Methods and assays for analyzing secretome-containing compositions

Abstract

The present disclosure provides methods for producing and / or purifying secretomes, extracellular vesicles, and fractions thereof from cells, such as progenitor cells, and methods for analyzing the activity, function, 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 analyzed using such methods.

Description

[Technical Field]

[0001] The present disclosure generally relates to methods and assays for analyzing the activity, functionality, properties, and / or efficacy of conditioned medium, or secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions. [Background technology]

[0002] Cells, including those in vitro or ex vivo culture, secrete a wide 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 into membrane-bound extracellular vesicles such as exosomes. Extracellular vesicles have the ability to alter the biology of other cells through signal transduction or by delivering their cargo (e.g., including proteins, lipids, and nucleic acids). The membrane-enclosed cargo of extracellular vesicles allows, among other things, specific targeting (e.g., targeting cells) via specific markers on the membrane; and increased stability during transport in biological fluids, such as through the bloodstream or across the blood-brain barrier (BBB).

[0003] Exosomes fulfill a wide variety of important physiological functions, for example, by acting as molecular messengers communicating between different cell types. For example, exosomes deliver proteins, lipids, and soluble factors, including RNA and microRNA, that participate in signaling pathways that can affect apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, immunity by directing T cells toward immune activation, immunosuppression, growth, division, survival, differentiation, stress response, and apoptosis, depending on their source. 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, in some instances, the terminology applied to the cell of origin can be used simply to refer to secreted exosomes.

[0004] The ability of progenitor cells to proliferate and differentiate into mature cells makes them attractive for therapeutic applications, such as regenerative medicine in the treatment of myocardial infarction and congestive heart failure. Extracellular vesicles secreted by stem cell-derived cardiovascular progenitor cells have been reported to produce therapeutic effects similar to those of the cells that secrete them 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 (with benefits such as simplicity, stability, and improved operator accessibility). See El Harane et al. (Eur. Heart J., 2018;39:1835-1847). However, there is currently a need for more accurate and reliable methods for determining the activity, function, or potency of conditioned medium, or secretome-, extracellular vesicles-, and / or sEV-containing compositions.

[0005] For conditioned medium, or secretome, extracellular vesicles, and / or sEV-containing compositions, known cell viability assays involving serum deprivation can be used to measure their functionality or efficacy. In particular, a well-known cardiomyocyte viability assay uses serum-starved rat H9c2 cardiomyoblasts, which can be used to measure the effect of conditioned medium, or secretome, extracellular vesicles, and / or sEV-containing compositions on the viability of serum-starved cells. See, for example, El Harane et al. (Eur. Heart J., 2018, 39(20): 1835-1847).

[0006] However, H9c2 cardiomyoblast cells, commonly used in such assays, have been reported to exhibit variability in cell parameters with repeated passage. For example, Witek et al. (Cytotechnology, 2016, 68(6):2407-2415) reported that cell parameters (e.g., cell morphology, gene expression profile, oxidative stress response, sensitivity to cellular stress factors) fluctuate depending on the number of years of cell culture (which can lead to unreliable results, for example, when testing drugs for their effects on promoting or reducing cell viability). In addition, few options exist for screening the therapeutic potential of secretomes (e.g., obtained from different cell types and / or different cell culture conditions).

[0007] Thus, there is a need for improved and more reliable assays for determining the activity, functionality, and / or potency of conditioned medium, or secretome, extracellular vesicles, sEV-containing compositions. Summary of the Invention

[0008] The present disclosure addresses the above-mentioned limitations in the art by providing methods and assays for reliably determining the activity, function, and / or potency of conditioned medium, secretomes, extracellular vesicles, and fractions thereof. The present disclosure further provides reliable analytical methods and assays that can compare conditioned medium, secretomes, extracellular vesicles, and fractions thereof, enable high-throughput analysis, and produce consistent results in vitro.

[0009] Non-limiting embodiments of the present disclosure include:

[0010] [1] (a) contacting a culture of target cells with a preconditioning medium and culturing the target cells in the preconditioning medium under at least one stress-inducing condition; (b) administering a secretome to a cell culture and culturing the target cells in the presence of the secretome; and (c) measuring at least one characteristic of the cultured cells one or more times during the culturing of step (b); A method for analyzing secretome activity, comprising:

[0011] [2] The method of [1], further comprising removing the preconditioning medium from the cultured cells before step (b).

[0012] [3] The method of [2], wherein the target cells are cultured under at least one stress-inducing condition before administering the secretome to the cell culture, and the culturing of the target cells in step (b) is carried out in the absence of at least one stress-inducing condition.

[0013] [4] (a) contacting a culture of target cells with a pretreatment medium and culturing the target cells in the pretreatment medium; (b) administering a secretome to a cell culture and optionally culturing said target cells in the presence of the secretome; (c) culturing the target cells under at least one stress-inducing condition; and (d) measuring at least one characteristic of the cultured cells one or more times during the culturing of step (c); A method for analyzing secretome activity, comprising:

[0014] [5] The method of [4], wherein the target cells are cultured in the presence of a secretome prior to the culture in step (c).

[0015] [6] The method of [5], further comprising removing the secretome from the cultured cells before step (c).

[0016] [7] The method of [4], wherein the stress-inducing conditions are culture in the presence of a cell stress agent, the cell stress agent is co-administered with the secretome, and the target cells are cultured in the presence of the secretome and the cell stress agent.

[0017] [8] The method according to any one of [1] to [6], wherein at least one stress-inducing culture condition is culture in the presence of a cell stress agent.

[0018] [9] The method according to [7] or [8], wherein the cell stress agent is a chemotherapeutic agent and / or an apoptosis inducer.

[0019]

[10] The method of [9], wherein the apoptosis inducer is indolocarbazole.

[0020]

[11] The method according to [9], wherein the apoptosis inducer is indolo(2,3-a)pyrrole(3,4-c)carbazole or a derivative thereof.

[0021]

[12] The method of [9], wherein the apoptosis inducer is staurosporine or a derivative thereof.

[0022]

[13] The method of [9], wherein the apoptosis inducer is doxorubicin or a derivative thereof.

[0023]

[14] Any one of the methods [1] to

[13] , wherein at least one characteristic measured is selected from the group consisting of cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology.

[0024]

[15] The method of

[14] , further comprising measuring at least one characteristic of the cultured cells one or more times during the culture of step (a).

[0025]

[16] The method of [1] to [3], wherein at least one characteristic is measured multiple times during the culture in step (b).

[0026]

[17] The method of [4] to [7], wherein at least one characteristic is measured multiple times during the culture in step (c).

[0027]

[18] Methods

[16] or

[17] in which multiple measurements are taken at intervals of 5 minutes to 10 hours from each other.

[0028]

[19] The method of

[18] , wherein the measurements are taken at intervals of 10 minutes to 4 hours.

[0029]

[20] The method of

[19] in which multiple measurements are taken at intervals of 30 minutes to 2 hours from each other.

[0030]

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

[20] ], wherein the at least one property is selected from cell viability, cell adhesion, cell number, cell morphology, cell proliferation, and / or ATP content.

[0031]

[22] The method of

[21] , wherein at least one characteristic is the viability of cultured cells, and the viability is measured using a fluorescent DNA labeling dye or a fluorescent nuclear staining dye.

[0032]

[23] The method of

[21] , wherein at least one characteristic is adhesion, cell number, proliferation, and / or cell morphology of the cultured cells, and the adhesion, cell number, proliferation, and / or morphology of the cultured cells is determined by measuring electrical impedance across the surface of the culture vessel during culture.

[0033]

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

[23] , wherein the target cells are cultured in the pretreatment medium in step (a) for 30 minutes to 10 hours.

[0034]

[25] The method of

[24] , wherein the target cells are cultured in the pretreatment medium in step (a) for 1 hour to 5 hours.

[0035]

[26] The method of

[25] , wherein the target cells are cultured in the pretreatment medium in step (a) for about 4 hours.

[0036]

[27] The method according to any one of [1] to [3], wherein the target cells are cultured for at least 2 hours in step (b).

[0037]

[28] The method according to any one of [4] to [7], wherein the target cells are cultured for at least 2 hours in step (c).

[0038]

[29] Methods

[27] or

[28] , in which the incubation time is at least 5 hours.

[0039]

[30] The method of

[29] , in which the incubation time is at least 12 hours.

[0040]

[31] The method of

[30] , in which the incubation period is at least 24 hours.

[0041]

[32] The method of

[22] , in which the viability of cultured cells is measured by imaging DNA-labeling dyes or nuclear-staining dyes.

[0042]

[33] Any one of the methods [1]-

[20] and

[24] -

[31] , wherein at least one characteristic is the viability of the cultured cells and the viability is measured by live cell imaging.

[0043]

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

[33] , wherein the target cells are specialized cells.

[0044]

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

[33] , wherein the target cells include cardiomyocytes, cardiovascular progenitor cells, cardiac progenitor cells, and / or vascular cells.

[0045]

[36] The method of

[34] , wherein the specialized cells are obtained from induced pluripotent stem cells (iPSCs).

[0046]

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

[33] , wherein the target cells are frozen in advance.

[0047]

[38] Any one of the methods [1] to

[37] , wherein the secretome is isolated from a culture of one or more cells selected from totipotent progenitor cells, multipotent progenitor cells, and terminally differentiated cells.

[0048]

[39] The method of

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

[0049]

[40] Any one of the methods [1] to

[39] , wherein the secretome comprises a small extracellular vesicle-enriched fraction (sEV) isolated from a cell culture.

[0050]

[41] The method of

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

[0051]

[42] The method of

[40] , wherein the sEVs comprise one or more of exosomes, microparticles, extracellular vesicles, and secreted proteins.

[0052]

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

[42] , further comprising culturing the target cells for 1 to 21 days before step (a).

