acceptable excipient or carrier.

47. 10. A secretome-containing composition obtainable by the method of claim 1, wherein the 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.

48. 34. An sEV-containing composition obtainable by the method of claim 33, wherein the 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.

49. 48. A therapeutic composition comprising the secretome-containing composition of claim 47 and a pharmaceutically acceptable excipient or carrier.

50. 49. A therapeutic composition comprising the composition of claim 48 and a pharmaceutically acceptable excipient or carrier.

51. 51. 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 described in claim 49 or 50.

52. 51. A method of improving angiogenesis, comprising administering to a subject in need thereof the therapeutic composition of claim 49 or 50.

53. 51. A method of improving cardiac function comprising administering to a subject in need thereof the therapeutic composition of claim 49 or 50.

54. 12. The method of claim 11, wherein the culturing in step (a) is for 60 to 84 hours.

55. 15. The method of claim 14, wherein the final 12 to 36 hours of the culturing in step (c) is carried out under normoxic conditions.

56. 56. The method of claim 55, wherein said normoxic conditions comprise culturing in an atmosphere containing 20-21% oxygen.

57. 30. The method of claim 29, wherein the bioreactor is a vertical wheel bioreactor.

58. 40. The method of claim 39, further comprising cryopreserving, freezing or lyophilizing the secretome-containing composition.

59. 43. The method of claim 42, further comprising cryopreserving, freezing or lyophilizing the sEV-containing composition.

60. The method of claim 2, wherein the first serum-free medium contains 0.1 to 10 μg / mL of FGF-2.

61. 61. The method of claim 60, wherein the first serum-free medium comprises 0.5 to 5 μg / mL of FGF-2.

62. 62. The method of claim 61, wherein the first serum-free medium comprises 0.5 to 2.5 μg / mL of FGF-2.

63. 63. The method of claim 62, wherein the first serum-free medium comprises about 1 μg / mL FGF-2.

64. 64. The method of any one of claims 1 to 36, 39 to 44 or 54 to 63, which complies with Good Manufacturing Practice (GMP).

65. 38. The secretome-containing composition of claim 37, which corresponds to GMP.

66. 39. The sEV-containing composition of claim 38, which complies with GMP.

67. 15. The method of claim 14, wherein the final 12 to 36 hours of the culturing in step (c) is carried out under normoxic conditions.

68. 68. The method of claim 67, wherein the normoxic conditions comprise culturing in an atmosphere containing 20-21% oxygen.

69. 31. The method of claim 30, wherein the pre-clarification comprises at least three filtration steps.

70. 35. The method of claim 34, wherein separation of the sEVs from the collected medium comprises tangential flow filtration.

71. 38. The secretome-containing composition of claim 37, comprising trehalose and L-histidine.

72. The sEV-containing composition of claim 3, comprising trehalose and L-histidine.

73. 66. The secretome-containing composition of claim 37 or 65, which is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

74. 67. The sEV-containing composition of claim 38 or 66, which is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

75. The secretome-containing composition of claim 37 or 65, which is at least one of: a composition enriched in extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm, preferably about 50-150 nm or 50-150 nm; a composition that is substantially free of or free of whole cells; and / or a composition that is substantially free of one or more culture medium components.

76. The sEV-containing composition of claim 38 or 66, which is at least one of: a composition enriched in extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm, preferably about 50-150 nm or 50-150 nm; a composition that is substantially free of or free of whole cells; and / or a composition that is substantially free of one or more culture medium components.

77. 52. The method of claim 51, 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.

78. 78. The method of claim 77, 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.

79. 66. The secretome-containing composition of claim 37 or 65, capable of promoting cardiomyocyte viability in an in vitro chemotherapy-induced cardiomyopathy viability assay.

80. 67. The sEV-containing composition of claim 38 or 66, which is capable of promoting cardiomyocyte viability in an in vitro chemotherapy-induced cardiomyopathy viability assay.

81. 80. The secretome-containing composition of claim 79, wherein in the in vitro chemotherapy-induced cardiomyopathy viability assay, the chemotherapy is an anthracycline.

82. 82. The secretome-containing composition of claim 81, wherein the anthracycline is doxorubicin.

83. 81. The sEV-containing composition of claim 80, wherein in the in vitro chemotherapy-induced cardiomyopathy viability assay, the chemotherapy is an anthracycline.

84. 84. The sEV-containing composition of claim 83, wherein the anthracycline is doxorubicin.

85. 52. The method of claim 51, wherein the chemotherapy-induced cardiomyopathy is caused by an anthracycline.

86. 86. The method of claim 85, wherein the anthracycline is doxorubicin.

87. 51. A method of maintaining physiological cardiac volume in a subject by administering to the subject a therapeutic composition of any one of claims 45, 46, 49 or 50.

