Extracellular vesicles that promote angiogenesis or neovascularization

Abstract

The present disclosure relates to extracellular vesicles (EVs) (and their contents) derived from pluripotent stem cell-derived endothelial cell products (ECPs); and their isolation; uses and methods of using EVs in therapy, for example, in promoting angiogenesis and / or neovascularization.

Description

[Technical Field]

[0001] The present disclosure relates to extracellular vesicles (EVs) (and their contents) derived from pluripotent stem cell-derived endothelial cell products (ECPs); and their isolation; uses and methods of using EVs in therapy, for example, in promoting angiogenesis and / or neovascularization. [Background technology]

[0002] Cardiovascular disease (CVD) remains the most common cause of death worldwide, with the World Health Organization (WHO) reporting that 17.9 million people died from CVD in 2017. Of these deaths, an estimated 7.4 million were attributable to coronary heart disease (CHD) alone (WHO-Cardiovascular Diseases (CVDs), 2017). CHD is characterized by narrowing of coronary arteries due to the gradual formation and subsequent rupture of plaque within the vessel walls. Blockage of these arteries results in oxygen and nutrient deprivation in downstream tissues. This results in ischemic injury and myocardial death in the affected area of ​​the heart, a phenomenon known as myocardial infarction (MI) (Thygesen et al., 2019). Therapeutic angiogenesis, which generates new myocardial vascular networks and reduces the degree of myocardial damage, has been proposed as a potential strategy. Paracrine cellular communication plays a crucial role in regulating this process (Gnecchi et al., 2008). Paracrine cellular communication is regulated by several mechanisms, including extracellular vesicles (EVs), which carry and transport a variety of bioactive molecules, such as small non-coding RNAs, proteins, and lipids, that regulate signaling pathways in recipient cells ( Thery et al., 2018 ).

[0003] Although somewhat controversial, preclinical studies have demonstrated the promise of EVs in regulating complex processes, such as postischemic neovascularization ( Kesidou et al., 2020 ), but the optimal source of these EVs, their respective cargo and function, and their application in clinical opportunities remain relatively in their infancy.

[0004] Endothelial cell (EC) injury and activation are important factors in the cellular release of EVs. Generally, EC-EVs are present at lower concentrations under physiological conditions and are released at higher levels from ECs upon activation (Koga et al., 2005). Circulating EVs released from ECs have been shown to play a role in vascular EC activation (Ridger et al., 2017). However, emerging evidence suggests that endothelial-derived EVs may play diverse roles in neovascularization, as their effect depends not only on the EV donor cells but also on the dose or number of EVs to which recipient cells are exposed (Lacroix et al., 2007). Nevertheless, a potential role for the endothelial secretome in EC activation and neovascularization, despite limited retention in ischemic tissues, has been proposed by EC transplantation studies showing improved capillary density and blood flow after EC transplantation (Chekanov et al., 2003). We previously demonstrated that transplantation of human embryonic stem cell-derived EC products (hESC-to-ECP) after left femoral artery occlusion resulted in increased capillary density 21 days after ischemia in mice. Furthermore, we demonstrated improved cardiac function after intracardiac cell injection following myocardial ischemia (Ana-Mishel Spiroski et al., 2022), demonstrating the broad relevance of these cells as a therapeutic approach. Despite their ability to increase capillary density, these cells showed poor retention (MacAskill et al., 2018), suggesting the involvement of an acute paracrine mechanism of action. These findings were consistent with previous cell transplantation studies that revealed very few transplanted cells were able to engraft at the injury site (Tompkins et al., 2018). Summary of the Invention

[0005] Because the increased rate of neovascularization after hESC-ECP transplantation suggests a paracrine mechanism of action, we investigated the effects of hESC-ECP-derived EVs (hESC-eEVs) on angiogenesis. As described herein, we demonstrate that very low concentrations of hESC-eEVs promote EC tube formation and wound healing, and that hESC-eEVs are enriched in known angiogenic miRNAs and other miRNAs whose role in angiogenesis has not been reported. Furthermore, we demonstrated that hESC-eEVs induce angiogenesis at low doses, which may be due to selected miRNA molecules present in hESC-eEVs, as well as other components present in EVs.

[0006] In a first aspect, there is provided an isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents obtainable from pluripotent stem cell-derived endothelial cell product (ECP).

[0007] Also provided is an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents obtainable from pluripotent stem cell-derived ECP for use in therapy, e.g., for use in promoting angiogenesis and / or neovascularization, in a subject in need thereof. In one explanation, the isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents is obtained from pluripotent stem cell-derived ECP.

[0008] Also provided are methods of treatment, e.g., by promoting angiogenesis and / or neovascularization, comprising administering to a subject in need of such treatment an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents obtained from pluripotent stem cell-derived ECPs.

[0009] In one embodiment, the present disclosure relates to the use of intact EVs as described herein. Angiogenesis primarily refers to the formation of new blood vessels from pre-existing vessels, while neovascularization is the process of de novo formation of blood vessels or the formation of new blood vessels from pre-existing vessels. The present disclosure may relate to both angiogenesis and / or neovascularization. That is, the present disclosure may relate to promoting only angiogenesis, only neovascularization, or both angiogenesis and neovascularization. The remaining description may refer to either term alone, but unless the context clearly dictates otherwise, reference to angiogenesis is understood to extend to neovascularization, and vice versa.

[0010] The term "therapy" or "treatment" as used herein may be understood to relate to the prevention and / or treatment of a condition or disease.

[0011] As used herein, the phrase "pluripotent stem cells" refers to cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm, and mesoderm). The phrase "pluripotent stem cells" includes embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPS cells).

[0012] The phrase "embryonic stem cells," as used herein, refers to cells obtained from embryonic tissue formed after conception (e.g., a blastocyst) and before fertilization (i.e., a pre-fertilized blastocyst); expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / pre-gastrulation stage (see WO2006 / 040763); and / or embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably before 10 weeks of gestation.

[0013] According to some embodiments, the pluripotent stem cells are embryonic stem cells, e.g., of human or primate (e.g., monkey) origin. Embryonic stem cells can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in-vivo preimplantation embryos or from in-vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. For the isolation of human ES cells, the zona pellucida is removed from the blastocyst, and the inner cell mass (ICM) is isolated by immunosurgery, in which trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated into tissue culture flasks containing an appropriate medium that allows for explants. After 9-15 days, the ICM-derived explants are dissociated into clumps either by mechanical dissociation or enzymatic digestion, and the cells are then replated onto fresh tissue culture medium. Colonies showing undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are then routinely split every 4 to 7 days. For further details on human ES cell preparation methods, see Thomson et al., U.S. Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995; Bongso et al., Hum Reprod 4: 706, 1989; and Gardner et al., Fertil. Steril. 69: 84, 1998.

[0014] It will also be appreciated that commercially available stem cells can also be used. Human ES cells can be purchased from commercial sources, including the NIH Human Embryonic Stem Cell Registry (www: / / escr(dot)nih(dot)gov). Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE04, and TE06.

[0015] The phrase "induced pluripotent stem (iPS) cells" (or embryonic-like stem cells), as used herein, refers to proliferative and pluripotent stem cells obtained by dedifferentiation of somatic cells (e.g., adult somatic cells). According to some embodiments, iPS cells are characterized by a proliferative capacity similar to that of ESCs and can therefore be maintained and expanded in culture for an almost unlimited period of time.

[0016] iPS cells can be pluripotent by genetic manipulation to reprogram cells to acquire embryonic stem cell properties. For example, iPS cells of the present invention can be generated from somatic cells by inducing the expression of Oct-4, Sox2, Kfl4, and c-Myc in somatic cells, essentially as described in Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Meissner et al., 2007; and Okita K., et al., 2007, Nature 448: 313-318. Additionally or alternatively, iPS cells of the present invention can be generated from somatic cells by inducing the expression of Oct4, Sox2, Nanog, and Lin28, essentially as described in Yu et al., 2007; and Nakagawa et al., 2008. Genetic manipulation (reprogramming) of somatic cells can be carried out using any known method, using plasmid or viral vectors, or by induction without any integration into the genome [Yu J, et al., Science. 2009, 324: 797-801].

[0017] iPS cells can be obtained by inducing dedifferentiation of embryonic fibroblasts [Takahashi and Yamanaka, 2006; Meissner et al, 2007], fibroblasts formed from hESCs [Park et al, 2008], fetal fibroblasts [Yu et al, 2007; Park et al, 2008], foreskin fibroblasts [Yu et al, 2007; Park et al, 2008], adult dermal and skin tissues [Hanna et al, 2007; Lowry et al, 2008], B-lymphocytes [Hanna et al, 2007], and adult liver and stomach cells [Aoi et al, 2008].

[0018] iPS cell lines are also available through cell banks such as the WiCell bank. Non-limiting examples of commercially available iPS cell lines include iPS foreskin clone 1 [WiCell catalog number iPS(foreskin)-1-DL-1], iPSIMR90 clone 1 [WiCell catalog number iPS(IMR90)-1-DL-1], and iPSIMR90 clone 4 [WiCell catalog number iPS(IMR90)-4-DL-1].

[0019] According to some embodiments, the iPS cells are human induced pluripotent stem cells.

[0020] The term "pluripotent stem cell-derived ECP," as used herein, is understood to refer to a cell product obtained from differentiated or partially differentiated pluripotent stem cells and containing at least 40%, 50%, 60%, or 70% endothelial cells. hESC-derived ECP can be a mixed population composed of endothelial cells and other differentiated cell types, including those that typically express markers indicative of mesenchymal or pericyte lineages (see, e.g., MacAskill et al., 2018).

[0021] As used herein, the term "population of extracellular vesicles" refers to a population of extracellular vesicles that have pro-angiogenic and / or pro-angiogenic properties as a result of being produced from pluripotent stem cell-derived ECP. The terms "extracellular vesicle population" and "extracellular vesicles" can be used interchangeably to refer to a population of extracellular vesicles that have pro-angiogenic and / or pro-angiogenic properties.

[0022] As used herein, the term "extracellular vesicles," abbreviated EV, includes exosomes. The terms "extracellular vesicles" and "EVs," as used herein, in some embodiments, refer to membranous particles having a diameter (or maximum diameter if the particle is not a spheroid) of about 10 nm to about 5,000 nm, more typically 30 nm to 1,000 nm, and most typically about 50 nm to 750 nm. Most commonly, EVs have a size (average diameter) that is up to 5% of the size of the donor cell. Therefore, EVs specifically intended include those shed from cells.

[0023] As used herein, isolated EVs (or EV populations) are those that are physically separated from their natural environment. Isolated EVs may be completely or partially physically separated from the pluripotent stem cell-derived ECPs that secrete the EVs. In some embodiments of the present disclosure, the composition of isolated extracellular vesicles may not include the ECPs from which they are produced. In some embodiments, the EVs may be present in the medium used to culture the ECPs, or may be free of or substantially free of such medium. In some embodiments, isolated EVs may be provided at a higher concentration than the EVs present in the medium from which they are derived.

[0024] The extracellular vesicles (EVs) of the present disclosure may be obtained from ECPs derived from pluripotent stem cells. Thus, the pluripotent stem cells may be instructed to differentiate into endothelial cell products using suitable techniques known in the art (see, for example, Ana-Mishel Spiroski et al., 2022; MacAskill et al., 2018; McCracken et al., 2019; Zhang et al., 2017).