[0053]

[44] The method of

[43] , wherein the target cells are cultured for 5 to 14 days before step (a).

[0054]

[45] The method of

[43] , in which fresh culture medium is supplied to the target cells 12 to 36 hours before step (a).

[0055]

[46] Any one of the methods [1] to

[45] , wherein the target cells are cultured in a two-dimensional cell culture.

[0056]

[47] The method of

[46] , wherein the two-dimensional cell culture comprises culturing target cells on a surface of a culture vessel.

[0057]

[48] ​​The method of

[47] , wherein the surface of the culture vessel is coated with a substance that promotes cell adhesion.

[0058]

[49] The method according to

[48] , wherein the substance that promotes cell adhesion is fibronectin.

[0059]

[50] Any one of the methods [1] to

[49] , further comprising culturing positive control cells in parallel, wherein the positive control cells are not administered with the secretome and are not subjected to stress-inducing conditions.

[0060]

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

[50] , further comprising culturing negative control cells, wherein the negative control cells are not administered with secretome.

[0061]

[52] The method of

[51] , wherein the negative control cells are subjected to the same steps as the target cells, except that the negative control cells are not administered with the secretome.

[0062]

[53] The method of

[51] or

[52] , wherein the negative control cells comprise negative control cells cultured in a pre-conditioning medium under at least one stress-inducing condition, and the method comprises measuring at least one property of the negative control cells during or after culturing in the pre-conditioning medium under at least one stress-inducing condition.

[0063]

[54] The method of

[51] , wherein the negative control cells include negative control cells to which a simulated secretome composition has been added, and the simulated secretome composition is produced by omitting cells from the step of producing a secretome.

[0064]

[55] Any one of the methods [1] to

[54] , wherein the amount of secretome to be added to the target cells is determined 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 amount of exosomes in the secretome; and the number of particles in the secretome.

[0065]

[56] The method of

[50] , wherein the target cells and the positive control cells are cultured in duplicate.

[0066]

[57] The method of

[56] in which the number of positive control cells in replicate cultures is averaged to obtain the mean maximum cell number, and the number of target cells in each replicate culture is normalized to the mean maximum cell number.

[0067]

[58] Any one of the methods

[51] to

[54] , wherein at least one characteristic is the viability of cultured cells, and the secretome is determined to be effective and / or exhibit a therapeutic effect when the viability of target cells is higher than the viability of negative control cells.

[0068]

[59] The sEV is at least one of the following: sEVs enriched for extracellular vesicles having a diameter of between about 50-200 nm or between 50-200 nm, preferably between about 50-150 nm or between 50-150 nm; sEVs that are substantially free of or free from whole cells; and / or sEVs that are substantially free of one or more culture medium components;

[40] method.

[0069]

[60] (a) contacting a culture of target cells with a pretreatment medium and culturing the target cells in the pretreatment medium in the presence of a small molecule or a chemotherapeutic agent; (b) administering a secretome to a cell culture and culturing the target cells in the presence of the secretome; and (c) measuring at least one characteristic of the cultured cells one or more times during the culturing of step (b); 1. A method for analyzing the activity of a small molecule or chemotherapeutic agent, comprising:

[0070] 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. [Brief explanation of the drawings]

[0071] The patent or application file contains at least one color drawing. Copies of any color drawing(s) in the patent or published patent application file will be provided by the Office upon request and payment of the necessary fee.

[0072] [Figure 1] Figure 1 shows representative images of cells incubated with and without staurosporine in the staurosporine cardiomyocyte viability assay. Healthy cell nuclei appear round and of moderate red intensity (seen in panel 1), while dead or dying nuclei appear shrunken and intensely red (seen in panel 3). Incucyte image analysis software identifies and counts viable nuclei (blue "masking" in panels 2 and 4) in each image.

[0073] [Figure 2] Figure 2 shows the time course of cell viability in the staurosporine cardiomyocyte viability assay. After staurosporine pretreatment, cell viability was measured at various time points after administration of small extracellular vesicle-enriched fraction (sEV) secretomes from human cardiovascular progenitor cells (CPCs); mock sEV preparations (virgin medium control); or complete medium without sEVs. The complete medium control (positive control) was not pretreated with staurosporine.

[0074] [Figure 3] Figure 3 shows histograms depicting the 24-hour results of the staurosporine cardiomyocyte viability assay time course. Values ​​for these histograms are first calculated by normalizing the number of viable cells at 24 hours to the number of viable cells at time 0 for the same well. The normalized baseline results (determined from the negative control wells) are then subtracted from each result. Finally, the results for each condition are expressed as a percentage of the mean value for the positive control wells. The mean and standard deviation for each condition are shown.

[0075] [Figure 4]Figure 4 shows histograms depicting cell viability data (obtained using a biocompatible dye and measured using an Incucyte at the 13 hour time point) and ATP quantification data (obtained using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and measured using a CLARIOStar® (BMG Labtech) and Tecan for Life Science® plate reader at the 24 hour time point, i.e., after the end of the Incucyte time course) for the staurosporine assay described in Example 2 using sEVs produced from CPCs.

[0076] [Figure 5] Figure 5 shows histograms depicting cell viability data (obtained using a biocompatible dye and measured using an Incucyte at 12 hours) and ATP content data (obtained using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and measured using a CLARIOStar® (BMG Labtech) and Tecan for Life Science® plate reader at the 23 hour time point, i.e., after the end of the Incucyte time course) for the staurosporine assay described in Example 2 using sEVs generated from MSCs.

[0077] [Figure 6]Figure 6 shows a histogram depicting ATP content data (obtained using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and measured on a Tecan for Life Science® plate reader at the 23-hour time point) for the staurosporine assay described in Example 2 using sEVs produced from MSCs. sEV treatment was performed in either serum-containing medium (iCell Cardiomyocyte Maintenance Medium M1003) or serum-free medium (iCell Cardiomyocyte Serum-Free Medium M1038).

[0078] [Figure 7] Figure 7 shows a comparison of the results of a staurosporine cardiomyocyte viability assay and a HUVEC scratch wound healing assay to determine the effect of sEV preparations on angiogenesis. Samples tested in parallel in these two assays showed similar trends in efficacy, indicating that cardiovascular progenitor cell-sEVs have an effect on both cardiac and vascular target cells in vitro.

[0079] [Figure 8] Figure 8 shows the results of a scratch wound healing assay, where sEVs isolated from MSCs from three different donors (62, 64, and 82) appear to have widely different effects on wound healing when administered based on particle number (left graph). However, when administered based on secretory cell number (right graph), sEVs from different MSC donors appear to have much more similar effects in this assay.

[0080] [Figure 9]Figure 9 shows the time course of electrical impedance measurements in the staurosporine-induced cardiomyocyte adhesion, number, and proliferation assay. After pretreatment (with or without staurosporine), cell adhesion, number, and proliferation were continuously analyzed (by measuring electrical impedance) before and after administration of either sEVs derived from human cardiovascular progenitor cells ("sEVs," conditions 3-5); mock sEV preparations (virgin medium control ("MV," conditions 1 and 7); or sEV-free medium / culture control (conditions 2, 6, and 8).

[0081] [Figure 10] FIG. 10 shows the results of the time course experiment shown in FIG. 9, normalized to the treatment ("Tx") time point. DETAILED DESCRIPTION OF THE INVENTION

[0082] It should 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, the reference to a "cell" includes one or more cells.

[0083] 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 pertains. 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.

[0084] As used herein, the terms "subject," "individual," or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, including, without limitation, humans and other primates, including non-human primates such as rhesus monkeys, chimpanzees, and other monkey and ape species; agricultural animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rabbits, mice, rats, and guinea pigs; and birds, including poultry, wild birds, and game birds such as chickens, turkeys, and other pheasants, ducks, and geese. The term does not denote a particular age or sex. Thus, the term includes adult, juvenile, and newborn individuals, 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.

[0085] As used herein, "differentiation" refers to the process by which an unspecialized cell (such as a pluripotent stem cell or other stem cell), or for example, a multipotent or oligopotent cell, acquires specialized structural and / or functional characteristics characteristic of a more mature or fully mature cell. "Transdifferentiation" is the process by which one differentiated cell type transforms into another differentiated cell type.

[0086] As used herein, "embryoid body" refers to a three-dimensional aggregate of pluripotent stem cells that can undergo differentiation into cells of the three germ layers: endoderm, mesoderm, and ectoderm. This three-dimensional structure allows for differentiation and morphogenesis, including the establishment of complex cell adhesion bodies and paracrine signaling within the embryoid body microenvironment.

[0087] As used herein, "stem cell" refers to a cell that has the capacity to self-renew, i.e., undergo numerous cell division cycles, while remaining non-terminally differentiated. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells can be, for example, embryonic stem cells, fetal stem cells, amniotic stem cells, adult stem cells, or induced pluripotent stem cells.

[0088] As used herein, "pluripotent stem cells" (PSCs) refer to cells that have the ability to replicate themselves indefinitely and differentiate into any other cell type in an adult organism. Generally, pluripotent stem cells are stem cells that are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; have the potential to differentiate into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal cell types); and express one or more markers characteristic of PSCs. Examples of such markers expressed by PSCs, including embryonic stem cells (ESCs) and iPSCs, include Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and REX1.

[0089] As used herein, "induced pluripotent stem cells" (iPSCs) refer to a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell, typically a somatic cell. In some embodiments, the somatic cell is a human somatic cell. Examples of somatic cells include, but are not limited to, skin fibroblasts, bone marrow-derived mesenchymal cells, cardiomyocytes, keratinocytes, hepatocytes, gastric 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.

[0090] iPSCs may 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 embryonic, 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 may be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors to reprogram the somatic cells into pluripotent stem cells. Cells may be reprogrammed by introducing the 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, piggyback, minicircle vectors, and episomal plasmids. iPSCs can also be generated by introducing reprogramming factors or activating endogenous programming genes using, for example, CRISPR-Cas9-based techniques.