88. 88. The method of any one of claims 51-53 or 87, wherein the left ventricular end-systolic volume (LVESV) is maintained within 15% of the pre-treatment LVESV.

89. 88. The method of any one of claims 51-53 or 87, wherein the LVEDV is maintained within 2% of the pre-treatment volume.

90. 88. The method of any one of claims 51 to 53 or 87, wherein progressive post-ischemic heart failure is prevented.

91. 88. The method of any one of claims 51 to 53 or 87, which improves the survival, health and function of endothelial cells in said subject.

92. 88. The method of any one of claims 51-53 or 87, wherein the method reduces fibrosis in stimulated cardiac fibroblasts.

93. 93. The method of claim 92, wherein the expression of the pro-fibrotic marker POSTN in TGF-β1-stimulated cardiac fibroblasts is reduced to below the level prior to stimulation with TGF-β1.

94. 88. The method of any one of claims 51-53 or 87, which does not induce an alloinflammatory response in the subject.

95. 88. The method of any one of claims 51 to 53 or 87, which does not induce allogeneic peripheral blood mononuclear cell (PBMC) activation.

96. 88. The method of any one of claims 51 to 53 or 87, which does not induce a significant increase in the percentage of IFNg or IL-2 expressing PBMCs.

97. 88. The method of any one of claims 51-53 or 87, which does not induce allogeneic natural killer (NK) cell degranulation.

98. 88. The method of any one of claims 51 to 53 or 87, which does not induce a significant increase in the percentage of CD107-expressing NK cells.

99. 51. A method of improving cardiac function in a patient experiencing heart failure by administering a therapeutic composition of any one of claims 45, 46, 49 or 50.

100. 100. The method of claim 99, which improves survival of stressed cardiomyocytes.

101. 100. The method of claim 99, wherein one or more of the seeding, survival, viability and proliferation of stressed endothelial cells are improved in vitro.

102. 100. The method of claim 99, which improves cell migration and / or wound healing capacity in stressed endothelial cells.

103. 103. The method of any one of claims 99 to 102, wherein the method improves wound healing in the subject.

104. 104. The method of any one of claims 99 to 103, wherein the method reduces signs of fibrosis in the subject's fibroblasts.

105. 105. The method of claim 104, wherein the fibroblasts are activated with TGF-β1.

106. The method of any one of claims 99 to 105, which does not stimulate allogeneic human PBMC activation.

107. 107. The method of any one of claims 99 to 106, which does not induce NK degranulation of allogeneic human NK cells.

108. The composition has a potency of 4×10 in mice and rats. １１ 108. The method of any one of claims 99 to 107, which is non-toxic at a dose of particles / kg.

109. The composition was administered in mice at a dose of 4×10 １１ 109. The method of any one of claims 99 to 108, which is not tumorigenic at a dose of particles / kg.

110. 110. The method of any one of claims 99 to 109, wherein the composition does not contain DNA fragments in the range of 179 to 742 pb at concentrations in the μg / mL range.

111. 111. The method of any one of claims 51-53 or 87-110, wherein the therapeutic composition is administered as an intravenous infusion, direct cardiac injection, or administered intra-arterially.

112. 112. The method of claim 111, wherein the therapeutic composition is administered in a dose containing secretomes from 100 to 10 million cells per kg of body weight of the subject per dose.

113. 112. The method of claim 111, wherein the therapeutic composition is administered in a dose containing secretomes from 0.5 to 5 million cells per kg of body weight of the subject per dose.

114. 112. The method of claim 111, wherein the therapeutic composition is administered in a dose containing secretomes from 1 to 3 million cells per kg of body weight of the subject.

115. 112. The method of claim 111, wherein the therapeutic composition is administered in a dose containing secretomes from 1 to 2 million cells per kg of body weight of the subject.

116. The therapeutic composition is administered to a subject in an amount of 1 x 10 nanoparticles per kg of body weight as measured by nanoparticle tracking analysis (NTA). ９ ~60 x 10 ９ 112. The method of claim 111, wherein the dose is administered in a dose containing particles.

117. The therapeutic composition is administered at a dose of 10 x 10 per kg of the subject's body weight as measured by NTA. ９ ~60 x 10 ９ 112. The method of claim 111, wherein the dose is administered in a dose containing particles.

118. The therapeutic composition is administered at a dose of 10 x 10 per kg of the subject's body weight as measured by NTA. ９ ~40 x 10 ９ 112. The method of claim 111, wherein the dose is administered in a dose containing particles.

119. The therapeutic composition is administered at a dose of 20 x 10 per kg of the subject's body weight as measured by NTA. ９ ~40 x 10 ９ 112. The method of claim 111, wherein the dose is administered in a dose containing particles.