[0025] Typically, suitable endothelial cells can be characterized as expressing one or more (ideally both) cell surface markers, CD31 and CD144. Preferably, at least 40%, 50%, or 60% of pluripotent stem cell-derived cells co-express CD31 and CD144. Furthermore, typically, less than 10%, 5%, 2.5%, or 1% of ECPs express the pluripotency marker SSEA-3 / TRA-1-60. Generally, any suitable method for isolating, purifying, and / or concentrating EVs may be used, including, for example, magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and / or chromatography. In some embodiments, extracellular vesicles are isolated by centrifugation and / or ultracentrifugation. EVs may also be purified by ultracentrifugation of clarified conditioned medium. They may also be purified by ultracentrifugation through a sucrose cushion. Protocols are described, for example, in Thery et al. Current Protocols in Cell Biol. (2006) 3.22, which is incorporated herein by reference (Thery et al., 2006). In some embodiments, extracellular vesicles are isolated by step size exclusion chromatography. Protocols are described, for example, in Boing et al. Journal of Extracellular Vesicles (2014) 3:23430, which is incorporated herein by reference.For examples of other protocols that can be followed, see also Paganini, C., Capasso Palmiero, U., Pocsfalvi, G., Touzet, N., Bongiovanni, A. and Arosio, P. (2019), Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses. Biotechnol. J., 14: 1800528 and Thanaporn Liangsupree, Evgen Multia, Marja-Liisa Riekkola, Modern isolation and separation techniques for extracellular vesicles, Journal of Chromatography A, Volume 1636, 2021.

[0026] A detailed method for harvesting EVs from ECP derived from pluripotent stem cells comprises, consists essentially of, or consists of a combination of centrifugation, ultrafiltration, and size exclusion chromatography (SEC). A detailed protocol is provided in the Examples. Thus, in a further embodiment, a method for isolating EVs from ECP derived from pluripotent stem cells comprises: a) subjecting a liquid containing EVs resulting from pluripotent stem cell-derived endothelial cell products to centrifugation and / or ultrafiltration to separate EVs from larger molecular weight substances and obtain an EV-enriched solution; b) subjecting the EV concentrate to size exclusion chromatography to obtain a liquid fraction containing isolated EVs; The method includes:

[0027] The liquid containing EVs produced by pluripotent stem cell-derived ECP is conditioned medium, from which the ECP has been removed by centrifugation. (Conditioned medium is medium to which cells have been exposed for a specific period of time, e.g., 12-36 hours, e.g., 24 hours. This medium contains factors secreted by the cells, including extracellular vesicles.) Centrifugation may be performed to remove cells and cell debris from the medium while retaining EVs.

[0028] Ultrafiltration may involve centrifugation through a filter of appropriate molecular weight, for example 100 KDa, to separate lower molecular weight material from the EVs.

[0029] Size exclusion chromatography (SEC) separates molecules based on size by filtration through a resin. The resin consists of spherical beads containing pores of specific sizes. Separation occurs when molecules of different sizes are included in or excluded from the pores within the matrix. Small molecules diffuse into the pores and their flow through the column is retarded according to their size, while larger molecules do not enter the pores and elute in the void volume of the column. As a result, molecules are separated based on their size as they pass through the column, eluting in order of decreasing molecular weight (MW). Operating conditions and gel selection depend on the application and desired resolution. Exemplary SEC media include sephacryl, sephadex, superose, superdex, and sepharose.

[0030] Further provided is an isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or contents thereof prepared according to the methods as described herein for use in promoting angiogenesis and / or neovascularization as described herein.

[0031] A suitable isolated population of pro-angiogenic EVs may be determined as being positive for the markers CD63 and CD81 and negative for the cellular contamination marker calnexin. Additionally, the isolated population of pro-angiogenic EVs is desirably free of non-EV protein contaminants.

[0032] The angiogenic activity of EVs can be determined using known techniques (Irvin et al., 2014). For example, EVs can be applied to tube formation and / or wound healing assays, such as those described herein. Other suitable assays include in vitro proliferation assays, but also ex vivo and in vivo assays, such as aortic ring assays, chorioallantoic membrane assays (CAMs), Matrigel plug assays, fluorescent zebrafish assays, dorsal air sac models, and chamber assays.

[0033] A disadvantage of pro-angiogenic EVs previously known in the art is that they need to be used in high doses to provide their angiogenic effect. Advantageously, the pro-angiogenic and / or pro-angiogenic EVs of the present disclosure have been observed to exert their pro-angiogenic and / or pro-angiogenic effects at much lower doses (typically 50, 25, 10, 5, e.g., as little as 1 EV / cell). Furthermore, increasing the EV dose does not increase the angiogenic effect. This demonstrates that the pro-angiogenic and / or pro-angiogenic EVs of the present disclosure are clearly different from the pro-angiogenic and / or pro-angiogenic EVs previously known in the art.

[0034] EVs can be used immediately or stored for short or long periods, e.g., frozen, before use. Proteinase inhibitors are typically included in the freezing medium because they ensure the integrity of extracellular vesicles during long-term storage. Freezing at -20°C is not preferred because it is associated with increased loss of extracellular vesicle activity. Rapid freezing at -80°C is more preferred because it preserves activity. See, for example, Kidney International (2006) 69, 1471-1476 (Zhou et al., 2006), which is incorporated herein by reference. Additives can be used in the freezing medium to enhance the retention of extracellular vesicle biological activity. These additives are similar to those used for cryopreservation of intact cells and may include, but are not limited to, DMSO, glycerol, and polyethylene glycol.

[0035] The present disclosure further relates to pharmaceutical compositions comprising an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents obtainable or derived from pluripotent stem cell-derived endothelial cell products. Preferably, the pharmaceutical composition comprises an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents obtainable or derived from pluripotent stem cell-derived ECPs, together with a pharmaceutically acceptable carrier or excipient. The composition may include carriers and excipients as are well known to those skilled in the art. Such compositions may find use in promoting angiogenesis and / or neovascularization.

[0036] Isolated populations of pro-angiogenic and / or pro-angiogenic EVs and / or their contents, and compositions as described herein, may find use in the treatment of ischemic tissue damage, cardiovascular disease, e.g., pulmonary hypertension and / or wound healing. As used herein, the term ischemic tissue damage includes disorders and complications, including post-myocardial infarction cardiac injury and progression to heart failure and cardiovascular ischemic disorders, including limb ischemia, mesenteric ischemic disorders, and / or renovascular ischemic disorders.

[0037] Isolated populations of pro-angiogenic and / or pro-angiogenic EVs and / or their contents, as well as compositions as described herein, may find use in therapies where new / improved blood vessel formation is beneficial, for example, in tissue transplantation therapy and / or graft transplantation.

[0038] In further embodiments, the present disclosure extends to an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents as described herein, or to a medical device coated with a composition as described herein. The medical device may be a stent, suture, bandage, dressing, prosthesis, biomaterial, etc., engineered for wound healing, etc.

[0039] The terms "ischemic" and "cardiovascular" disorders and diseases, as used herein, can include interruptions in the blood supply to an organ or tissue. Ischemic events are often the result of blood clots, most frequently occurring in patients with atherosclerotic stenosis during embolic removal from atherosclerotic lesions. The narrowing, or stenosis, or blockage of an artery or other blood vessel caused by this obstruction can result in numerous adverse conditions, many of which have serious consequences for the subject. As referred to herein, ischemic and cardiovascular disorders / diseases include, but are not limited to, stroke / transient ischemic attack or cerebrovascular accident, myocardial infarction, myocardial ischemia (angina pectoris), any myocardial disease complicated by myocardial ischemia (e.g., symptomatic aortic stenosis, HOCM), cerebral hemorrhage, peripheral (unstable) angina, peripheral atherosclerotic vascular disease, and other major abnormalities occurring in blood vessels. The term "abnormalities occurring in blood vessels" includes reference to peripheral vascular disease, as well as coronary and cerebrovascular events. The term "ischemic cardiovascular or cerebrovascular event" is often the acute stage of medical conditions broadly encompassed by the terms "cardiovascular, cerebrovascular, and peripheral arterial disease" (collectively referred to herein as "cardiovascular disease"). Such diseases include cerebrovascular and also peripheral arterial disease.

[0040] The term "ischemia," as used herein, refers to an absolute or relative deficiency in blood supply to an organ, body part, or tissue, or an inadequate flow of blood. Relative deficiency refers to a discrepancy between blood supply (oxygen delivery) and blood demand (oxygen consumption by tissues). Restriction of blood supply, generally due to factors within the blood vessels, is most frequently, but not exclusively, caused by narrowing or blockage of blood vessels due to thromboembolism (blood clots) or atherosclerosis (lipid-laden plaques that obstruct the lumen of arteries). Ischemia results in tissue damage or dysfunction. Ischemia of the myocardium results in angina pectoris and is referred to herein as ischemic heart disease.

[0041] The term "cardiovascular disease" (CVD) generally refers to many diseases that affect the heart and circulatory system, including aneurysms; angina pectoris; arrhythmias; atherosclerosis; myocardial disease; cerebrovascular accidents (stroke); cerebrovascular disease; congenital heart disease; congestive heart failure; coronary artery disease (CAD), also known as coronary heart disease (CHD), ischemic heart disease or atherosclerotic heart disease; dilated cardiomyopathy; diastolic dysfunction; endocarditis; heart failure; hypertension (high blood pressure); hypertrophic cardiomyopathy; myocardial infarction (heart attack); myocarditis; peripheral vascular disease; small vessel disease; and venous thromboembolism. As used herein, the term "cardiovascular disease" also refers to ischemia; arterial damage (damage to the endothelial cell lineage) due to physical injury (endarterectomy, balloon angiogenesis) or as a result of chronic injury (including atherosclerosis); myocardial injury (myocardial necrosis); and myonecrosis. Generally, as used herein, any physiological or pathophysiological condition that induces an angiogenic or neovascularization response is included within the term "cardiovascular disease."

[0042] Unless otherwise specified, "a" or "an" means "one or more."

[0043] Unless otherwise defined, all technical and scientific terms used herein should be construed to have the same meaning as commonly understood by one of ordinary skill in the art.

[0044] As used herein, the term "subject" (also referred to herein as "patient") includes warm-blooded animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In even more preferred embodiments, the subject is a human.

[0045] As used herein, the terms "treating," "treat," or "treatment" include reducing, alleviating, or eliminating at least one symptom of a disease or condition.

[0046] As used herein, the terms "preventing," "prevent" or "prevention" include stopping or impeding the appearance or presence of at least one symptom of a disease or condition. Alternatively, the terms "preventing," "prevent" or "prevention" may include stopping or impeding the appearance or presence of at least one symptom of a disease or condition.

[0047] The present disclosure is based, at least in part, on the discovery of a cohort of miRNAs present in EVs. Without wishing to be bound by theory, the pro-angiogenic and / or pro-angiogenic effects of EVs disclosed herein are due, at least in part, to the miRNA content of EVs. Each member (or combinations thereof) of the cohort of miRNAs regulates cell proliferation (and migration), influences (or regulates) vascular remodeling, and represents a target for treating a variety of vascular complications, injuries, diseases, and disorders, diseases, syndromes, and / or conditions affecting (e.g.,) human or animal blood vessels and / or the vasculature.

[0048] Within the context of the present disclosure, any of the disclosed miRNAs may be targeted to, for example, regulate the expression level of pro-angiogenic and / or pro-angiogenic gene(s) in a cell. For example, the expression level of any of the disclosed miRNAs may be increased or decreased as needed. Without wishing to be bound by theory, the expression level of any of the disclosed miRNAs is associated with potentially beneficial therapeutic effect(s). For example, and again without wishing to be bound by theory, targeting one or more of the disclosed miRNAs can promote angiogenesis and / or neovascularization. This may help modify vascular wall architecture / structure after injury. As such, the present disclosure provides a cohort of miRNAs, each of which can be targeted as a means of regulating (e.g., promoting) angiogenesis and / or neovascularization, and / or for the treatment or prevention of (vascular) complications, (vascular) injuries, and / or cardiovascular disease.