[0091] As used herein, "embryonic stem cells" are embryonic cells derived from embryonic tissue, preferably the inner cell mass or morula of a blastocyst, 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 from mammalian embryos at the zygote, blastomere, or blastocyst stage, for example, produced by sperm and egg fusion, nuclear transfer, or parthenogenesis. Human ESCs include, without limitation, 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- or morula-stage embryo. These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone cannot develop into fetuses or adult animals when transplanted in utero because they lack the ability to contribute to all extraembryonic tissues (e.g., placenta in vivo or trophoblast in vitro).

[0092] As used herein, the term "progenitor cell" refers to the descendants of stem cells that have the potential to further differentiate into one or more specialized cell types but cannot divide and replicate indefinitely. That is, unlike stem cells (which have unlimited self-renewal potential), progenitor cells have only limited self-renewal potential. Progenitor cells may be multipotent, oligopotent, or unipotent and are typically classified based on the specialized cell type into which they can differentiate. 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. In addition, "cardiovascular progenitor cells" have the potential to differentiate into cells of the cardiac and vascular lineages. Mesenchymal stem cells (MSCs) are another type of progenitor cell.

[0093] As used herein, "expanding" or "growing" can refer to the process by which the number of cells in a cell culture increases due to cell division.

[0094] "Multipotent" implies that a cell has the ability, through its progeny, to give rise to several different cell types found in the adult animal.

[0095] "Pluripotent" implies that a cell has the ability to give rise, through its progeny, to all cell types contained in an adult animal, including germ cells. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.

[0096] The term "autologous cells," as used herein, refers to donor cells that are genetically identical to the recipient.

[0097] As used herein, the term "allogeneic cells" refers to cells derived from another genetically non-identical individual of the same species.

[0098] The term "totipotent," as used herein, 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 every cell of a particular animal. A totipotent cell can give rise to every cell of an animal when it is utilized in a procedure to develop an embryo from one or more nuclear transfer steps.

[0099] 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, shedding vesicles, membrane particles, shedding microvesicles, oncosomes, exomers, and apoptotic bodies.

[0100] Extracellular vesicles can be classified, for example, based on 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 greater than approximately 200 nm and less than 400 nm may be referred to as "intermediate extracellular vesicles," and extracellular vesicles having a diameter greater 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 is enriched and / or enriched in small extracellular vesicles having a diameter of approximately 50-200 nm. Such enrichment and / or enrichment may be achieved using one or more of the purification, isolation, enrichment, and / or enrichment techniques disclosed herein.

[0101] The term "exosomes," as used herein, refers to extracellular vesicles that are released from cells when multivesicular bodies (MVBs), intermediate endocytic compartments, fuse with the plasma membrane.

[0102] "Exosome-like vesicles," which share a common origin with exosomes, are typically described as having size and sedimentation properties that distinguish them from exosomes, and in particular, as lacking lipid raft microdomains. "Ectosomes," as used herein, are typically neutrophil- or monocyte-derived microvesicles.

[0103] "Microparticles," as used herein, are typically about 100-1000 nm in diameter and originate from cell membranes. "Extracellular membrane structures" also include linear or folded membrane fragments, e.g., from necrosis, as well as membrane structures from other cellular sources, including secreted lysosomes and nanotubes.

[0104] 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.

[0105] Within the class of extracellular vesicles, a key component is the "exosome" itself, which can be membrane vesicles of endocytic origin, i.e., vesicles surrounded by a phospholipid bilayer, with a diameter of between about 40-50 nm and about 200 nm, and arise from the exocytic fusion of multivesicular bodies (MVBs), i.e., "exocytosis." In some cases, exosomes can be between about 40-50 nm and up to about 200 nm in diameter, such as between 60 nm and 180 nm.

[0106] As used herein, the terms "secretome" and "secretome composition" refer synonymously 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, without limitation, 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 remain unpurified or can be further processed (e.g., components of the secretome or secretome composition can be present in culture medium, such as in conditioned medium; or alternatively, components of the secretome or secretome composition can be purified, isolated, and / or enriched from culture medium or an extract, portion, or fraction thereof). Secretomes or secretome compositions can further include one or more substances not secreted by cells (e.g., culture medium, additives, nutrients, etc.). Alternatively, the secretome or secretome composition does not contain (or contains only trace amounts of) one or more substances that are not secreted by the cell (eg, culture medium, additives, nutrients, etc.).

[0107] 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. When cells are cultured in a culture medium, one or more molecules and / or biological factors (including, but 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) may be secreted and / or accumulated; a medium containing such one or more molecules and / or biological factors is a conditioned medium. An example of a method for preparing a conditioned medium is described, for example, in U.S. Pat. No. 6,372,494, which is incorporated herein by reference in its entirety.

[0108] As used herein, the term "cell culture" refers to cells grown under one or more controlled conditions outside the cells' natural environment. For example, cells can be grown completely 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 grow in number, depending, for example, on the specific 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 a suitable liquid medium or agar.

[0109] The term "cell line," as used herein, may refer to a culture of cells that can be passaged at least once without being terminated.

[0110] The term "suspension," as used herein, may refer to a cell culture condition in which the cells are not attached to a solid support. Cells grown in suspension can be agitated during growth using equipment well known to those skilled in the art.

[0111] The term "monolayer," as used herein, may refer to cells that are attached to a solid support while growing under suitable culture conditions. A small portion of cells growing in a monolayer under suitable growth conditions may be attached to the cells in the monolayer rather than to the solid support.

[0112] The terms "plated" or "plating," as used herein with respect to cells, can refer to the establishment of 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 plated at various concentrations and / or cell densities.

[0113] 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 involving (1) the release of cells from a solid support or substrate and dissociation of those cells, and (2) the dilution of the cells into 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 to the original culture vessel to dilute the cells for further cell growth. Additionally, the cells can also be added to a new culture vessel supplemented with a medium suitable for further cell growth.

[0114] 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 also be used, such as, for example, solids, semi-solids, gels, suspensions, etc.

[0115] 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 present therein are particularly useful for cell survival, growth, and division.

[0116] As used herein, the term "basal medium" refers to an unsupplemented synthetic 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 example, for promoting the growth of a particular cell type or for maintaining or altering the differentiation state (e.g., fibroblast growth factor-basic (bFGF), also known as fibroblast growth factor 2 (FGF-2)).

[0117] As used herein, the terms "wild-type," "naturally occurring," and "unmodified" are used herein to refer to the typical (or most common) form, appearance, phenotype, or strain found in nature; e.g., the typical form of a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA, or genome as it appears 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, recombinant, and modified forms are not wild-type forms.

[0118] As used herein, the term "isolated" refers to material that has been removed from its original environment and has thus been altered "by the hand of man" from its natural state.

[0119] As used herein, the term "enriched" means to selectively concentrate or increase the amount of one or more components in a composition relative to one or more other components. For example, enrichment may include reducing or diminishing (e.g., removing or eliminating) the amount of undesirable material; and / or may include specifically selecting or isolating a desired material from a composition.

[0120] 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. These terms encompass genome modification methods, including genome editing as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, codon optimization, and the like. Genetic engineering methods are known in the art.

[0121] As used herein, the terms "nucleic acid sequence," "nucleotide sequence," and "oligonucleotide" all refer to a polymeric form of nucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides that, when linear, has one 5' end and one 3' end and can contain one or more nucleic acid sequences. The nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length. Polynucleotides may perform any function and may have a variety of secondary and tertiary structures. The term encompasses known analogs of natural nucleotides as well as nucleotides modified in the base, sugar, and / or phosphate moieties. Analogs of a particular nucleotide have the same base-pairing specificity (e.g., an analog of A base pairs with T). A polynucleotide may contain one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. The nucleotide structure may be modified before or after assembly of the polymer. After polymerization, polynucleotides may be further modified, for example, by conjugation with a labeling moiety or a target-binding moiety. Nucleotide sequences may incorporate non-nucleotide components. These terms also encompass nucleic acids containing synthetic, naturally occurring, and / or non-naturally occurring modified backbone residues or groups that have similar binding properties to a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA), locked nucleic acid (LNA™) (Exiqon, Inc., Woburn, MA) nucleosides, glycol nucleic acid, bridged nucleic acid, and morpholino structures. Peptide nucleic acid (PNA) is a synthetic nucleic acid homolog 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.

[0122] As used herein, "sequence identity" generally refers to the percent identity of nucleotide bases or amino acids comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms 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 at sites on the World Wide Web, including, but not limited to, 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 of 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 between two sequences where only a portion of the two sequences can be aligned.

[0123] 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, e.g., 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 over the length of the reference polynucleotide or polypeptide or query sequence, but less than 90%.

[0124] 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.

[0125] 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 to the second macromolecule in a non-covalent manner). A portion 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). A binding interaction can be characterized by a dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. Increased binding affinity correlates with a lower Kd.

[0126] "Gene," as used herein, refers to a polynucleotide sequence comprising exons and associated regulatory sequences. A gene may further comprise introns and / or untranslated regions (UTRs).

[0127] As used herein, "expression" refers to transcription of a polynucleotide from a DNA template to give, for example, messenger RNA (mRNA) or other RNA transcripts (e.g., non-coding RNA, such as structural or scaffolding RNA). The term also refers to the process by which the transcribed mRNA is translated into a peptide, polypeptide, or protein. The transcript and encoded polypeptide are sometimes collectively referred to as the "gene product." Expression can also include splicing of the mRNA in eukaryotic cells if the polynucleotide is derived from genomic DNA.

[0128] 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 when placed under the control of appropriate regulatory sequences in vitro or in vivo. 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.