120. The therapeutic composition is administered at a dose of 20 x 10 per kg of the subject's body weight as measured by NTA. ９ ~200 x 10 ９ 112. The method of claim 111, wherein the compound is administered in a cumulative daily dose containing particles.

121. The therapeutic composition is administered at a dose of 30×10 per kg of the subject's body weight as measured by NTA. ９ ~100 x 10 ９ 112. The method of claim 111, wherein the compound is administered in a cumulative daily dose containing particles.

122. The therapeutic composition is administered at a dose of 60 x 10 per kg of the subject's body weight as measured by NTA. ９ 112. The method of claim 111, wherein the compound is administered in a cumulative daily dose containing particles.

123. The therapeutic composition is administered at a dose of 40 x 10 per kg of the subject's body weight as measured by NTA. ９ 112. The method of claim 111, wherein the compound is administered in a cumulative daily dose containing particles.

124. 124. The method of any one of claims 111 to 123, wherein the composition is administered 1 to 10 times per day.

125. 124. The method of any one of claims 111-123, wherein the composition is administered 3-6 times per day.

126. 124. The method of any one of claims 111-123, wherein the composition is administered 1 to 5 times per day.

127. 124. The method of any one of claims 111-123, wherein the composition is administered three times per day.

128. 124. The method of any one of claims 111-123, wherein the composition is administered twice daily.

129. 129. The method of any one of claims 51-53 or 87-128, wherein the treatment period is 60 days or less.

130. 130. The method of claim 129, wherein the treatment period is 5 to 50 days.

131. 130. The method of claim 129, wherein the treatment period is 10 to 50 days.

132. 130. The method of claim 129, wherein the treatment period is 20 to 45 days.

133. 130. The method of claim 129, wherein the treatment period is 42 days.

134. 134. The method of any one of claims 111 to 133, wherein the therapeutic composition is administered daily.

135. 134. The method of any one of claims 111 to 133, wherein the therapeutic composition is administered every other day.

136. 134. The method of any one of claims 111-133, wherein the therapeutic composition is administered at a frequency of from every day to every 30 days.

137. 134. The method of any one of claims 111 to 133, wherein the therapeutic composition is administered every 7 days to every 21 days.

138. 134. The method of any one of claims 111-133, wherein the therapeutic composition is administered every 21 days.

139. 139. The method of any one of claims 111-138, wherein the therapeutic composition is formulated into a solution comprising one or more pharmaceutically acceptable excipients.

140. A secretome-containing composition obtainable by the method of any one of claims 1 to 32, comprising extracellular vesicles secreted from the progenitor cells.

141. The extracellular vesicles contain 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- 141. The secretome-containing composition of claim 140, comprising one or more miRs selected from 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.

142. The extracellular vesicles contain 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-4761-5p, hsa-miR-4762-5p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4765-5p, hsa-miR-4765-5p, hsa-miR-4760-5p, hsa-miR-4761-5p, hsa-miR-4762-5p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4765 ...5-5p, hsa-miR-4760-5p, hsa-miR-4761-5p, hsa-miR-4761-5p, hsa-miR-4761-5p, hsa-miR-4762-5p, hsa-miR-4763-5p, hsa-miR-4761-5p, hsa-miR-4761-5p, hsa-mi 141. The secretome-containing composition of claim 140, comprising one or more miRs selected from 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.

143. The extracellular vesicles contain 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-4760-5 ...

141. The secretome-containing composition of claim 140, comprising at least five miRs selected from a-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.

144. 144. The secretome-containing composition of any one of claims 141 to 143, wherein the expression level of the one or more miRs is in the range of -5 to +5 units.

145. A secretome-containing composition comprising a secretome derived from a progenitor cell, wherein the secretome comprises extracellular vesicles secreted from the progenitor cell.

146. The secretome-containing composition of claim 145, wherein the progenitor cells are cardiovascular progenitor cells.

147. The extracellular vesicles contain 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-101-3 p, 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-mi R-191-5p, hsa-miR-26b-5p, hsa-miR-151a-3p, hsa-miR-103a-3p / 107, hsa-miR-660-5p, hsa-miR-32 0a-3p / 320b / 320c / 320d / 320e, hsa-miR-130a-3p, hsa-miR-19b-3p, hsa-miR-27a-3p / 27b-3p, hsa-miR 147. The secretome-containing composition of claim 145 or 146, comprising one or more miRs selected from 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.

148. The extracellular vesicles contain 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 147. The secretome-containing composition of claim 145 or 146, comprising one or more miRs selected from: 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.

149. The extracellular vesicles contain 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- 147. The secretome-containing composition of claim 145 or 146, comprising at least five miRs selected from iR-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.

150. The secretome-containing composition of any one of claims 147 to 149, wherein the expression level of the one or more miRs is in the range of -5 to +5 units.

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