[0049] The present disclosure provides compounds and compositions for a variety of therapeutic uses, uses of the compounds and compositions for the manufacture of therapeutically effective medicaments, and methods of treating a variety of diseases, disorders, and conditions. Within the context of the present disclosure, such compounds and compositions include EVs and / or their contents that contain one or more miRNAs disclosed herein.

[0050] One particular application of the compounds / compositions, medicaments, uses and methods described herein may, for example, be in the treatment or prevention of cardiovascular diseases, such as coronary heart disease, ischemic tissue damage and pulmonary hypertension. In the context of the present invention, the terms "comprise" and "comprising" include embodiments in which the invention "consists essentially of" or "consists of" the relevant feature.

[0051] In one explanation, there is provided an isolated EV or an isolated population of EVs and / or their contents obtainable from pluripotent stem cell-derived ECP for use in therapy, said EVs comprising one or more pro-angiogenic and / or pro-angiogenic properties, e.g., including any one or more of the miRNAs disclosed herein. The isolated EVs or isolated population of EVs of the present disclosure may be for use in treating or preventing cardiovascular disease in a subject in need thereof.

[0052] The disclosed EVs or isolated EVs (or isolated populations of EVs) and / or their contents can comprise any one or more of the miRNAs disclosed herein. For example, the disclosed EVs or isolated EVs (or isolated populations of EVs) and / or their contents can comprise at least one, two, or more of the miRNAs disclosed herein.

[0053] Those skilled in the art are familiar with the term "microRNA" (or "miR"). MicroRNAs are small non-coding RNA molecules that affect gene expression regulation. They are produced either from gene sequences or intron / exon sequences; many are encoded by intergenic sequences.

[0054] Within the context of the present disclosure, the term microRNA or "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-5p; (iv) miR-92a-3p (v) miR-7-1-5p; (vi) miR-196b-5p; (vii) miR-302b-3p; (viii) miR-222-3p; (ix)miR-6087; (x)miR-10a-5p; (xi) miR-99b-5p; (xii) miR-184; (xiii) miR-302a-3p; (xiv)miR-423-3p; (xv)miR-3184-5p; (xvi)miR-7-2-5p; (xvii)miR-3529-3p; (xviii) miR-92b-3p; (xix)miR-342-3p; (xx)miR-23a-3p; (xxi)miR-302d-3p; (xxii)miR-483-3p; (xxiii)miR-302c-3p; (xxiv)miR-151a-5p; (xxv)miR-26a-5p; (xxvi)miR-149-5p; (xxvii)miR-miR-27b-3p; (xxviii)miR-302a-5p.

[0055] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p (iv) miR-7-1-5p; (v) miR-196b-5p; (vi) miR-302b-3p; (vii) miR-222-3p; (viii) miR-6087; (ix) miR-10a-5p; (x)miR-99b-5p; (xi) miR-184; (xii) miR-302a-3p; (xiii) miR-423-3p; (xiv)miR-3184-5p; (xv)miR-7-2-5p; (xvi)miR-3529-3p; (xvii) miR-92b-3p; (xviii)miR-342-3p; (xix)miR-23a-3p; (xx)miR-302d-3p.

[0056] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3p; (v) miR-222-3p; (vi) miR-92b-3p; (vii) miR-196-5p; (viii) miR-10a-5p; (viv)miR-483-3p; (x)miR-302a-3p; (xi) miR-302c-3p; (xii) miR-7-1-5p; (xiii) miR-99b-5p; (xiv)miR-151a-5p; (xv)miR-26a-5p; (xvi)miR-149-5p; (xvii) miR-23a-3p; (xviii) miR-27b-3p; (xiv)miR-302d-3p; (xx)miR-302a-5p.

[0057] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; (v) miR-196b-5p; (vi) miR-302b-3p; (vii) miR-222-3p; (viii) miR-10a-5p; (viv)miR-99b-5p; (x)miR-302a-3p; (xi) miR-92b-3p; (xii) miR-23a-3p; (xiii) miR-302d-3p.

[0058] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p (iv) miR-7-1-5p; (v) miR-196b-5p; (vi) miR-302b-3p; (vii) miR-126-3p; (viii) miR-222-3p.

[0059] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; and (v) miR-196b-5p.

[0060] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3-p; and (v) miR-222-5p.

[0061] In one explanation, the term "miR" may include any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii) miR-21-5p; and (iii) miR-92a-3p.

[0062] The miRs described herein may each individually have a pro-angiogenic and / or pro-angiogenic effect on a target cell or tissue. Combinations of any two or more of the miRs detailed herein may produce additive or synergistic effects on a target cell or tissue to promote angiogenesis and / or neovascularization.

[0063] Without wishing to be bound by theory, a combination of two, three, four, five, ten, fifteen or twenty of the miRs detailed herein may constitute at least 50%, 60%, 65%, 70% or 75% of the total miR content of the disclosed EVs or isolated EVs (or isolated populations of EVs).

[0064] In one explanation, the miR content of the disclosed EVs or isolated EVs (or isolated populations of EVs) may include combinations of two, three, four, or five or more of the miRs disclosed herein. Without wishing to be bound by theory, the disclosed EVs or isolated EVs (or isolated populations of EVs) that include combinations of two or more of the miRs disclosed herein may produce enhanced pro-angiogenic and / or pro-angiogenic effects on target cells or tissues.

[0065] Of the miRs listed above, the following specific combinations of miRs, or EVs / isolated EVs containing such miRs, are now known to be involved in angiogenesis and / or neovascularization: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; and (v) miR-196b-5p; or (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3-p; and (v) miR-222-5p; or (i) miR-126-3p; (ii) miR-21-5p; and (iii) miR-92a-3p.

[0066] As such, by targeting one or more or combinations of these miRs or by using EVs / isolated EVs containing such miRs, it may be possible to treat or prevent conditions or diseases in which an enhanced pro-angiogenic and / or pro-neovascular effect on target cells or tissues may be necessary or beneficial.

[0067] The present disclosure further provides modulators of any of the miRs described herein.

[0068] An miR-modulating agent of the present disclosure can be any molecule or compound that can either increase or inhibit (decrease) the expression of a particular miR (e.g., one or more miRs described herein).

[0069] As such, an "miR-modulating agent" is any compound or molecule that increases or inhibits (decreases) the expression of any one or more of the miRs listed herein (or any combination of the miRs listed herein), including, for example, any of those listed as (i) through (xxviii) above (and any combination thereof).

[0070] Accordingly, the present invention provides a miR modulator (e.g., a modulator that increases expression) of any one or more of the miRs listed herein (or any combination of the miRs listed herein), including, for example, any of those listed as (i) through (xxviii) above (and any combination thereof), that: For use in medicine or as a drug; Treating or preventing cardiovascular disease; Treating or preventing coronary heart disease; Treating or preventing ischemic tissue damage; Treating or preventing pulmonary hypertension; modulating or promoting angiogenesis and / or neovascularization; and / or Regulating or promoting wound healing The present invention provides the modulator for any one or more of the therapeutic applications described herein, including the application of

[0071] In one aspect, the present disclosure provides: In medicines or as drugs; in treating or preventing cardiovascular disease; in treating or preventing coronary heart disease; in treating or preventing ischemic tissue damage; in treating or preventing pulmonary hypertension; in regulating or promoting angiogenesis and / or neovascularization; and / or In regulating or promoting wound healing miR-modulating agents for use are provided.

[0072] In another aspect, the present disclosure provides: Treating or preventing cardiovascular disease; Treating or preventing coronary heart disease; Treating or preventing ischemic tissue damage; Treating or preventing pulmonary hypertension; modulating or promoting angiogenesis and / or neovascularization; and / or Regulating or promoting wound healing The present invention provides a method comprising administering to a subject in need thereof an miR-modulating agent. The modulating agent may be administered in a therapeutically effective or modulating amount. The subject in need thereof may be a human or animal subject.

[0073] In another aspect, the present disclosure provides: Treating or preventing cardiovascular disease; Treating or preventing coronary heart disease; Treating or preventing ischemic tissue damage; Treating or preventing pulmonary hypertension; modulating or promoting angiogenesis and / or neovascularization; and / or Regulating or promoting wound healing The present invention provides the use of a miR-modulating agent for the manufacture of a medicament for use in treating a disease.

[0074] Thus, the present disclosure: Treating or preventing cardiovascular disease; Treating or preventing coronary heart disease; Treating or preventing ischemic tissue damage; Treating or preventing pulmonary hypertension; modulating or promoting angiogenesis and / or neovascularization; and / or Regulating or promoting wound healing The following modulators (e.g., modulators that increase expression) are provided for use in: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p (iv) miR-7-1-5p; (v) miR-196b-5p; (vi) miR-302b-3p; (vii) miR-126-3p; (viii) miR-222-3p one or more of; or: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; and (v) miR-196b-5p; one or more of; or: (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3-p; and (v)miR-222-5p one or more of; or: (i) miR-126-3p; (ii) miR-21-5p; and (iii) miR-92a-3p One or more of.

[0075] The degree of modulation affected by a miR-modulating agent of the invention may be assessed in comparison to a "normal" or "control" level of miR expression and / or miR target gene expression, such as might occur in normal / healthy tissue not exhibiting pathology associated with a cardiovascular disease or condition.

[0076] An miR-modulating agent for any of the uses or methods described herein can take the form of an inhibitor of one or more of the miRs described herein. The term "miR inhibitor" can include, for example, a compound or molecule that inhibits or reduces the expression, function, and / or activity of a miR, including one or more of the miRs described herein.

[0077] Modulating agents of the present disclosure may include miR promoters, which are molecules that increase the expression (in a cell) of an appropriate miR. The term "miR promoter" may include, for example, compounds or molecules that increase the expression, function, and / or activity of a miR, including one or more of the miRs described herein.

[0078] An miR promoter may include, for example, an miR mimic, which is a nucleic acid encoding a miR suitable for expression in a cell. One skilled in the art will understand that when introduced into a cell, a nucleic acid encoding a particular miR provides additional copies of that miR, i.e., additional copies that supplement any natural copy(s) expressed by the cell, such that the net result is overexpression of that miR in the cell.

[0079] A suitable miR mimic can comprise a double-stranded RNA molecule that mimics the mature miR duplex.

[0080] Suitable miR-encoding nucleic acids may include stem-loop miRNAs.

[0081] The nucleic acid can encode any of the miRs described herein (including any of the miRs listed herein as (i) through (xxviii)).

[0082] For expression, a nucleic acid encoding an miR may further comprise (or be operably linked to) a promoter element and a polyA element. The promoter element and polyA element may be "adjacent" to the miR-encoding nucleic acid sequence.

[0083] The nucleic acid may be provided in the form of a vector for delivery to a cell.

[0084] The vector may comprise a viral vector.

[0085] The vector may comprise an adenoviral vector, such as HAdV5 or an adeno-associated virus (e.g., AAV1, AAV2, AAV3, AAV4 or AAV5), or a lentivirus.

[0086] The miR promoter, eg, a nucleic acid encoding a miR, can be packaged or included within a viral, adenoviral, or AAV5 vector.

[0087] Suitable miR inhibitors for use in the present disclosure may include, for example, organic / inorganic small molecules, proteins, peptides, amino acids, nucleic acids (including RNA, DNA and / or synthetic or peptide-based nucleic acids, including PNAs), carbohydrates, lipids, antibodies (including antigen-binding fragments thereof), and the like.