[0129] As used herein, a "different" or "altered" level, for example, a characteristic or property, is measurably different and preferably statistically significant (e.g., cannot be attributed to the standard error of the assay). In some embodiments, the difference, for example, when compared with a control or reference sample, can be, for example, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, such as greater than 2-fold difference; greater than 5-fold difference; greater than 10-fold difference; greater than 20-fold difference; greater than 50-fold difference; greater than 75-fold difference; greater than 100-fold difference; greater than 250-fold difference; greater than 500-fold difference; greater than 750-fold difference; or greater than 1,000-fold difference.

[0130] As used herein, the term "between" includes the endpoints of a given range (e.g., between about 1 and about 50 nucleotides in length includes 1 nucleotide and 50 nucleotides).

[0131] As used herein, the term "amino acid" refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.

[0132] As used herein, the terms "peptide," "polypeptide," and "protein" are synonymous and refer to a polymer of amino acids. A polypeptide can be of any length. It can be branched or linear, it can be interrupted by non-amino acids, and it can contain modified amino acids. These terms also refer 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 indicated. Polypeptides and polynucleotides can be made using routine techniques in the field of molecular biology.

[0133] "Moiety," as used herein, refers to a portion of a molecule. A moiety may be a functional group or may describe a portion of a molecule that has multiple functional groups (e.g., shares a common structural aspect). The terms "moiety" and "functional group" are typically used interchangeably; however, "functional group" can more specifically refer to a portion of a molecule that includes some common chemical behavior. "Moiety" is often used as a structural description.

[0134] The term "effective amount" or "therapeutically effective amount" of a composition or agent, such as a therapeutic composition as provided herein, refers to a sufficient amount of the composition or agent to bring about a desired response, which will depend on the particular disease at issue.

[0135] "Transformation," as used herein, 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.

[0136] Obtaining progenitor cells

[0137] The present disclosure relates, in part, to methods and assays for analyzing the activity, function, and / or efficacy of conditioned medium; or secretome-, extracellular vesicles-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions. In some embodiments, the conditioned medium, or secretome-, extracellular vesicles-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions can be obtained from one or more cells in culture, such as cultured progenitor cells. However, other types of cells can also be used, including, for example, terminally differentiated cells, pluripotent stem cells, and the like.

[0138] Progenitor cells suitable for producing conditioned medium, or secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions, may be isolated, for example, from a subject or tissue, or may be generated from pluripotent stem cells, such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs). In some embodiments, activity, function, and / or efficacy can be assessed for conditioned medium, or secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions produced from progenitor cells differentiated from iPSC cells.

[0139] Generation of iPSC cells

[0140] iPSC cells may be obtained from somatic cells, including, for example, human somatic cells. The somatic cells may be derived from human 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; agricultural animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rabbits, mice, rats, and guinea pigs; and birds, including poultry, wild birds, and game birds, such as chickens, turkeys, and other pheasants, ducks, and geese.

[0141] In some embodiments, the somatic cells are selected from keratinized squamous 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 less than fully differentiated. For example, undifferentiated progenitor cells that are not PSCs, including somatic stem cells, and terminally differentiated mature cells can be used. Somatic cells may be from animals of any age, including adult and fetal cells.

[0142] The somatic cells may be of mammalian origin. For example, if 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 not MHC- / HLA-matched to the subject. In some embodiments, the iPSCs are MHC- / HLA-matched to the subject. In some embodiments, the somatic cells may be obtained from the subject to be treated or from another subject of the same or substantially the same HLA type as the subject, for example, if the iPSCs are to be used to generate PSC-derived progenitor cells (to obtain secretomes or extracellular vesicles for therapeutic use in the subject). The somatic cells can be cultured before nuclear reprogramming, or can be reprogrammed without culture, e.g., after isolation.

[0143] To introduce reprogramming factors into somatic cells, viral vectors can be used, 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 (HHV) 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 have the ability to 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 linking viral proteins, such as envelope proteins, to binding agents such as antibodies or to specific ligands (e.g., for targeting to receptors or proteins on or within specific cell types).

[0144] In some embodiments, viral vectors, such as lentiviral vectors, may integrate into the genome of a host cell. This transferred genetic material is then transcribed and, in some cases, 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.

[0145] 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.

[0146] Somatic cell can be reprogrammed by methods known to those skilled in the art to produce induced pluripotent stem cell (iPSC).Those skilled in the art can easily make induced pluripotent stem cell, for example, see US Patent Application Publication No. 2009 / 0246875, US Patent Application Publication No. 2010 / 0210014; US Patent Application Publication No. 2012 / 0276636; US Patent No. 8,058,065; US Patent No. 8,129,187; and US Patent No. 8,268,620 (all of which are incorporated herein by reference).

[0147] Generally, reprogramming factors that can be used to generate induced pluripotent stem cells 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, either alone, in combination, or as a fusion with a transactivation domain. 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: NM_028016; mouse Lin28: NM_145833; human MyoD: NM_002478; human Oct4 (POU5F1): NM_002701, NM_203289, NM_001173531; human Sox2: NM_003106; human Klf4: NM_004235; human c-Myc: NM_002467; human Nanog: NM_024865; and / or human Lin28: NM_024674. Also contemplated are sequences similar thereto, including those having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0148] Exemplary reprogramming factors for the generation 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 (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).

[0149] 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. In addition, at the functional level, PSCs, such as ESCs and iPSCs, also demonstrate the ability to differentiate into lineages from all three embryonic germ layers and to form teratomas in vivo (e.g., in SCID mice).

[0150] Generation of progenitor cells by differentiating PSCs

[0151] The present disclosure further contemplates differentiating PSCs, including ESCs and iPSCs, into progenitor cells, which can then be used to generate the secretomes (and extracellular vesicles) of the present disclosure.

[0152] Progenitor cells of the present disclosure include, for example, hematopoietic progenitor cells, myeloid progenitor cells, neural progenitor cells; pancreatic progenitor cells, cardiac progenitor cells, cardiomyocyte progenitor cells, cardiovascular progenitor cells, renal progenitor cells, skeletal myoblasts, satellite cells, intermediate progenitor cells formed in the subventricular zone, radial glial cells, bone marrow stromal cells, periosteal cells, endothelial progenitor cells, blast cells, boundary cap cells, and mesenchymal stem cells. Methods for differentiating pluripotent stem cells into progenitor cells, and methods for culturing and maintaining progenitor cells are known in the art, such as those described in U.S. Provisional Patent Application No. 63 / 243,606, entitled "Methods for the Production of Committed Cardiac Progenitor Cells," which is incorporated herein by reference in its entirety.

[0153] The resulting progenitor cells are then cultured, and the resulting culture medium can be collected (and optionally further processed) to provide a conditioned medium, or a secretome, extracellular vesicles, and / or a small extracellular vesicle-enriched fraction (sEV)-containing composition. The present disclosure also contemplates engineered extracellular vesicles. For example, a conditioned medium, or a secretome, extracellular vesicles, and / or a small extracellular vesicle-enriched fraction (sEV)-containing composition can be collected from engineered cells. Additionally, or alternatively, the extracellular vesicles themselves can be manipulated before, during, or after their collection.

[0154] For example, the harvested culture medium may be concentrated, purified, refrigerated, frozen, cryopreserved, lyophilized, sterilized, etc. In some embodiments, the harvested conditioned medium may be pre-clarified to remove particulates above a certain size. For example, the harvested conditioned medium may be pre-clarified by one or more centrifugation and / or filtration techniques.

[0155] In some embodiments, the collected conditioned medium is further processed to obtain a specific extract or fraction of the collected conditioned medium. For example, the collected conditioned medium may be further processed to separate a small extracellular vesicle-enriched fraction (sEV) therefrom. The sEV fraction can be separated from the collected 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.

[0156] Any of the above processing techniques can be performed, for example, on harvested conditioned medium (or pre-processed extracts or fractions thereof) that is fresh or that has been previously frozen and / or refrigerated.

[0157] In some embodiments, the sEV fraction to be analyzed by the methods and assays herein is CD63 +, CD81 + , and / or CD9 + The sEV fraction may contain one or more types of extracellular vesicles, such as one or more of exosomes, microparticles, and extracellular vesicles. The sEV fraction may also contain secreted proteins (enveloped and / or non-enveloped). The extracellular vesicles present 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.

[0158] In some embodiments, the presence of a desired type of extracellular vesicles in a fraction may be determined, for example, by nanoparticle tracking analysis (determining the size of particles in the fraction); and / or by confirming the presence of one or more markers associated with the desired type of extracellular vesicles. For example, fractions of collected conditioned medium can be analyzed for the presence of the desired type of extracellular vesicles by detecting the presence of one or more markers in the fraction, such as, for example, CD9, CD63, and / or CD81.

[0159] 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 contains a reduced 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 relative 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 of, or is negative for, one or more markers selected from the group consisting of CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69 (e.g., by MACSPlex assay, by immunoassay, etc.).

[0160] 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 of 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).

[0161] Assays for determining the activity, function, and / or potency of secretomes and extracellular vesicles

[0162] The present disclosure also encompasses methods for analyzing the activity, function, and / or efficacy of conditioned medium; or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions. The activity, function, and / or efficacy of conditioned medium; or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions can be assessed by a variety of techniques, depending, for example, on the type of progenitor cells used to generate the conditioned medium or composition and the desired use of the conditioned medium or composition.

[0163] For example, the activity, function, and / or efficacy of the conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions may 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 characteristics of the target cells, such as, for example, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology, can then be analyzed to determine the activity, function, and / or efficacy of the conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions.

[0164] In the disclosed methods and assays, the activity, function, characteristics, and / or efficacy of conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions, can be assessed by a method comprising administering the conditioned medium or secretome-, extracellular vesicle-, and / or 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 may be selected from, for example, cell migration, cell viability, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology. In some embodiments, 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 characteristics of the target cells are measured.

[0165] In the first method, target cells are cultured in a preconditioning medium under at least one stress-inducing condition, followed by administration of a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition to the cell culture. The target cells are then cultured in the presence of the conditioned medium or 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). In some embodiments, the at least one property is measured at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, or at least 500 times during culture in the presence of conditioned medium or secretome, extracellular vesicles, and / or sEV-containing compositions.

[0166] In some embodiments of this first method, culturing in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, 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, extracellular vesicle-containing, and / or sEV-containing composition is performed in the absence of at least one stress-inducing condition.

[0167] In some embodiments of this first method, the preconditioning medium is removed from the cells prior to culturing in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition. Thus, in embodiments of the first method in which at least one stress-inducing condition is imparted by the preconditioning medium (e.g., by a stress-inducing agent present in the preconditioning medium), culturing in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition is performed in the absence of the at least one stress-inducing condition.

[0168] In other embodiments of this first method, the preconditioning medium is not removed from the cells prior to culturing in the presence of the conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition. Thus, in embodiments of the first method in which at least one stress-inducing condition is imparted by the preconditioning medium (e.g., by a stress-inducing agent present in the preconditioning medium), culturing in the presence of the conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition is performed in the presence of at least one stress-inducing condition.

[0169] In a second method, target cells are cultured in a preconditioning medium, followed by administration of conditioned medium or a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition (and optionally thereafter, the target cells are cultured in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition). The target cells are then cultured under at least one stress-inducing condition, and at least one property of the cultured cells is measured one or more times during culture under the at least one stress-inducing condition (which is also performed in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or 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, extracellular vesicle-containing, 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. In some embodiments, the at least one property is measured at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, or at least 500 times during culture under at least one stress-inducing condition.

[0170] In some embodiments of this second method, the target cells are cultured in the presence of conditioned medium or a secretome-containing, extracellular vesicle-containing, and / or 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-containing, extracellular vesicle-containing, 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 secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition is removed from the target cells before being cultured under at least one stress-inducing condition.

[0171] In some embodiments of the first and second methods described above, the stress-inducing condition is culturing 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, extracellular vesicle-containing, and / or sEV-containing composition.

[0172] In some embodiments of the first and second methods above, the cell stress agent is one or more chemotherapeutic agents and / or one or more apoptosis-inducing agents.

[0173] Chemotherapeutic agents may be selected from, for example, antimetabolites, alkylating agents, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, kinase inhibitors (including protein kinase inhibitors), anthracyclines, platinum, plant alkaloids, nitroureas, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, nucleotide and precursor analogs, peptide antibiotics, and retinoids.

[0174] The one or more apoptosis inducers may be selected from, for example, doxorubicin, staurosporine, etoposide, camptothecin, paclitaxel, vinblastine, gambogic acid, daunorubicin, tyrphostins, thapsigargin, okadaic acid, mifepristone, colchicine, ionomycin, 24(S)-hydroxycholesterol, cytochalasin D, brefeldin A, raptinal, carboplatin, C2 ceramide, actinomycin D, rosiglitazone, kaempferol, berberine chloride, bioimifi, betulinic acid, tamoxifen, embelin, phytosphingosine, mitomycin C, birinapant, anisomycin, genistein, cycloheximide, and the like, and derivatives thereof, and combinations thereof.

[0175] 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.

[0176] In some embodiments of the first and second methods, the at least one characteristic measured is viability of the cultured cells. Viability may be measured, for example, using a DNA labeling dye or a nuclear staining dye. In some embodiments thereof, the DNA labeling dye or the nuclear staining dye is a fluorescent dye, such as a near-infrared fluorescent dye.

[0177] In some embodiments of the first and second methods, the at least one property 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 the culture vessel during culture.

[0178] In embodiments of the first and second methods, the target cells can be cultured in the pretreatment medium for various lengths of time, for example, the target cells can be cultured in the pretreatment medium for 30 minutes to 10 hours, for 1 hour to 5 hours, or for more than, less than, or about 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.

[0179] In embodiments of the first and second methods, the target cells are cultured with the conditioned medium or the secretome-containing, extracellular vesicle-containing, 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.

[0180] 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, less than 18 days, less than 16 days, less than 14 days, less than 12 days, less than 10 days, less than 8 days, less than 6 days, less than 4 days, or less than 2 days 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.

[0181] In the first and second method embodiments, the culture of target cells may be a two-dimensional or three-dimensional cell culture. For example, in some embodiments, the culture vessel used for the culture may be, for example, a flask, tissue culture flask, hyperflask, dish, Petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multi-plate, multiwell plate, microslide, chamber slide, tube, tray, CellSTACK® chamber, culture bag, roller bottle, bioreactor, stirred culture vessel, spinner flask, microcarrier, or vertical wheel bioreactor.

[0182] In embodiments in which the culture comprises a two-dimensional cell culture, such as on the surface of a culture vessel, the culture surface (to which the cells are intended to adhere) may be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, 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.

[0183] In the first and second method embodiments, the at least one property may also be analyzed relative to one or more control samples.

[0184] For example, the first and second methods may further include culturing (e.g., in parallel) positive control cells, where the positive control cells are not administered conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or 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.

[0185] The first and second methods may include culturing (e.g., in parallel) negative control cells, where the negative control cells are not administered conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions. In some embodiments, the negative control cells include negative control cells that are subjected to the same steps as the target cells, except that they are not administered conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions.

[0186] In certain embodiments, the negative control cells comprise negative control cells cultured in a pre-conditioning medium under at least one stress-inducing condition, and then the at least one property measured in the target cells can also be measured in the negative control cells either while they are being cultured in a pre-conditioning medium under at least one stress-inducing condition or thereafter.

[0187] In some embodiments, negative control cells include negative control cells to which mock conditioned medium or a mock secretome-, extracellular vesicle-, and / or sEV-containing composition is added, hi specific embodiments thereof, the mock conditioned medium or mock secretome-, extracellular vesicle-, and / or sEV-containing composition is produced by omitting cells from a process for producing a conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition, such as a process of the present disclosure.

[0188] The use of such one or more negative controls allows for the determination of the activity, function and / or efficacy of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition.

[0189] For example, if at least one property measured is viability of cultured cells, the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be determined to have activity, function, efficacy (and / or exhibit a therapeutic effect) when the viability of the target cells is greater than the viability of negative control cells subjected to at least one stress-inducing condition.

[0190] Alternatively, for example, if the at least one property measured is cell adhesion, cell growth, and / or cell number, and the cell adhesion, cell growth, and / or cell number are determined by measuring the electrical impedance of the culture vessel surface in the culture, the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be determined to have activity, function, efficacy (and / or exhibit a therapeutic effect) when the electrical impedance of the culture vessel surface in the culture is higher than the electrical impedance of the culture vessel surface in a culture of negative control cells that have been subjected to at least one stress-inducing condition.

[0191] 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 determine an average maximum cell number (and cell viability may be calculated by normalizing the number of target cells in each replicate test culture by this average maximum cell number).

[0192] In some embodiments, assays known in the art can be used in combination with the methods and assays of the present disclosure to further determine, verify, and / or confirm the activity, function, and / or potency of the conditioned medium, or secretome, extracellular vesicles, and / or sEV-containing compositions.

[0193] For example, the activity, function, and / or efficacy of conditioned medium; or secretome, extracellular vesicles, and / or sEV-containing compositions obtained from cardiomyocyte progenitor cells may be further measured using known cardiomyocyte viability assays, such as those described in El Harane et al. (Eur. Heart J., 2018, 39(20): 1835-1847).

[0194] Specifically, serum-deprived cardiac myoblasts (e.g., H9c2 cells) can be contacted with conditioned medium; or secretome, extracellular vesicles, and / or sEV-containing compositions, and then cell viability can be measured. In some embodiments of this assay, the cells are serum-deprived before administering the conditioned medium or secretome, extracellular vesicles, and / or sEV-containing compositions. In other embodiments of this assay, the cells are serum-deprived after administering the conditioned medium or secretome, extracellular vesicles, and / or sEV-containing compositions. In some embodiments of this assay, the cells are serum-deprived before and after administering the conditioned medium or secretome, extracellular vesicles, and / or sEV-containing compositions.

[0195] Another assay known in the art that can be used with the methods and assays of the present disclosure is HUVEC scratch wound healing assay.In HUVEC scratch wound healing assay, HUVEC cells are cultured on a culture surface, and then the cultured cell layer is scratched;Then, the angiogenic activity of conditioned medium or secretome, extracellular vesicles, and / or sEV-containing composition can be determined by the ability of conditioned medium or secretome, extracellular vesicles, and / or sEV-containing composition to cause wound closure under serum-free conditions.

[0196] To more accurately compare the activity, function, characteristics, and / or potency of different conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions, it may be beneficial to determine the amount of conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition that is (or should be) 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 number of particles in the secretome.

[0197] Therapeutic Compositions and Applications

[0198] Conditioned medium, or secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions, can be analyzed by the methods and assays of the present disclosure to identify whether the medium or composition contains a desired activity, function, and / or efficacy. If the medium or composition exhibits a desired activity, function, and / or efficacy using the methods and / or assays of the present disclosure, such medium or composition can be formulated or adapted for therapeutic use.

[0199] For example, the tested medium or composition may be a sample from a larger batch of conditioned medium, or from a larger batch of secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing composition. In such cases, all or a portion of the remaining medium or composition from the batch can be formulated or adapted for therapeutic use.

[0200] Alternatively, the tested medium or composition may be representative of other batches of conditioned medium or other batches of secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions (e.g., because they were produced in parallel under similar or identical conditions). In such cases, the analysis of the tested medium or composition may indicate that the other batches of conditioned medium or other batches of secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing compositions are suitable for therapeutic formulation or adaptation.