[0088] Any miR modulating agent of the present disclosure can be administered directly to the vessel wall to be treated (e.g., a vessel wall that has been repaired through surgery and / or shows signs of disease and / or injury or damage). The miR modulating agent of the present disclosure can also be packaged and administered in a vector, e.g., a viral (adenoviral) vector.

[0089] The present disclosure provides adenoviral vectors that include sequences for expression in a cell of one or more of the following miRs: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-5p; (iv) miR-92a-3p; (v) miR-7-1-5p; (vi) miR-196b-5p; (vii) miR-302b-3p; (viii) miR-222-3p; (ix)miR-6087; (x)miR-10a-5p; (xi) miR-99b-5p; (xii) miR-184; (xiii) miR-302a-3p; (xiv)miR-423-3p; (xv)miR-3184-5p; (xvi)miR-7-2-5p; (xvii)miR-3529-3p; (xviii) miR-92b-3p; (xix)miR-342-3p; (xx)miR-23a-3p; (xxi)miR-302d-3p; (xxii)miR-483-3p; (xxiii)miR-302c-3p; (xxiv)miR-151a-5p; (xxv)miR-26a-5p; (xxvi)miR-149-5p; (xxvii)miR-miR-27b-3p; (xxviii)miR-302a-5p.

[0090] The present disclosure provides compositions comprising a miRNA modulating agent of the present disclosure and one or more excipients.

[0091] The present disclosure further provides pharmaceutical compositions comprising a miR modulating agent of the present disclosure and one or more pharmaceutically acceptable excipients.

[0092] Compositions or pharmaceutical compositions of the disclosure may include miR-modulating agents that are mimetics of one or more of the following miRs: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-5p; (iv) miR-92a-3p; (v) miR-7-1-5p; (vi) miR-196b-5p; (vii) miR-302b-3p; (viii) miR-222-3p; (ix)miR-6087; (x)miR-10a-5p; (xi) miR-99b-5p; (xii) miR-184; (xiii) miR-302a-3p; (xiv)miR-423-3p; (xv)miR-3184-5p; (xvi)miR-7-2-5p; (xvii)miR-3529-3p; (xviii) miR-92b-3p; (xix)miR-342-3p; (xx)miR-23a-3p; (xxi)miR-302d-3p; (xxii)miR-483-3p; (xxiii)miR-302c-3p; (xxiv)miR-151a-5p; (xxv)miR-26a-5p; (xxvi)miR-149-5p; (xxvii)miR-miR-27b-3p; (xxviii)miR-302a-5p.

[0093] The composition or pharmaceutical composition comprises: Treating or preventing cardiovascular disease; Treating or preventing coronary heart disease; Treating or preventing ischemic tissue damage; Treating or preventing pulmonary hypertension; modulating or promoting angiogenesis and / or neovascularization; and / or Regulating or promoting wound healing It may be for (i) use in or (ii) use in a method.

[0094] Where the composition or pharmaceutical composition is for use in therapy, the composition or pharmaceutical composition may be administered to a subject (as defined herein) in need thereof.

[0095] Additionally, the composition or pharmaceutical composition comprises: Treating or preventing cardiovascular disease; Treating or preventing coronary heart disease; Treating or preventing ischemic tissue damage; Treating or preventing pulmonary hypertension; modulating or promoting angiogenesis and / or neovascularization; and / or Regulating or promoting wound healing The compounds may be used in the manufacture of pharmaceuticals for

[0096] In a further embodiment, the present disclosure also provides a method for identifying pro-angiogenic and / or pro-angiogenic EVs or their contents, comprising detecting the presence and / or measuring the expression level of one or more miRNAs disclosed herein. Detecting the presence or expression of any one of the miRs disclosed herein in an EV indicates that the EV may be pro-angiogenic or pro-angiogenic.

[0097] In a preferred example, the method comprises detecting the presence of and / or measuring the expression level of any one or more miRNAs selected from the group consisting of or consisting essentially of: (i) miR-126-3p; (ii)miR-21-5p; (iii)miR-92a-5p; (iv)miR-92a-3p; (v)miR-7-1-5p; (vi)miR-196b-5p; (vii)miR-302b-3p; (viii)miR-222-3p; (ix)miR-6087; (x)miR-10a-5p; (xi)miR-99b-5p; (xii)miR-184; (xiii)miR-302a-3p; (xiv)miR-423-3p; (xv)miR-3184-5p; (xvi)miR-7-2-5p; (xvii)miR-3529-3p; (xviii)miR-92b-3p; (xix)miR-342-3p; (xx)miR-23a-3p; (xxi)miR-302d-3p; (xxii)miR-483-3p; (xxiii)miR-302c-3p; (xxiv)miR-151a-5p; (xxv)miR-26a-5p; (xxvi)miR-149-5p; (xxvii)miR-miR-27b-3p; (xxviii)miR-302a-5p; または: (i)miR-126-3p; (ii)miR-21-5p; (iii)miR-92a-3p (iv)miR-7-1-5p; (v)miR-196b-5p; (vi)miR-302b-3p; (vii)miR-126-3p; (viii) miR-222-3p; or: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; and (v) miR-196b-5p; or: (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3-p; and (v) miR-222-5p; or: (i) miR-126-3p; (ii) miR-21-5p; and (iii) miR-92a-3p.

[0098] Without wishing to be bound by theory, the miRs disclosed herein may target any one or more of the following genes to induce pro-angiogenic and / or pro-angiogenic effects: [Table 1] TIFF2026501121000002.tif62164 [Table 2] TIFF2026501121000004.tif244164TIFF2026501121000005.tif248164TIFF2026501121000006.tif57165 [Table 3] TIFF2026501121000008.tif248164TIFF2026501121000009.tif249164TIFF2026501121000010.tif122164

[0099] One or more of the disclosed genes may each represent a target for regulating cell proliferation (and migration), affecting (or modulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and disorders, diseases, syndromes, and / or conditions affecting the blood vessels and / or vasculature of (e.g.) the human or animal body. Thus, the expression, function, and / or activity of any one or more of the genes may be modulated individually or collectively to regulate cell proliferation (and migration), affecting (or modulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and treating or preventing disorders, diseases, syndromes, and / or conditions affecting the blood vessels and / or vasculature of (e.g.) the human or animal body. [Brief explanation of the drawings]