[0201] The disclosed methods and assays may also be used to determine whether a particular process for producing a conditioned medium or for producing a secretome-, extracellular vesicle-, and / or small extracellular vesicle-enriched fraction (sEV)-containing composition produces a medium or composition with a desired activity, functionality, or efficacy.

[0202] Thus, based on the analytical results of the disclosed methods and assays, secretome-, extracellular vesicle-, and sEV-containing compositions may be identified, selected, and / or formulated for use as therapeutic agents (e.g., for administration of an effective amount thereof to a subject in need thereof).

[0203] The identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions can be used to treat various tissues, including, but 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 tissue to be treated can be damaged or completely or partially non-functional due to, for example, injury, age-related degeneration, acute or chronic disease, cancer, or infection. Such tissues can be treated, for example, by intravenous administration of secretome-, extracellular vesicle-, and / or sEV-containing compositions. Alternatively, the identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions can be used to pretreat patients before tissue damage is predicted or expected. For example, the identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions can be administered prior to chemotherapy, for example, to help prevent cardiac damage.

[0204] The identified, selected, and / or formulated secretome-, extracellular vesicles-, and / or sEV-containing compositions may be used to treat diseases such as myocardial infarction, stroke, heart failure, and critical limb ischemia. In some embodiments, the identified, selected, and / or formulated secretome-, extracellular vesicles-, and / or sEV-containing compositions may be used to treat heart failure having one or more of the following characteristics: acute, chronic, ischemic, non-ischemic, with ventricular dilation, or without ventricular dilation. In some embodiments, the compositions of the present disclosure may be used to treat heart failure selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy-induced heart failure. In some embodiments, the compositions of the present disclosure may be used to treat diseases such as congestive heart failure, heart disease, ischemic heart disease, valvular heart disease, connective tissue disease, viral or bacterial infections, cardiomyopathies, myopathies, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dystrophinopathy, liver disease, kidney disease, sickle cell disease, diabetes, and neurological diseases. It will be appreciated that the type or types of suitable progenitor cells may be selected depending on the disease to be treated or the tissue to be targeted.

[0205] For example, in some embodiments, the identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions can be used to treat a subject with a cardiac disease, such as acute myocardial infarction or heart failure. The identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions can be generated, for example, from cardiomyocyte progenitor cells, cardiac progenitor cells, mesenchymal stem cells, and / or cardiovascular progenitor cells.

[0206] The identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions can also be used to improve tissue function or performance. For example, improved angiogenesis or cardiac performance can be achieved by delivering secretome-, extracellular vesicle-, and / or sEV-containing compositions made from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells to a subject in need thereof.

[0207] Administration includes administration at a tissue or organ site that is the same as the target tissue. Administration may additionally or alternatively include administration at a tissue or organ site that is different from the target tissue. Such administration may include, for example, intravenous administration.

[0208] The identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions 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 salts, buffers, preservatives, or other therapeutic agents. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other non-toxic substances compatible with use in pharmaceutical formulations. For example, in some embodiments, the secretome-, extracellular vesicle-, and / or sEV-containing compositions 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.

[0209] The identified, selected, and / or formulated secretome-, extracellular vesicle-, and / or sEV-containing compositions may be administered in an effective amount, such as a therapeutically effective amount, depending on the intended purpose. The effective amount will depend on a variety of factors, including the material selected for administration, whether administration is in a single dose or multiple doses, and individual patient parameters, including age, health, size, weight, and stage of disease. These factors are well known to those skilled in the art.

[0210] 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, intracisternal, intraperitoneal, intranasal, intramyocardial, intracoronary, aerosol, suppository, epicardial patch, oral administration, or by 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 drops, or aerosol. For example, in some embodiments, a subject with a cardiac disease such as acute myocardial infarction or heart failure can be treated with a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition made from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells, wherein the composition is administered intravenously.

[0211] A single dose of the identified, selected, and / or formulated secretome-, extracellular vesicles-, and / or sEV-containing composition may be administered. In other embodiments, multiple doses are administered to a subject over one or more daily, weekly, or monthly periods. Single or repeated administrations of the secretome-, extracellular vesicles-, and / or sEV-containing composition may be administered, including two, three, four, five, or more administrations. The composition may also be administered continuously. Repeated or continuous administration may occur over a period of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, or 1-7 days), or several weeks (e.g., 1-2 weeks, 1-3 weeks, or 1-4 weeks), depending on the nature and / or severity of the condition being treated. When administration is repeated but not continuously, the time between administrations can be a few hours (e.g., 4 hours, 6 hours, or 12 hours), a few days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days), or a few weeks (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks). The time between administrations can be the same or they can be different. For example, if symptoms worsen or do not improve, the composition may be administered more frequently. Conversely, if symptoms stabilize or become less severe, the composition may be administered less frequently.

[0212] In some embodiments, the secretome-, extracellular vesicle-, and / or sEV-containing composition is administered intravenously in three doses, approximately two weeks apart. In some embodiments, the composition may be diluted, formulated with, and / or administered in conjunction with a carrier, diluent, or suitable material (e.g., saline). [Example]

[0213] The following examples illustrate non-limiting embodiments of the present invention. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, concentrations, rates of change, etc.), some experimental error and deviation must be accounted for. It should be understood that these examples are provided as illustrations 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 shown in each example are required, nor are the steps in each example necessarily in the exact order presented.

[0214] Example 1 Reproducibility of the H9c2 viability assay To test the reproducibility of the H9c2 viability assay, the assay was essentially performed 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 Media), but cease proliferation and lose viability when serum is deprived (e.g., cultured in H9c2 Poor Media). Therefore, by supplementing H9c2 Poor Media with EVs, we can investigate whether extracellular vesicle (EV) preparations containing sEVs can promote the viability of H9c2 cardiomyocytes.

[0215] Briefly, H9c2 cells were thawed and seeded at 4250 viable cells per well into a 96-well plate. After 24 hours of incubation in complete growth medium, the cells were refed. After another 24 hours of incubation, the culture medium was removed, and either serum-containing medium (H9c2 Complete Media; for positive control) or serum-free medium (H9c2 Poor Media; for test samples and negative controls) was added to the appropriate wells (along with a nuclear dye to allow for quantification of cell viability). The sEV preparation (or mock sEV preparation) was then added to the appropriate wells to examine the effect of sEVs on H9c2 viability. H9c2 cells were then cultured and imaged at several time points during culture using an Incucyte (Essen BioSciences).

[0216] Using this assay, we found that different banks of serum-starved H9c2 cells produced different cell viability results, even when using the same sEV preparation and culture conditions. Some sEV preparations had a positive effect on cell viability in one (high-passage) H9c2 cell bank but not in another (low-passage) H9c2 cell bank. This phenomenon was confirmed by manual cell counting of live cells (cells were harvested and counted manually using a hemocytometer) and by using a ViCell XR cell viability analyzer (Beckman Coulter). Analysis of different banks of H9c2 cells revealed that H9c2 cells from different banks exhibited different morphologies depending on the passage number. These results suggest that the variability between H9c2 lots and passages in this H9c2 cell viability assay is too great to be used as a standard method for evaluating the secretome or sEV efficacy. Furthermore, these rat cells may have limited applicability for the development and testing of human sEV products for human therapeutic development.

[0217] Example 2 Staurosporine cardiomyocyte viability assay In light of the results of the H9c2 cell viability assay in Example 1, another cell viability assay was developed.

[0218] Specifically, human iCell Cardiomyocytes 2 (Fujifilm Cellular Dynamics, Inc., ref: CMC-100-012-001) were plated at 50,000 cells / well in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001) onto fibronectin-coated (5 μg / mL) 96-well plates and cultured for 4 hours at 37°C (atmospheric oxygen, 5% CO2). After this 4-hour incubation, the medium was replaced with 100 μL (per well) of iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc.: M1003). The cells were cultured for up to 12 days, with complete medium changes every 2–3 days.

[0219] After at least 4 days, cells were exposed to iCMM supplemented with NucSpot Live 650 dye (Biotium, ref: 40082, final concentration 0.125%), which served as a live cell (positive) control, or iCMM supplemented with NucSpot Live 650 dye (final concentration 0.125%) and staurosporine (Abcam, ref: ab146588) at a final well concentration of 2 μM, which also served as an apoptotic cell (negative) control. The concentrations of dye, PBS, DMSO (0.325%), and the final well volume (120 μL) were equivalent in all wells. Cells were then cultured at 37°C (atmospheric oxygen, 5% CO2) for 4 hours.

[0220] After this 4-hour incubation, plates were imaged using an Incucyte (Essen BioSciences), and the pre-incubation medium was removed and the wells were washed with 100 μL of iCMM. Representative images of cells with and without staurosporine are shown in Figure 1. Cells were then fed with 120 μL (per well) of iCMM containing NucSpot Live 650 dye (final concentration 0.125%), DMSO (final concentration 0.325%), and 15 μL of DPBS (in a final volume of 120 μL), or with 120 μL (per well) of iCMM containing NucSpot Live 650 dye (final concentration 0.125%), DMSO (final concentration 0.325%), and increasing concentrations of sEV or mock-sEV preparations adjusted to 15 μL with DPBS. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2). Wells were imaged in an Incucyte® system every hour for 24 hours to determine nuclei counts. Time course results were obtained by comparing % Positive NucSpot Live 650 normalized to time 0 (T0). Figure 2 shows this time course, demonstrating that the sEV preparation improved cardiomyocyte viability, whereas the mock sEV preparation did not, demonstrating the functionality of the sEV preparation.