[0100] The present disclosure will now be further described, by way of example and with reference to the figures. [Figure 1]Isolation and characterization of extracellular vesicles (EVs) from human embryonic stem cell-derived endothelial cell product (hESC-ECP) conditioned medium. A. Schematic presentation of human embryonic stem cell-derived endothelial cell product (hESC-ECP) differentiation and extracellular vesicle (EV) secretion from cells at the mesoderm (day 4) and endothelial enrichment (day 8) stages. B. Workflow for particle isolation from hESC-ECP conditioned medium by a combination of ultrafiltration and size-exclusion chromatography (SEC). Generated with BioRender.com. C. Representative graph from nanoparticle tracking analysis (NTA) showing the size distribution of EVs in pooled fractions 5 and 6. D. EV surface marker characterization of pooled fractions 5 and 6 by Western blot. Cell lysates were used as positive controls. Calnexin detection appears as a 90 kDa band, CD63 detection appears at 30-65 kDa, and CD81 detection appears at 22-26 kDa. E. Transmission electron microscopy (TEM) imaging of particles in pooled fractions 5 and 6. Arrows indicate EVs. Scale bar = 100 nm (left photo) and scale bar = 1 µm (right photo). The scatter plot (bottom) shows the EV size distribution and circularity ratio (scale 0-1) as determined by 40 total TEM images using the "TEM Exosome Analyzer" tool. Each point (pink) corresponds to one EV. Error bars correspond to the median with interquartile range. [Figure 2]Human embryonic stem cell-derived endothelial cell product-derived extracellular vesicles (hESC-eEVs) induce tube formation at low concentrations. A. Tube formation assay on human cardiac microvascular endothelial cells (HCMECs) using increasing concentrations of human umbilical vein endothelial cell (HUVEC) hypoxic EVs (dose range: 1–10 EVs / cell, n=3). Cells cultured in basal medium and treated with sterile-filtered PBS (vehicle control) served as the negative control (GF-). Cells in fully supplemented medium served as the positive control (GF+). The graph (right) shows the number of meshes and total tube length formed in 4 hours. Images were analyzed using the "Angiogenesis Analyzer" tool in ImageJ. Control samples (GF- and GF+) are indicated by black circles, and HUVEC hypoxic EV-treated samples (HUVEC hypEVs) are indicated by purple triangles. Statistical significance (p values) was determined by one-way analysis of variance with Dunnett's multiple comparison test. Error bars indicate SD. Scale bar = 1 mm. B. Quantification of tube formation assay on HCMECs (n = 4) using increasing concentrations of hESC-eEVs (dose range: 1–10 EVs / cell). Cells cultured in basal medium and treated with sterile 0.1 μm-filtered PBS (vehicle control) served as a negative control (GF-). Cells in fully supplemented medium (GF+) or cells treated with 10 HUVEC hypoxic EVs / cell served as positive controls. Graphs show the number of meshes and total tube length formed over 4 hours. Data were analyzed using the "Angiogenesis Analyzer" tool. Control samples (GF- and GF+) are shown with black circles, HUVEC hypoxic EV-treated samples (HUVEC hyp EVs) are shown with purple triangles, and hESC-eEV-treated samples are shown with green circles. Statistical significance (shown as p-values) was determined by one-way ANOVA with Dunnett's multiple comparison test. Error bars indicate SD. C. Calcein AM staining of tube formation assay on HCMECs using increasing concentrations of hESC-eEVs (dose range: 1–10 EVs / cell, n=4). Cells cultured in basal medium and treated with sterile 0.1 μm-filtered PBS (vehicle control) served as a negative control (GF-).Cells in fully supplemented medium (GF+) or cells treated with 10 HUVEC hypoxic EVs / cell served as positive controls. Scale bar = 500 µm. D. Tube formation assay on HCMECs treated with human embryonic stem cell-derived mesodermal cell-product extracellular vesicles (hESC-mEVs) (dose range: 1–10 EVs / cell, n = 3). Cells cultured in basal medium and treated with sterile-filtered PBS (vehicle control) served as negative controls (GF-). Cells in fully supplemented medium (GF+) or cells treated with 1 hESC-eEV / cell served as positive controls. Images were analyzed using the "Angiogenesis Analyzer" tool in ImageJ. Control samples (GF- and GF+) are indicated by black circles, hESC-eEV-treated samples are indicated by green circles, and hESC-mEV-treated samples are indicated by red triangles. Statistical significance (shown as p-values) was determined by one-way analysis of variance with Dunnett's multiple comparison test. The graph (right) shows the number of meshes and total tube length formed over 4 hours. Error bars indicate SD. Scale bar = 1 mm. E. Schematic presentation of samples collected at different stages of particle isolation from hESC-EC cell culture medium. Generated with BioRender.com. F. Tube formation assay using samples collected at different stages of particle isolation from hESC-EC cell culture medium. Cells cultured in basal medium and treated with PBS (vehicle control) served as negative controls (GF-). Cells in fully supplemented medium (GF+) or treated with human recombinant VEGFA165 or cells cultured in basal medium and treated with 1 hESC-eEV / cell served as positive controls. [Figure 3]Human embryonic stem cell-derived endothelial cell product-derived extracellular vesicles (hESC-eEVs) promote endothelial cell wound healing at low concentrations. Wound healing assays were performed on human cardiac microvascular endothelial cells (HCMECs) treated with increasing concentrations of human embryonic stem cell-derived endothelial cell product-derived extracellular vesicles (hESC-eEVs) (dose range: 1–10 EVs / cell) or 10 human umbilical vein endothelial cell (HUVEC) hypoxic EVs / cell (n=4). Cells cultured in basal medium and treated with sterile-filtered PBS (vehicle control) served as a negative control (GF-). Cells in fully supplemented medium (GF+) served as a positive control. Graphs (bottom) show the percentage of wound closure 6, 12, and 24 hours after wound induction. Data were analyzed using the "MRI Wound Healing" tool. Control samples (GF- and GF+) are shown as black circles, HUVEC hypoxic EV-treated samples (HUVEC hyp EV) are shown as purple triangles, and hESC-eEV-treated samples are shown as green circles. Statistical significance (shown as p-values) was determined by one-way ANOVA with Dunnett's multiple comparison test. Error bars indicate SD. Scale bar = 100 μm. [Figure 4-1]Extracellular vesicles derived from human embryonic stem cell-derived endothelial cell products (hESC-ECPs) are enriched in pro-angiogenic miRNAs. A. Graph showing the number of mapped reads per million of small RNA biotypes in EV samples and human embryonic stem cell-derived endothelial cell products (hESC-ECPs) (n=3), normalized to the total number of small RNA reads (total RPMMs). B. Plot showing the percentage of reads accounted for by the top 20 miRNAs in each EV and cellular RNA sample. In all datasets, the top 20 miRNAs represent 68–86% of the total miRNA reads. The top five miRNAs in each group are highlighted. In the stacked bars, each miRNA is depicted in a different color to highlight the differences and similarities between the top 20 miRNAs in each group. C. Plot showing the RPMMs of the top 20 miRNAs in each group (representing 68–86% of total RPMMs) and their role in angiogenesis. The top five miRNAs in each group are separated by a vertical red dotted line. Angiogenic miRNAs are highlighted in green, and anti-angiogenic miRNAs are highlighted in red. Literature search was performed on PubMed in July 2022. [Figure 4-2]Extracellular vesicles derived from human embryonic stem cell-derived endothelial cell products (hESC-ECPs) are enriched in pro-angiogenic miRNAs. A. Graph showing the number of mapped reads per million of small RNA biotypes in EV samples and human embryonic stem cell-derived endothelial cell products (hESC-ECPs) (n=3), normalized to the total number of small RNA reads (total RPMMs). B. Plot showing the percentage of reads accounted for by the top 20 miRNAs in each EV and cellular RNA sample. In all datasets, the top 20 miRNAs represent 68–86% of the total miRNA reads. The top five miRNAs in each group are highlighted. In the stacked bars, each miRNA is depicted in a different color to highlight the differences and similarities between the top 20 miRNAs in each group. C. Plot showing the RPMMs of the top 20 miRNAs in each group (representing 68–86% of total RPMMs) and their role in angiogenesis. The top five miRNAs in each group are separated by a vertical red dotted line. Angiogenic miRNAs are highlighted in green, and anti-angiogenic miRNAs are highlighted in red. Literature search was performed on PubMed in July 2022. [Figure 4-3]Extracellular vesicles derived from human embryonic stem cell-derived endothelial cell products (hESC-ECPs) are enriched in pro-angiogenic miRNAs. A. Graph showing the number of mapped reads per million of small RNA biotypes in EV samples and human embryonic stem cell-derived endothelial cell products (hESC-ECPs) (n=3), normalized to the total number of small RNA reads (total RPMMs). B. Plot showing the percentage of reads accounted for by the top 20 miRNAs in each EV and cellular RNA sample. In all datasets, the top 20 miRNAs represent 68–86% of the total miRNA reads. The top five miRNAs in each group are highlighted. In the stacked bars, each miRNA is depicted in a different color to highlight the differences and similarities between the top 20 miRNAs in each group. C. Plot showing the RPMMs of the top 20 miRNAs in each group (representing 68–86% of total RPMMs) and their role in angiogenesis. The top five miRNAs in each group are separated by a vertical red dotted line. Angiogenic miRNAs are highlighted in green, and anti-angiogenic miRNAs are highlighted in red. Literature search was performed on PubMed in July 2022. [Figure 4-4]Extracellular vesicles derived from human embryonic stem cell-derived endothelial cell products (hESC-ECPs) are enriched in pro-angiogenic miRNAs. A. Graph showing the number of mapped reads per million of small RNA biotypes in EV samples and human embryonic stem cell-derived endothelial cell products (hESC-ECPs) (n=3), normalized to the total number of small RNA reads (total RPMMs). B. Plot showing the percentage of reads accounted for by the top 20 miRNAs in each EV and cellular RNA sample. In all datasets, the top 20 miRNAs represent 68–86% of the total miRNA reads. The top five miRNAs in each group are highlighted. In the stacked bars, each miRNA is depicted in a different color to highlight the differences and similarities between the top 20 miRNAs in each group. C. Plot showing the RPMMs of the top 20 miRNAs in each group (representing 68–86% of total RPMMs) and their role in angiogenesis. The top five miRNAs in each group are separated by a vertical red dotted line. Angiogenic miRNAs are highlighted in green, and anti-angiogenic miRNAs are highlighted in red. Literature search was performed on PubMed in July 2022. [Figure 4-5] Supplemental Figure 1. Representative flow cytometry analysis of hESC-ECPs. Cells were stained for endothelial (CD31 and CD144) (right panel), pluripotency markers (TRA-1-60 and SSEA-3) (left panel), and their corresponding isotype controls. [Figure 4-6] Supplemental Figure 2. The combination of ultrafiltration and SEC results in excellent separation of particle- and protein-containing fractions. The graph shows the particle and protein concentrations of the resulting hESC-ECP conditioned medium fractions after SEC, as determined by NTA and spectrophotometry, respectively. Particles elute in fractions 5 and 6, while proteins elute in fractions 12 and 13. [Figure 4-7] Supplementary Figure 3. Small RNA sequencing revealed a diverse composition of small RNA classes in EV samples. Venn diagram showing unique and common small RNA molecules present in different EV samples. Molecules with an average RPMM total count <10 were excluded from the analysis. [Figure 4-8]Supplementary Figure 3. Small RNA sequencing revealed a diverse composition of small RNA classes in EV samples. Venn diagram showing unique and common small RNA molecules present in different EV samples. Molecules with an average RPMM total count <10 were excluded from the analysis. [Figure 4-9] Supplemental Figure 4. Principal component analysis (PCA) of total reads from small RNA sequencing datasets. [Figure 4-10] Supplemental Figure 5. Small RNA sequencing read characteristics. A. RNA input reads (n=3) used for alignment after UniVec / rRNA / low-quality reads were removed. B. Stacked bar plot showing read composition per sample. Reads are normalized to the total number of reads (total RPMMs). C. Correlation between the percentage of unmapped reads and the number of particles used for small RNA sequencing of EV samples. [Figure 4-11] Supplemental Figure 5. Small RNA sequencing read characteristics. A. RNA input reads (n=3) used for alignment after UniVec / rRNA / low-quality reads were removed. B. Stacked bar plot showing read composition per sample. Reads are normalized to the total number of reads (total RPMMs). C. Correlation between the percentage of unmapped reads and the number of particles used for small RNA sequencing of EV samples. [Figure 5]Extracellular vesicles derived from RC11 human embryonic stem cell-derived endothelial cell product (RC11-eEV) induce endothelial cell (EC) tube formation at low concentrations. Tube formation assay was performed on human cardiac microvascular endothelial cells (HCMEC) treated with two EV doses: 1 and 10 EVs / cell (n=3). Cells cultured in basal medium and treated with sterile-filtered PBS (vehicle control) served as a negative control (GF-). Cells in fully supplemented medium (GF+) or treated with human embryonic stem cell-derived endothelial cell product-derived extracellular vesicles (hESC-eEV) served as positive controls. Quantification of the number of meshes formed and the total length of tubes formed over 4 hours was performed. Images were analyzed using the "Angiogenesis Analyzer" tool in ImageJ. Control samples (GF+ and GF-) are indicated by black circles, hESC-eEV-treated samples are indicated by green circles, and RC11-eEV-treated samples are indicated by blue circles. Statistical significance (shown as p-values) was determined by one-way analysis of variance with Dunnett's multiple comparison test. Error bars indicate standard deviation. Scale bar = 1 mm. [Figure 6] Extracellular vesicles derived from RC11 human embryonic stem cell-derived endothelial cell product (RC11eEV) promote endothelial cell (EC) wound healing at low concentrations. Wound healing assay (n=3) on EV-treated human cardiac microvascular endothelial cells (HCMEC). Cells cultured in basal medium and treated with PBS (vehicle control) served as a negative control (GF-). Cells in fully supplemented medium (GF+) or cells treated with human embryonic stem cell-derived endothelial cell product-derived extracellular vesicles (hESC-eEV) served as positive controls. Quantification of wound closure percentage 6, 12, and 24 hours after wound induction. Control samples (GF+ and GF-) are shown with black circles, hESC-eEV-treated samples are shown with green circles, and RC11-eEV-treated samples are shown with blue circles. Statistical significance (p values ​​shown) was determined by one-way analysis of variance with Dunnett's multiple comparison test. Error bars indicate SD. Scale bar = 100 μm. [Figure 7] The top 20 microRNA cargoes of RC11-eEV and hESC-eEV show 65% overlap. DETAILED DESCRIPTION OF THE INVENTION

[0101] material and method hESC-ECP differentiation The H9 hESC line was differentiated into endothelial cell products using our previously reported protocol (MacAskill et al., 2018). Human ESC lines were used in accordance with UK Stem Cell Bank Steering Committee guidelines (project approvals SCS11-51 and SCSC17-26). Briefly, on day 0 (d0), hESCs were plated on a fibronectin matrix. On day 1, lateral mesoderm was induced with GSK3 inhibitor (CHIR99021) (7 μM) and BMP4 (25 ng / ml) in N2B27 / Neurobasal / DMEM:F12 medium. This was followed by endothelial induction on day 4 with forskolin (2 μM) and vascular endothelial growth factor (VEGF) (200 ng / ml) in StemPro34 medium. Finally, cells were replated without matrix and cultured until day 8. For EV isolation, the medium was replaced with EV-free medium on day 7, and conditioned medium was collected 24 hours later. To prepare EV-free medium, human serum was ultrafiltered using an Amicon-Ultra15 centrifugal filter device with a 100 kDa cutoff (Merck Millipore) after centrifugation at 3,000 x g for 55 minutes (4°C) and supplemented to the cell culture medium.

[0102] hESC-ECP flow cytometry Antibodies were used to stain cells on day 8 to determine the proportion of pluripotent (SSEA-4+ / TRA-181+) and endothelial (CD31+ / CD144+) cells within the population. Flow cytometry was performed on a BD LDR Fortessa system (Becton) or an Attune NxT system (Thermo Fisher Scientific), and data were analyzed using FlowJo software (FlowJo LLC, Ashland, USA).

[0103] EV isolation To isolate EVs, conditioned medium was obtained from cells at 70–90% confluence and centrifuged at 3,000 x g for 15 min (4°C) to remove cellular debris. The supernatant was collected and concentrated to 1 ml using an Amicon-Ultra 15 centrifugal filter device (Merck Millipore) with a 100 kDa cutoff by centrifugation at 4,000 x g for 15 min (4°C). The concentrated sample was loaded onto a 15 ml Sepharose CL-6B (GE Healthcare Bio-Sciences AB) size-exclusion chromatography column. Once the entire sample had entered the column matrix, successive additions of 0.1 μm-filtered DPBS (Gibco) supplemented with 1% penicillin / streptomycin (Gibco) ensured complete drainage of the sample. The eluate was collected by gravity in 15 successive 1 ml fractions. For each fraction, the protein amount was determined by spectrophotometry (absorbance 280 nm, Nanodrop, Thermo Fisher Scientific).

[0104] Nanoparticle Tracking Analysis (NTA) Particle concentration and size exclusion were determined using a NanoSight LM 10 instrument (NanoSight Ltd, Amesbury, UK). The above samples were diluted 10-40 times in 0.1 μm filtered DPBS (Gibco) supplemented with 1% penicillin / streptomycin (Gibco) to obtain a 2x10 8 ~1x10 9 Concentrations of particles / ml were obtained. The camera screen gain was set to 2 and the camera level was set to 16. Settings were kept constant between samples, and each video was analyzed to obtain the mean, mode, median, and estimated concentration for each particle size. Analysis was performed with NTA software (version 3.3) using 60 seconds of video capture per sample (five replicates per sample). For video processing, the screen gain was set to 14 and the detection threshold was set to 3.

[0105] Western blot The EV-enriched fraction was concentrated using Amicon-Ultra 15 centrifugal filter devices (Merck Millipore) with a 100 kDa cutoff by centrifugation at 4,000 x g for 15 min (4 °C). For a positive control, cells were lysed in RIPA lysis buffer (50 mM Tris HCl pH 8, 1% NP-40, 0.2% sodium deoxycholate, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS) supplemented with protease inhibitors (Roche Diagnostics) and centrifuged at 12,000 x g for 10 min at 4 °C. The supernatant was collected, and the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 5 x 10 10 Particles and 15 mg of protein were separated on a NuPAGE™ 4-12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific) and electrophoretically transferred to a nitrocellulose membrane (Invitrogen). The membrane was probed for CD63 (Santa Cruz Biotechnology), CD81 (Santa Cruz Biotechnology), and calnexin (Abcam).

[0106] Transmission electron microscope (TEM) The isolated particles were diluted 100-fold in 0.1 μm-filtered DPBS and mixed 1:1 with 4% methanol-free formaldehyde solution (Thermofisher Scientific). A drop of this solution was plated on a Petri dish, and then a Formcar-coated 200-mesh gold grid (Taab, Aldermaston, UK) was floated on top and left for 20 minutes. The grid was transferred to PBS and washed twice for 5 minutes. EVs were then re-fixed on the grid using a 1% glutaraldehyde solution and again washed twice with PBS. Finally, the grid was transferred to a drop of 0.5% uranyl acetate-2% 25 centipoise methylcellulose (Sigma-Aldrich). After 5 minutes of staining, excess liquid was removed, and the grid was air-dried and then examined on a JEOL JEM-1400 series 120 kV transmission electron microscope. TEM photographs were analyzed using a “TEM Exosome Analyzer” ( Kotrbova et al., 2018 ).

[0107] endothelial cell culture HCMEC and HUVEC were obtained from PromoCell, UK. Concentrated medium containing 5% fetal calf serum (FCS), 5 ng / ml epidermal growth factor (recombinant human), 10 ng / ml basic fibroblast growth factor (recombinant human), 20 ng / ml insulin-like growth factor (long-chain R3 IGF), and 0.5 ng / ml VEGF was used. 165 HCMECs were cultured at 37°C in a humidified atmosphere containing 5% CO and 95% O in MV2 medium (Promocell) supplemented with recombinant human VEGF, 1 μg / ml ascorbic acid, 0.2 μg / ml hydrocortisone, 2% fetal calf serum (FCS), 5 ng / ml epidermal growth factor (recombinant human), 10 ng / ml basic fibroblast growth factor (recombinant human), 20 ng / ml insulin-like growth factor (long-chain R3 IGF), and 0.5 ng / ml VEGF. 165HUVECs were cultured in EBM2 medium (Promocell) supplemented with recombinant human erythrocyte colony stimulating factor (HRS), 1 μg / ml ascorbic acid, 22.5 μg / ml heparin, and 0.2 μg / ml hydrocortisone. To isolate EVs from hypoxic HUVECs, once the cells reached confluence, the medium was replaced with EV-free medium and transferred to a sealed chamber containing 5% CO2, 92% N2, and 3% O2. To prepare EV-free medium, FBS was ultrafiltered using an Amicon-Ultra 15 centrifugal filter device (Merck Millipore) with a 100 kDa cutoff after centrifugation at 3,000 x g for 55 minutes (4°C) and supplemented to the cell culture medium.

[0108] Tube formation assay The angiogenic effects of hESC-eEVs, hESC-mEVs, and HUVEC hypoxic EVs on HCMECs were evaluated by tube formation assay. Cells were incubated at 37°C for 30 minutes and then stained with 2 μM / ml calcein AM (Invitrogen). Growth factor-reduced ECM gel (Sigma) was thawed overnight at 4°C on ice and plated at 10 μl per well onto a μ-Plate Angiogenesis 96-well (Ibidi, UK). After a 30-minute gelation period at 37°C, 1.5x10 cells were cultured. 4 HCMECs were seeded onto each well. HCMECs were cultured in fully supplemented MV2 medium (Promocell), or in basal MV2 medium (Promocell, UK) containing DPBS+P / S, or in increasing concentrations of EVs (1–10 5 The cells were incubated with either basal MV2 medium containing EVs / cells. Brightfield micrographs were taken at 4x magnification using an EVOS XL imaging system (Invitrogen). The number of meshes and branches, as well as the total tube length, were analyzed using the "Angiogenesis analyzer" plugin (Gilles Carpentier) (Carpentier et al., 2020) in ImageJ software.

[0109] Wound healing assay The ability of hESC-eEVs to promote wound healing in vitro was assessed by scratch assay. HCMECs were seeded into 6-well plates to generate confluent monolayers. These were then incubated for 70 min. Upon reaching ~90% confluence, scratch the cell monolayer with a sterile P200 pipette tip and replace the medium with fully supplemented MV2 medium (Promocell), or basal MV2 medium (Promocell, UK) containing DPBS+P / S, or increasing concentrations of hESC-eEVs (1–10 2 EV / cell) or 10 5 The wound was replaced with basal MV2 medium containing HUVEC hypoxic EVs / cells for 24 hours. At 0, 6, 12, and 24 hours after scratching, HCMEC wound closure was monitored using bright-field microscopy. The percentage of wound coverage was calculated using the MRI wound healing tool (Montpellier Resources) in ImageJ software, with 0% coverage at time 0.

[0110] RNA extraction Total cellular RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA was recovered from EV samples using the Total Exosomal RNA and Protein Isolation Kit (Invitrogen) according to the manufacturer's instructions. RNA quantification and quality were further analyzed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Inc.) using a total RNA Pico Series II chip.

[0111] Small RNA sequencing EV isolation by a combination of SEC and ultrafiltration produced highly pure EV preparations, but yields were lower than other methods (Thery et al., 2018). To counteract this, the resulting input for our libraries was low, and a minimum total RNA amount of 1 ng provided the maximum possible input for sequencing each EV library (n=3). First, small RNAs of 18-30 nt were selected by 15% urea-PAGE gel electrophoresis and gel extraction. After gel purification, adenylated 3' adapter ligation to the small RNA fragments was performed. A barcode-bearing reverse transcription (RT) primer was used to anneal the 3' adenylated adapter and combine it with a redundant, unligated 3' adenylated adapter. 5' adapter ligation and RT reaction were then performed. After cDNA first-strand synthesis, the product was amplified for 15 cycles. A second size selection of 103-115 bp fragments from the gel was performed. This step was performed to purify the PCR product and remove any nonspecific products. After gel purification, PCR yields were quantified using Qubit (Invitrogen, catalog number Q33216). Samples were pooled together to generate single-stranded DNA circles (ssDNA circles), which resulted in the final small RNA library. DNA nanoballs (DNBs) were generated in the ssDNA circles by rolling circle replication (RCR) to enhance the fluorescent signal during the sequencing process. The DNBs were loaded into patterned nanoarrays, and 50-bp single-end reads were read on a BGISEQ-500 platform (sequencing depth: ≥ 20 million reads per sample) for the following data analysis studies.

[0112] Sequencing read mapping and small RNA annotation BGI-SEQ500 raw data were in fastq format. We used two mapping and quantification approaches to ensure robust analysis of EV miRNA profiles. The first was the exceRpt small RNA-seq processing pipeline (Rozowsky et al., 2019). For quality control and read mapping against the human genome and default small RNA libraries, files were exported to Genboree Workbench's exceRpt small RNA-seq pipeline (version 4.6.2), which allowed for single mismatches up to 18 nucleotides. Reads that failed clipping (0%), did not meet quality filters (<1.2%), were UniVec contaminants (<0.6%), and were rRNA (5–51.4%) were excluded from the analysis. After these initial filters, the average number of reads used for alignment ranged from 24.7 to 28.3 million reads for each group (Supplementary Figure 5A). A high percentage of reads could not be aligned to either the genomic or small RNA libraries, likely a result of background nonspecific amplification in lower-input libraries (particularly in hESC-eEVs and HUVEC hypoxic EVs, where 30.4–78% of the reads used for alignment were unmappable) (Supplementary Figure 5B). We found a negative correlation (rho −0.7, p < 0.05, Sperman's rank test) between the percentage of unmapped reads and the number of particles used for RNA extraction (as measured by NTA and Agilent Pico Chip Bioanalyzer, respectively), suggesting that higher background amplification may occur when the initial input is low for a particular sample (Supplementary Figure 5C). We view this low input into the sequencing library as an acceptable tradeoff for the high purity of our EV preparation. Unmapped reads were excluded from all further analysis. Principal component analysis (PCA) of the remaining reads for each sample suggested distinct small RNA profiles (Supplementary Figure 4).

[0113] Alignment and quantification of reads to a recent release of the reference human genome (GRCh38) was performed using the Shortstack alignment tool (Axtell, 2013; Rozowsky et al., 2019) mapping exclusively to miRbase. ShortStack offers the ability to provide counts for the same miRNA across multiple genomic loci. Both approaches provided nearly identical read percentages to miRbase V21. We then used the default (RPMM) alignment provided by the exceRpt pipeline, which normalizes miRNA counts to the total number of reads mapped to all small RNA libraries or genomes. 総数 We compared the normalization of miRNAs to the total counts assigned to miRNAs through Shortstack (RPMM) for all miRNAs (exceRpt default). These measurements therefore quantify individual miRNAs relative to the total pool of miRNAs or small RNAs present in EVs, respectively. We compared the normalization of miRNAs to RPMM and RPMM. 総数 We observed a strong overall positive correlation between values ​​(rho, 0.76-0.93, p<0.0001, Spearman's rank test). However, the variability between replicates was not significant. 総数miRNAs were more highly expressed in hESC-eEVs than in other conditions (RPMM > 100), especially for the highest-expressing miRNAs. Therefore, unless otherwise specified, we used RPMM values ​​for our analyses. MiRNAs enriched in hESC-eEVs compared to other conditions were determined using ShortStack-derived counts processed by DESeq2 (v1.34.0) (Love et al., 2014). We also used the ashr package within DESeq2 (Stephens, 2017) to mitigate overestimation of LFC due to noise and high variability in miRNA counts. Comparisons to hESC-mEVs were treated separately from other comparisons because the greater inter-replicate variability seen through PCA could unduly affect normalization for differential expression analysis. Another differential expression analysis was also performed using the edgeR tool in a "classical" approach (Robinson et al., 2010), again separating the hESC-mEV comparison from the others.