[0221] Because the absolute number of cells per well may vary between assays, the data were processed as follows to obtain comparable values. First, normalization was performed by comparing the average number of cells at a particular time point for each condition to the average number of cells at time point 0. Next, the normalized negative control values ​​were subtracted from each condition, and the resulting values ​​were compared to the difference between the positive and negative controls. Figure 3 shows a histogram showing the results at the 24-hour time point of the staurosporine cardiomyocyte viability assay time course.

[0222] Regarding sEV dosage, we determined that protein concentration, RNA concentration, and sEV particle number can vary considerably depending on the medium, culture conditions, cell type, sEV isolation / enrichment technique, and donor. Therefore, to ensure accurate dosing and accurate comparison between different sEV samples, we calculated the dosage based on the number of secreting cells that generated the secretome. This proved to be a reliable measure in these cases, where the active component is unknown and the inactive and co-isolated components can vary considerably (see Example 4).

[0223] Additionally, as a confirmatory measure of cell viability, cell cultures in 96-well plates that were first subjected to the above time course (Incucyte and biocompatible dye) experiments were subsequently analyzed for ATP content (i.e., at the end of the Incucyte and biocompatible dye time course).

[0224] Specifically, cells from each well were lysed and ATP content was quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. The resulting signal was analyzed using a CLARIOStar® (BMG Labtech) and a Tecan for Life Science® plate reader. The results are shown in Figure 4. Figure 4 shows cell viability data obtained using the Incucyte at the 13-hour time point and ATP quantification data obtained using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) with a CLARIOStar® (BMG Labtech) and a Tecan for Life Science® plate reader (at the 24-hour time point, i.e., the end of the Incucyte time course). As Figure 4 shows, the results from the three data sets were remarkably similar to each other, confirming the applicability of the staurosporine assay to a variety of detection methods and readout devices.

[0225] Figure 5 shows the results of a further set of experiments. Here, cell viability data (using an Incucyte and showing data from the 12-hour time point) were obtained essentially as described above, and ATP quantification data were obtained using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) on a CLARIOStar® (BMG Labtech) and a Tecan for Life Science® plate reader. (The data shown is from the 23-hour time point, i.e., after the Incucyte time course had ended.) Figure 5 also shows experimental results for a positive control (no staurosporine added ("No-stress")), a negative control without sEVs ("Staurosporine"), and sEVs produced from mesenchymal stem cells (used at a 1x dose ("MSC-sEV, 1x")). As can be seen, the results are consistent with those shown in Figure 4. In a further series of experiments, human iCell cardiomyocytes (2) were plated, cultured, and pretreated with staurosporine (in iCMM medium containing serum) essentially as described above. However, after staurosporine pretreatment, the cells were washed with either iCMM or iCell Cardiomyocyte Serum-Free Medium (iCSFM, Fujifilm Cellular Dynamics, Ref: M1038). The cells were then treated with PBS (vehicle), sEV, or MV in the same medium (i.e., either iCMM or iCSFM) in which the cells were washed and incubated at 37°C (atmospheric oxygen, 5% CO). At 23 hours (i.e., 23 hours after the start of staurosporine pretreatment), the cells in each well were lysed, and their ATP content was quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. The resulting signal was analyzed using a Tecan for Life Science® plate reader.The results are shown in Figure 6 and indicate that at least the EV processing step may be performed in the absence of serum with comparable results.

[0226] Example 3 Comparison of the staurosporine cardiomyocyte viability assay and the HUVEC scratch assay To validate the staurosporine cardiomyocyte viability assay described in Example 2, a HUVEC scratch wound healing assay (developed by Essen Biosciences for Incucyte) was performed using the same sEV and mock sEV preparations. Briefly, HUVEC cells were grown in HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C-22210) supplemented with Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210). After growth, cells were cultured at 1-2 × 10 per aliquot in CS10 (Cryostore, Ref: 210102). 6Cells were cryopreserved at 100°C (enough to fill half to a full 96-well plate). Two days before the assay, an aliquot of HUVECs was thawed and plated at 10,000 cells / well into ImageLock 96-well plates (EssenBio, Ref: 4379) and grown in HUVEC Complete medium for 2 days. Cultures were then maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. Wells were scratched using a Wound Maker (EssenBio, Ref: 4493) according to the manufacturer's instructions, and cells were washed with endothelial cell basal medium and cultured overnight (either HUVEC Complete medium alone as a positive control, endothelial cell basal medium alone as a negative control, or endothelial cell basal medium supplemented with sEVs or mock sEV preparations). Plates were imaged every 3 hours for a total of 18 hours using an Incucyte Scratch Wound Healing Module. Wound closure was measured using the manufacturer's software, and baseline (negative control) subtracted values ​​were normalized to the positive control. Figure 7 compares the results of the staurosporine cardiomyocyte viability assay and the HUVEC scratch wound healing assay, demonstrating that the staurosporine cardiomyocyte viability assay provides consistent results comparable to the HUVEC scratch assay, but here for a cell type that may be more relevant to cardiac disease: cardiomyocytes.

[0227] Example 4 Dosing of sEVs by particle number compared to cell number As discussed in Example 2, a dosing strategy was required to ensure accurate comparisons between different sEV samples. Initially, sEV doses were calculated based on the number of particles per sEV sample, with a "1x" dose corresponding to a target value of 1 billion particles identified by NanoSight. The mock sEV dosing strategy was to match the dose to the "1x" dose of the sEV sample. When multiple sEV samples were present, mock sEVs were matched to the largest volume of the "1x" sEV sample.

[0228] When comparing 1 billion particles from different MSC donor lots with each other in a HUVEC scratch wound healing assay, we found variability in the functionality of sEV samples. These MSC donors were cultured and harvested under similar conditions, and sEV samples were prepared. To examine the functional differences between these lots, we explored an alternative dosing strategy. In this new strategy, the dose was calculated based on the number of secretory cells that generated the sEV secretome. As assessed in a HUVEC scratch wound healing assay, sEV samples from each MSC lot were administered at a dose of 1.85x10 5 Dosing at a "1x" amount equal to the cell secretome resulted in more similar functional results compared to dosing at particle numbers (see Figure 8).

[0229] Example 5 Considerations when matching simulated sEVs to sEV preparations As described in Example 4, sEV sample dosing was originally based on particle number, and simulated sEV dosing relied on a "1x" volume-to-volume matching strategy for the sEV sample. However, when we adopted a new sEV sample dosing strategy that determines the dose based on the number of secretory cells that generated the sEV secretome, a new simulated sEV dosing strategy was also required. In this strategy, the volume of the simulated sEVs is matched to the sEV sample as follows:

[0230] For matched sEV samples, calculate the ratio of the final sEV preparation volume to the volume of conditioned medium used in the isolation / enrichment method (µL sEV / mL MC). Similarly, for mock sEV samples, divide the volume of the final preparation by the volume of virgin medium used in the isolation / enrichment method (µL MV / mL MV). If the sEV sample will be used in a staurosporine assay or other in vitro or in vivo assay, calculate the "MC medium equivalent" for the dosage ("MC medium equivalent" = sEV dosage in assay / "µL sEV / mL MC"). Next, calculate the amount of MV needed as a control in the assay to match the MC medium equivalent, thereby controlling for potential effects from contaminating components from the medium itself (amount of MV used as control = "µL MV / mL MV" * "MC medium equivalent").

[0231] Example 6 Staurosporine cardiomyocyte attachment / proliferation / number assay (electrical impedance) Cell viability was determined (by live cell imaging using Incucyte and biocompatible dyes; and by measuring ATP content) in the staurosporine cardiomyocyte viability assay experiments described in Example 2. To demonstrate that the staurosporine assay described in Example 2 can be used with alternative detection methods or readouts to determine the functionality of sEV preparations, the staurosporine assay described in Example 2 was performed using cell proliferation / adhesion / number as indicators of sEV preparation functionality.

[0232] Specifically, we first developed human iCell cardiomyocytes. 2(Fujifilm Cellular Dynamics, Inc., ref: CMC-100-012-001) were seeded in iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., ref: M1003) at 37°C (atmospheric oxygen, 5% CO2) onto fibronectin-coated wells of a culture plate equipped with electrodes (CytoView Z-Plate, Axion Biosystems®) and allowed to adhere and form a monolayer ("0 h").

[0233] After 126 hours, the seeded cells adhered and formed a confluent monolayer (as determined by the plateau of impedance values ​​after increasing from zero at time 0). The cells were washed and exposed to preincubation medium containing either iCMM (which served as a viable cell (positive) control) or iCMM containing staurosporine (Abcam, ref. ab146588) at a final well concentration of 2 μM (which also served as an apoptotic cell (negative) control). The cells were then cultured at 37°C (atmospheric oxygen, 5% CO) for 4 hours. Impedance was measured continuously throughout the incubation period using an Axion BioSystems® (Maestro Z) instrument.

[0234] After this 4-hour incubation, the pre-incubation medium was removed (except for the control sample in which staurosporine was present during the subsequent incubation; see condition 8 in Figure 9), and the wells were washed with iCMM. Different wells of cells were then fed with iCMM (see conditions 2 and 8 in Figure 9) or iCMM supplemented with various concentrations of sEVs (see conditions 3–5 in Figure 9); mock sEV preparations (virgin medium control; see conditions 1 and 7 in Figure 9); or PBS (see condition 6 in Figure 9). Cultures were then maintained at 37°C (atmospheric oxygen, 5% CO2) for 72 hours, and impedance was again measured continuously. Figure 9 shows this time course, demonstrating that compared to the negative (staurosporine-treated) control (see conditions 6 and 8 in Figure 9), the sEV preparations (see conditions 3–5 in Figure 9) improved cardiomyocyte adhesion after staurosporine treatment, whereas the mock sEV preparation (see condition 7 in Figure 9) did not. Positive controls are shown as conditions 1 and 2 in Figure 9. These results, similar to those of the cardiomyocyte viability assay experiments described in Example 2, demonstrated the functionality of the sEV preparation. Figure 10 shows the experimental results normalized to the treatment ("Tx") time point.