[0114] statistical analysis All biological replicates using human primary cells represent separate expansion and passage-number independent experiments. All graphs (except ratios) are shown as data points with mean ± standard deviation (SD) for biological or technical replicates as detailed in the figure legends. qRT-PCR data in the graphs are shown as relative expression as described by Livak and Schmittgen (Livak & Schmittgen, 2001). Statistical analysis was performed using GraphPad Prism 9.0.0 (GraphPad Software, La Jolla, CA, USA). The tests used to assess significance are detailed in each figure legend, and the exact p-values ​​for significant changes are indicated on the graphs. A value of p<0.05 was the nominal significance level. Because each experimental data set is the average of a large number of cultured cells for in vitro experiments, we assumed the data were normally distributed based on the central limit theorem. To determine significant differences between samples in our in-vitro experiments (>2 groups), one-way analysis of variance with Dunnett's multiple comparison test was used.

[0115] result 1. EVs isolated from hESC-ECP-conditioned medium express EV markers and exhibit phenotypic characteristics of exosomes To isolate hESC-eEVs, pluripotent hESCs underwent an established 8-day differentiation protocol through the mesoderm stage (Figure 1A) as previously described. We confirmed that on day 8, two-thirds (65.7%) of the cells expressed the EC cell surface markers CD31 and CD144, and less than 0.05% co-expressed the pluripotency marker SSEA-3 / TRA-1-60, as shown by flow cytometry (Supplementary Figure 1). EVs were isolated from hESC-ECP-conditioned medium using a combination of ultrafiltration and size-exclusion chromatography (SEC) (Figure 1B). To select fractions enriched for EVs and free of protein contamination, the protein and EV concentrations of all fractions were measured by spectrophotometry and nanoparticle tracking analysis (NTA), respectively. Fractions 5 and 6, which contained significantly higher particle counts and lower protein concentrations (Supplementary Figure 2), were pooled and used for further analysis to confirm EV isolation.

[0116] The enrichment of EVs in the isolated fractions was assessed by characterization of EV size distribution, surface markers, and morphology. NTA showed that the size of particles in pooled fractions 5 and 6 ranged from 30 to 200 nm, with a mean value of 84 ± 7.3 (Figure 1C). Western blot analysis confirmed that the particles were positive for the EV markers CD63 and CD81, but negative for the endoplasmic reticulum marker, calnexin (Figure 1D). Transmission electron microscopy (TEM) identified structures of characteristic size and shape of EVs. Quantification of TEM images showed that the mean EV size was 100.42 ± 38.36 and the circularity ratio was 0.96 ± 0.06 (Figure 1E). Thus, EV preparation from hESC-ECP conditioned medium through a combination of ultrafiltration and SEC resulted in the isolation of EV particles of characteristic size, morphology, and markers (CD63 and CD81), while also being free of protein contamination. These highly pure EV preparations are referred to herein as hESC-eEVs.

[0117] 2. hESC-eEVs are internalized by ECs and, at low concentrations (<100EVs / cell), promote tube formation and wound closure Increased EC tube formation and wound closure are considered hallmarks of angiogenesis (S. Guo et al., 2014), and previous reports have demonstrated that the effects of EC-derived EVs on angiogenesis are dose-dependent (Lacroix et al., 2007). To evaluate the angiogenic potential of hESC-eEVs, we first performed tube formation and wound closure assays using increasing concentrations of hESC-eEVs isolated from four independent differentiations (i.e., n = 4). HCMECs cultured in fully supplemented medium served as a positive control for angiogenic / migration cells, while cells cultured in growth factor-free medium and treated with an equal volume of sterile 0.1 μm-filtered PBS (EV suspension solution) served as a negative control. Because hypoxia induces the release of angiogenic EVs (Bister et al., 2020), we also treated HCMECs with EVs obtained from hypoxic human umbilical vein ECs (HUVECs) as an EC-derived EV control. By performing dose-response experiments using HUVEC hypoxic EVs, we found that these EVs were ineffective in inducing EC tube formation at low doses (<100 EVs / cell) and were potent at high doses (10 5 We demonstrated that treatment with approximately 1 hESC-eEV per cell produced a significant increase in HCMEC tube formation ability compared to the negative control (p<0.0001) (Figure 2B). Therefore, treatment with HUVEC hypoxic EVs (10 5 A much lower dose of hESC-eEVs (1 EV / cell) was required to promote HCMEC tube formation at levels similar to those of HUVEC hypoxic EVs (1 EV / cell). We also showed that, in contrast to HUVEC hypoxic EVs, treatment with high hESC-eEV concentrations (≥100 EVs / cell) failed to reproduce this effect. Staining with calcein AM confirmed cell viability in all conditions during the tube formation assay (Figure 2C).

[0118] To understand whether the effect on angiogenesis was specific to EVs derived from the endothelial stage of the hESC-ECP differentiation system, we investigated the effect of EVs derived from the mesodermal stage of the differentiation protocol (hESC-mEVs) on HCMEC tube-forming ability (dose range: 1–10 5 Cells cultured in fully supplemented medium or in basal medium but treated with 1 hESC-eEV / cell served as positive controls, while cells cultured in basal medium and treated with PBS served as negative controls. Our results showed that, in contrast to treatment with 1 hESC-eEV / cell, treatment with hESC-mEV did not induce tube formation at any dose tested (Figure 2D). Thus, the ability to promote angiogenesis is specific to EVs derived from the endothelial stage of the ESC-ECP differentiation system (hESC-eEVs) and absent from EVs derived from the mesoderm stage (hESC-mEVs). To confirm that the angiogenic effect observed after hESC-eEV treatment is specific to EVs and not due to other soluble, non-EV-associated secreted factors, we subjected complete medium (not exposed to cells) to an EV purification process and collected samples at different stages of the purification (Figure 2E). These samples were tested for their ability to induce HCMEC tube formation (Figure 2F). Cells cultured in fully supplemented medium or cells cultured in basal medium but treated with 1 hESC-eEV / cell were used as positive controls, while cells cultured in basal medium and treated with PBS were used as negative controls. The cell culture medium contained a high concentration of human recombinant VEGFA. 165 Since VEGFA is a key driver of angiogenesis (Apte et al., 2019), human recombinant VEGFA was used. 165 Cells treated with EV-treated EVs were also used as a positive control for this experiment. Our results showed that treatment with complete medium subjected to an EV purification process did not improve HCMEC tube formation, whereas treatment with EVs isolated from conditioned medium (1 hESC-eEV / cell) promoted HCMEC tube formation. Therefore, soluble non-EV-associated secreted factors are unlikely to contribute to the observed angiogenic effect after hESC-eEV treatment.

[0119] Because we observed that hESC-eEVs induced HCMEC tube formation at low concentrations, we performed scratch assays using increasing concentrations of hESC-eEVs, with the maximum dose being 100 hESC-eEVs / cell, to evaluate the effect of hESC-eEVs on EC wound healing (Figure 3). Our results demonstrated that 6 to 24 hours after wound induction, HCMECs treated with low doses of hESC-eEVs (<100 EVs / cell) migrated significantly faster than cells cultured in basal medium and treated with PBS. Cells treated with high concentrations of hESC-eEVs (100 EVs / cell) or HUVEC hypoxic EVs did not exhibit significantly improved migration ability compared to the negative control. Taken together, these results demonstrate the efficacy of hESC-eEVs at low concentrations (<100 EVs / cell) to stimulate angiogenesis. Moreover, this angiogenic effect appears to be lost at higher hESC-eEV concentrations, suggesting a dose-specific effect. Furthermore, our data demonstrate that the ability to induce angiogenesis is specific to endothelial-derived EVs and not the mesodermal stage of hESC-eEV differentiation.

[0120] 3. hESC-eEVs are enriched in angiogenic miRNAs and other miRNAs with potential roles in angiogenesis Because the effects of EVs are largely attributable to their small RNA cargo (O'Brien et al., 2020), we sought to identify RNA molecules involved in the angiogenic activity of hESC-eEVs. Small RNAs were extracted from EVs derived from the following samples: hESC-eEVs, hESC-mEVs, and HUVEC hypoxic EVs (n = 3, independent stem cell differentiation). We also obtained RNA from hESC-ECPs to identify EV-enriched miRNAs. RNA size profiling showed enrichment of small RNAs (25-250 nt) in EVs, while ribosomal and longer RNAs were absent. Cellular RNA sample profiling showed two distinct ribosomal peaks corresponding to 18S and 28S eukaryotic RNAs. Small RNA sequencing was performed on these RNA samples. The majority of cellular RNA sample reads mapped to sense miRNAs, whereas EV RNA samples showed a more diverse composition of small RNA classes, with the majority mapping to tRNAs, miRNAs, miscellaneous RNAs (misc_RNAs), and protein-coding molecules (Figure 4A). To understand whether EVs from different conditions possess distinct RNA molecules, we compared the lists of unique miRNAs, piRNAs, tRNAs, and other RNAs between EV types (Supplementary Figure 3). tRNAs and piwiRNAs were less diverse, while miRNAs and other small RNAs (including miscellaneous RNAs, snRNAs, yRNAs, retained introns, protein-coding molecules, lincRNAs, etc.) represented the most heterogeneous populations among EV samples. Overall, different EV samples shared a total of 72 miRNAs and 172 other small RNAs. In this study, we focused on miRNAs for further investigation, as EV-miRNAs have a well-established role in regulating angiogenesis in cardiovascular disease (CVD) ( Keseidou et al., 2020 ).

[0121] We next evaluated the presence of EC-enriched miRNAs (miR-126-5p, miR-222-3p, miR-99b-5p, miR-22a-3p) (de Rie et al., 2017) in our dataset and found them to be abundant in hESC-eEVs and HUVEC hypoxic EVs, but not in hESC-mEVs. Consistent with previous findings on cellular RNA composition, which reported that for any given human cell type, the most abundant 3–5 miRNAs account for more than 50% of the total miRNA pool (de Rie et al., 2017), we found that the top three miRNAs in hESC-ECPs represented more than 50% of total miRNA reads. A similar profile was observed in EV samples, with the top five EV miRNAs representing approximately 50% of total miRNA reads (Figure 4B). A literature search of the 20 most abundant miRNAs in each sample (which represented 68–86% of the total reads) showed the presence of several miRNAs with no reported role in angiogenesis in hESC-eEVs. The top five miRNAs in hESC-eEVs contain molecules thoroughly studied for their role in driving angiogenesis, while the top five miRNAs in hESC-mEVs have anti-angiogenic effects (Cao et al., 2019; Fish et al., 2008; B. Guo et al., 2017; Jakob et al., 2012; Jansen et al., 2013; Li et al., 2016; Liu et al., 2019; Qiao et al., 2019; Qu et al., 2019; Q.-Z. Wang et al., 2021; S. Wang et al., 2008) (Figure 4C). Taken together, our data suggest that hESC-eEVs are enriched for angiogenic miRNAs as well as miRNAs with no reported role in angiogenesis.

[0122] Consideration Our previous data suggested that the improvements observed after hESC-ECP transplantation may be due to a paracrine mechanism (MacAskill et al., 2018). Here, we sought to investigate the effects of hESC-eEVs on angiogenesis. This study provides several novel insights into the EV field. We developed a reproducible protocol for isolating pure, biologically active EVs from hESC-ECP cultures and demonstrated that hESC-eEVs induce angiogenesis at low concentrations compared with other EC-derived EVs. We also demonstrated that hESC-eEVs are enriched in angiogenic miRNAs and other miRNAs whose role in angiogenesis has not been reported.

[0123] In this study, we isolated EVs by a combination of ultrafiltration and SEC, which led to the isolation of highly pure EV populations (Thery et al., 2018). Although EV yields are lower compared to other approaches (Thery et al., 2018), the combination of ultrafiltration and SEC has been proposed as a promising strategy for isolating biologically active EVs (Benedikter et al., 2017; Mol et al., 2017). Using this approach, we confirmed the isolation of EVs with intact ultrastructures, which were positive for EV markers CD63 and CD81 and negative for the cellular contamination marker calnexin (Kozlov & Gehring, 2020), and free of protein contamination. To examine the effects of hESC-eEVs on angiogenesis, we performed tube formation and scratch assays on ECs. We also compared the effects of our EVs to EVs isolated from the mesodermal stage of hESC-ECP differentiation (hESC-mEVs) and mature ECs. As a mature EC population, we chose HUVEC hypoxic EVs because hypoxia induces the release of angiogenic EVs (Bister et al., 2020). Our results show that while a low concentration of hESC-eEVs (<100 EVs / cell) significantly increased EC angiogenic potential, a much higher dose of HUVEC hypoxic EVs (10 5We demonstrated that a high concentration of hESC-eEVs (≥100 EVs / cell) is required for the angiogenic potential of ECs. We also demonstrated that treatment with high concentrations of hESC-eEVs (≥100 EVs / cell) did not affect EC angiogenic potential. This may suggest that hESC-eEV-mediated angiogenic effects have precise dosage requirements. Similar trends have been reported in other angiogenesis studies using increasing doses of EC-derived EVs (Lacroix et al., 2007; Ou et al., 2011). However, the use of different methodologies for EV isolation and characterization makes comparisons between individual EV studies difficult. Another important consideration is that, as a particle quantification technique, NTA has several disadvantages, including the potential for underestimation of particle concentrations (Bachurski et al., 2019). Therefore, accurate estimation of the EV dose to use in individual experiments remains a significant hurdle in this field. In contrast to EC-derived EVs, we showed that treatment with EVs derived from the mesoderm stage of hESC-ECP differentiation (hESC-mEVs) did not affect EC angiogenic potential, suggesting that the ability to induce angiogenesis is specific to EVs isolated from more mature stages of hESC-ECP differentiation. Taken together, the requirement for very low doses of hESC-eEVs, which resulted in improved efficacy, may be promising for the clinical application of hESC-eEVs for therapeutic angiogenesis.

[0124] One of the most useful tools for studying EV miRNA cargo is small RNA sequencing, likely because it provides an unbiased view of EV miRNA content. To identify potential miRNAs that may drive the angiogenic potential of hESC-eEVs, we performed small RNA sequencing and compared the small RNA cargo of hESC-eEVs to hESC-mEVs, HUVEC hypoxic EVs, and one of the EV donor cells. EV small RNA sequencing was performed using the DNBSEQ™ NGS technology platform. A potential limitation of our approach is the low RNA input in the EV samples, which was primarily due to two reasons: the hESC-eEV source was derived from a differentiated lineage, and the low yield of EV isolation by ultrafiltration and SEC (Thery et al., 2018), which was found to be associated with a high percentage of unmapped reads in our library. While our high-purity, low-depth approach to EV isolation and sequencing misses the opportunity for complete detection of all EV miRNAs, we provide sufficient coverage to profile highly abundant miRNAs that are most likely to affect angiogenesis at the low concentrations required for hESC-eEVs. Our results showed that the majority of EV RNA samples mapped to tRNAs, miRNAs, misc_RNAs, and protein-coding molecules, while the majority of cellular RNA sample reads mapped to sense miRNAs. We noted that miRNAs and other small RNAs (including promiscuous RNAs, snRNAs, yRNAs, retained introns, protein-coding molecules, lincRNAs, etc.) were the most heterogeneous populations across different EV samples. However, because the role of miRNAs in angiogenesis is better understood (Kesidou et al., 2020), we focused on miRNAs for the remainder of our analysis.

[0125] Our small RNA sequencing analysis revealed for the first time that the top five miRNAs also accounted for approximately 50% of total miRNA reads, consistent with previous findings on cellular RNA composition (de Rie et al., 2017), which reported that the top three to five miRNAs accounted for more than 50% of the total miRNA pool for any given human cell type. We demonstrated that the most abundant miRNAs in hESC-eEVs are molecules with well-established roles in inducing angiogenesis, while the most abundant miRNAs in hESC-mEVs have anti-angiogenic effects. This may explain why hESC-eEVs require low doses to induce angiogenesis and possibly explain why hESC-mEVs were ineffective at inducing angiogenesis in our in vitro experiments. Our results also suggest that the most abundant miRNAs in HUVEC hypoxic EVs are molecules with diverse roles in driving angiogenesis, which may explain why higher doses of HUVEC hypoxic EVs are required to observe similar effects on angiogenesis as hESC-eEVs. To assess whether the differences in EV angiogenic activity could be explained by their miRNA cargo, we performed differential expression analysis. Our results showed that several miRNAs are differentially expressed between angiogenic hESC-eEVs and non-angiogenic hESC-mEVs. The miRNA most enriched in hESC-eEVs compared to hESC-mEVs is the EC-specific (de Rie et al., 2017) miRNA, miR-126-3p, whose role in driving angiogenesis has been thoroughly studied (Qu et al., 2019).

[0126] Taken together, this study is the first to examine the effects of hESC-eEVs on angiogenesis, provides comprehensive insight into the miRNA content of hESC-eEVs, and compares the miRNA cargo of hESC-eEVs to hESC-mEVs and mature EC-EVs. Our data highlight several factors that will contribute to both the EV and CVD fields: (1) hESC-eEVs induce angiogenesis at low concentrations compared to other EC-derived EVs, whereas EVs derived from more immature stages of the hESC-ECP differentiation protocol are not angiogenic; (2) hESC-eEVs are enriched in angiogenic miRNAs, whereas hESC-mEVs are enriched in non-angiogenic miRNAs. The most abundant miRNAs in HUVEC hypoxic EVs are molecules with diverse roles in driving angiogenesis. References TIFF2026501121000011.tif237165TIFF2026501121000012.tif242165TIFF20265011210 00013.tif242165TIFF2026501121000014.tif251165TIFF2026501121000015.tif167165

Claims

1. An isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents, obtainable from pluripotent stem cell-derived endothelial cell products.

2. 10. An isolated population of pro-angiogenic and / or pro-neovascularization EVs and / or their contents according to claim 1 for use in therapy.

3. 10. A pharmaceutical composition comprising the isolated population of pro-angiogenic and / or pro-neovascularization EVs and / or their contents according to claim 1, optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

4. 10. A medical device coated with the isolated population of pro-angiogenic and / or pro-neovascularization EVs and / or their contents according to claim 1.

5. 10. An isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or contents thereof, pharmaceutical composition, or medical device according to any preceding claim for use in promoting angiogenesis and / or neovascularization in a subject in need thereof.

6. 6. An isolated population, pharmaceutical composition, or medical device of pro-angiogenic and / or pro-neovascularization EVs and / or their contents according to claim 5 for use in the treatment of ischemic tissue damage, cardiovascular disease, such as pulmonary hypertension and / or wound healing.

7. 5. The isolated population of pro-angiogenic and / or pro-neovascularization EVs and / or their contents, or pharmaceutical composition of claim 4, for use in an amount of no more than 50, 25, 10, 5, 2, or 1 EV / cell.

8. 1. A method for isolating EVs from endothelial cells derived from pluripotent stem cells, comprising: a) subjecting a liquid containing EVs produced by pluripotent stem cell-derived endothelial cells to ultrafiltration to separate EVs from larger molecular weight substances and obtain an EV-enriched liquid; b) subjecting the EV concentrate to size exclusion chromatography (SEC) to obtain a liquid fraction containing isolated EVs; The method comprising:

9. 9. The method of claim 8, wherein ultrafiltration comprises using a filter with a 100 KDa molecular weight cutoff.

10. 10. The method according to any one of claims 8 and 9, wherein SEC is carried out using a sephacryl, sephadex, superose, superdex or sepharose chromatography medium, in particular sepharose, such as sepharose CL-6B.

11. 11. An isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents prepared according to the method of claims 8 to 10 for use in promoting angiogenesis and / or neovascularization.

12. 12. The isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents according to claim 11 for use in the treatment of ischemic tissue damage, cardiovascular disease, such as pulmonary hypertension and / or wound healing.

13. An isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents, said isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents comprising one or more miRNAs.

14. 14. The isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents according to claim 13, wherein the one or more miRNAs are: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-5p; (iv) miR-92a-3p; (v) miR-7-1-5p; (vi) miR-196b-5p; (vii) miR-302b-3p; (viii) miR-222-3p; (ix)miR-6087; (x)miR-10a-5p; (xi) miR-99b-5p; (xii) miR-184; (xiii) miR-302a-3p; (xiv)miR-423-3p; (xv)miR-3184-5p; (xvi)miR-7-2-5p; (xvii)miR-3529-3p; (xviii) miR-92b-3p; (xix)miR-342-3p; (xx)miR-23a-3p; (xxi)miR-302d-3p; (xxii)miR-483-3p; (xxiii)miR-302c-3p; (xxiv)miR-151a-5p; (xxv)miR-26a-5p; (xxvi)miR-149-5p; (xxvii)miR-miR-27b-3p; (xxviii)miR-302a-5p; or: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p (iv) miR-7-1-5p; (v) miR-196b-5p; (vi) miR-302b-3p; (vii) miR-126-3p; (viii) miR-222-3p; or: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; and (v) miR-196b-5p; or: (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3-p; and (v) miR-222-5p; or: (i) miR-126-3p; (ii) miR-21-5p; and (iii) miR-92a-3p The isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents selected from the group consisting of or consisting essentially of:

15. (i) in treating or preventing cardiovascular disease; (ii) in treating or preventing coronary heart disease; (iii) in treating or preventing ischemic tissue damage; (iv) in treating or preventing pulmonary hypertension; (v) in regulating or promoting angiogenesis and / or neovascularization; and / or (vi) in regulating or promoting wound healing A miR modulator for use in

16. 16. The miR modulating agent for use of claim 15, wherein the miR modulating agent: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-5p; (iv) miR-92a-3p; (v) miR-7-1-5p; (vi) miR-196b-5p; (vii) miR-302b-3p; (viii) miR-222-3p; (ix)miR-6087; (x)miR-10a-5p; (xi) miR-99b-5p; (xii) miR-184; (xiii) miR-302a-3p; (xiv)miR-423-3p; (xv)miR-3184-5p; (xvi)miR-7-2-5p; (xvii)miR-3529-3p; (xviii) miR-92b-3p; (xix)miR-342-3p; (xx)miR-23a-3p; (xxi)miR-302d-3p; (xxii)miR-483-3p; (xxiii)miR-302c-3p; (xxiv)miR-151a-5p; (xxv)miR-26a-5p; (xxvi)miR-149-5p; (xxvii)miR-miR-27b-3p; (xxviii)miR-302a-5p; or: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p (iv) miR-7-1-5p; (v) miR-196b-5p; (vi) miR-302b-3p; (vii) miR-126-3p; (viii) miR-222-3p; or: (i) miR-126-3p; (ii) miR-21-5p; (iii) miR-92a-3p; (iv) miR-7-1-5p; and (v) miR-196b-5p; or: (i) miR-92a-3p; (ii) miR-21-5p; (iii) miR-302b-3p; (iv) miR-126-3-p; and (v) miR-222-5p; or: (i) miR-126-3p; (ii) miR-21-5p; and (iii) miR-92a-3p The miR-modulating agent increases the expression of one or more miRs selected from the group consisting of:

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