Claims

1. (a) contacting a culture of target cells with a preconditioning medium and culturing said target cells in said preconditioning medium under at least one stress-inducing condition, wherein said target cells are specialized cells derived from induced pluripotent stem cells (iPSCs); (b) administering a secretome to a cell culture and culturing the target cells in the presence of the secretome, wherein the amount of secretome added to the target cells is determined based on the amount of secretory cells that produced the secretome; and (c) measuring at least one characteristic of the cultured cells one or more times during the culturing of step (b); A method for analyzing secretome activity, comprising:

2. 10. The method of claim 1, wherein the method further comprises removing the conditioning medium from the cultured cells prior to step (b).

3. The method of claim 2, wherein the target cells are cultured under at least one stress-inducing condition prior to administering the secretome to the cell culture, and the culturing of the target cells in step (b) is performed in the absence of at least one stress-inducing condition.

4. (a) contacting a culture of target cells with a preconditioning medium and culturing the target cells in the preconditioning medium, wherein the target cells are specialized cells derived from induced pluripotent stem cells (iPSCs); (b) administering a secretome to a cell culture and optionally culturing said target cells in the presence of the secretome, wherein the amount of secretome added to said target cells is determined based on the amount of secretory cells that produced the secretome; (c) culturing the target cells under at least one stress-inducing condition; and (d) measuring at least one characteristic of the cultured cells one or more times during the culturing of step (c); A method for analyzing secretome activity, comprising:

5. The method of claim 4, wherein the target cells are cultured in the presence of a secretome prior to the culture in step (c).

6. 6. The method of claim 5, wherein the method further comprises removing the secretome from the cultured cells prior to step (c).

7. The method of claim 4, wherein the stress-inducing condition is culture in the presence of a cell stress agent, the cell stress agent being co-administered with the secretome, and the target cells being cultured in the presence of the secretome and the cell stress agent.

8. The method of any one of claims 1 to 6, wherein at least one stress-inducing culture condition is culture in the presence of a cell stress agent.

9. The method of claim 7 or 8, wherein the cell stress agent is a chemotherapeutic agent and / or an apoptosis-inducing agent.

10. 10. The method of claim 9, wherein the apoptosis-inducing agent is an indolocarbazole.

11. 10. The method of claim 9, wherein the apoptosis inducer is indolo(2,3-a)pyrrole(3,4-c)carbazole.

12. 10. The method of claim 9, wherein the apoptosis inducer is staurosporine.

13. The method of claim 9, wherein the apoptosis-inducing agent is doxorubicin.

14. 14. The method of any one of claims 1 to 13, wherein the at least one property measured is selected from the group consisting of cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology.

15. 15. The method of claim 14, wherein the method further comprises measuring at least one characteristic of the cultured cells one or more times during the culturing of step (a).

16. The method of claims 1 to 3, wherein at least one characteristic is measured multiple times during the culturing step (b).

17. The method of claims 4 to 7, wherein at least one characteristic is measured multiple times during the culturing step (c).

18. 18. The method of claim 16 or 17, wherein the measurements are taken 5 minutes to 10 hours apart from each other.

19. 20. The method of claim 18, wherein the measurements are taken between 10 minutes and 4 hours apart.

20. 20. The method of claim 19, wherein the multiple measurements are taken 30 minutes to 2 hours apart.

21. The method of any one of claims 1 to 20, wherein the at least one property is selected from cell viability, cell adhesion, cell number, cell morphology, cell proliferation, and / or ATP content.

22. 22. The method of claim 21, wherein the at least one characteristic is viability of the cultured cells, and the viability is measured using a fluorescent DNA labeling dye or a fluorescent nuclear staining dye.

23. 22. The method of claim 21, wherein the at least one characteristic is adhesion, cell number, proliferation, and / or cell morphology of the cultured cells, and the adhesion, cell number, proliferation, and / or morphology of the cultured cells is determined by measuring electrical impedance across the surface of the culture vessel during culture.

24. 24. The method of any one of claims 1 to 23, wherein the culturing of the target cells in the pretreatment medium in step (a) is for 30 minutes to 10 hours.

25. 25. The method of claim 24, wherein the target cells are cultured in the pretreatment medium in step (a) for 1 hour to 5 hours.

26. 26. The method of claim 25, wherein the culturing of the target cells in the preconditioning medium in step (a) is for about 4 hours.

27. The method of any one of claims 1 to 3, wherein the culturing of the target cells in step (b) is for at least 2 hours.

28. The method of any one of claims 4 to 7, wherein the culturing of the target cells in step (c) is for at least 2 hours.

29. 29. The method of claim 27 or claim 28, wherein the incubation is for at least 5 hours.

30. 30. The method of claim 29, wherein the incubation is for at least 12 hours.

31. 31. The method of claim 30, wherein the incubation is for at least 24 hours.

32. 23. The method of claim 22, wherein the viability of the cultured cells is measured by imaging a DNA-labeling dye or a nuclear-staining dye.

33. 32. The method of any one of claims 1 to 20 and 24 to 31, wherein at least one property is viability of the cultured cells, and viability is measured by live cell imaging.

34. 34. The method of any one of claims 1 to 33, wherein the target cells comprise cardiomyocytes, cardiovascular progenitor cells, cardiac progenitor cells, and / or vascular cells.

35. The method of any one of claims 1 to 33, wherein the target cells are previously frozen.

36. 36. The method of any one of claims 1 to 35, wherein the secretome is isolated from a culture of one or more cells selected from totipotent progenitor cells, multipotent progenitor cells, and terminally differentiated cells.

37. 37. The method of claim 36, wherein the one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, cardiovascular progenitor cells, and mesenchymal stem cells.

38. 38. The method of any one of claims 1 to 37, wherein the secretome comprises a small extracellular vesicle-enriched fraction (sEVs) isolated from a cell culture.

39. 39. The method of claim 38, wherein the sEV has one or more of the following characteristics: (a) CD63 ＋ , CD81 ＋ and / or CD9 ＋ (b) containing extracellular vesicles with a diameter of 50 to 200 nm; (c) containing CD49e, ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1), SSEA-4 (Stage-specific embryonic antigen 4), MSCP (Mesenchymal stem cell-like and / or (d) are negative for one or more of CD19, CD4, CD209, HLA-ABC (human leukocyte antigen-ABC), CD62P, CD42a, and CD69, contain extracellular vesicles of 50-150 nm in diameter, and / or are positive for one or more of CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142.

40. 39. The method of claim 38, wherein the sEVs comprise one or more of exosomes, microparticles, extracellular vesicles, and secreted proteins.

41. 41. The method of any one of claims 1 to 40, wherein the method further comprises culturing the target cells for 1 to 21 days prior to step (a).

42. 42. The method of claim 41, wherein the target cells have been cultured for 5 to 14 days prior to step (a).

43. 42. The method of claim 41, wherein the target cells are supplied with fresh culture medium 12 to 36 hours prior to step (a).

44. 44. The method of any one of claims 1 to 43, wherein the target cells are cultured in a two-dimensional cell culture.

45. 45. The method of claim 44, wherein the two-dimensional cell culture comprises culturing target cells on a culture vessel surface.

46. 46. ​​The method of claim 45, wherein the culture vessel surface is coated with a substance that promotes cell adhesion.

47. 47. The method of claim 46, wherein the cell adhesion promoting agent is fibronectin.

48. 48. The method of any one of claims 1 to 47, wherein the method further comprises culturing positive control cells in parallel, the positive control cells not administered the secretome and not subjected to stress-inducing conditions.

49. 49. The method of any one of claims 1 to 48, wherein the method further comprises culturing negative control cells, wherein the negative control cells are not administered with the secretome.

50. 50. The method of claim 49, wherein the negative control cells comprise negative control cells that have been subjected to the same steps as the target cells, except that they have not been administered a secretome.

51. 51. The method of claim 49 or 50, wherein the negative control cells comprise negative control cells cultured in pre-conditioning medium under at least one stress-inducing condition, and the method comprises measuring at least one characteristic of the negative control cells during or after culturing in pre-conditioning medium under at least one stress-inducing condition.

52. 50. The method of claim 49, wherein the negative control cells comprise negative control cells to which a simulated secretome composition has been added, the simulated secretome composition being produced by omitting cells from the step of producing a secretome.

53. 49. The method of claim 48, wherein the target cells and the positive control cells are cultured in duplicate.

54. 54. The method of claim 53, wherein the positive control cell numbers in the replicate cultures are averaged to form the mean maximum cell number, and the target cell numbers in each replicate culture are normalized to the mean maximum cell number.

55. 53. The method of any one of claims 49 to 52, wherein at least one characteristic is viability of cultured cells, and the secretome is determined to be efficacious and / or exhibit a therapeutic effect if the viability of target cells is higher than the viability of negative control cells.

56. The sEV is at least one of the following: sEVs enriched in extracellular vesicles having a diameter of between about 50-200 nm or between 50-200 nm, preferably between about 50-150 nm or between 50-150 nm; sEVs that are substantially free or free of whole cells; and / or sEVs that are substantially free of one or more culture medium components; 39. The method of claim 38.

57. (a) contacting a culture of target cells with a conditional medium and culturing the target cells in the conditional medium in the presence of a small molecule or a chemotherapeutic agent, wherein the target cells are specialized cells derived from induced pluripotent stem cells (iPSCs); (b) administering a secretome to a cell culture and culturing the target cells in the presence of the secretome, wherein the amount of secretome added to the target cells is determined based on the amount of secretory cells that produced the secretome; and (c) measuring at least one characteristic of the cultured cells one or more times during the culturing of step (b); A method for analyzing the activity of a small molecule therapeutic agent, comprising:

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