Extracellular vesicles to promote angiogenesis or neovascularization

Abstract

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

Description

[0001] field

[0002] The present disclosure relates to extracellular vesicles (EVs) (and their contents) from pluripotent stem cell-derived endothelial cell products (ECPs); and their isolation; and uses and methods of using EVs for therapy (e.g., promoting angiogenesis and / or neovascularization).

[0003] background

[0004] Cardiovascular disease (CVD) remains the most common cause of death worldwide, with the World Health Organization (WHO) reporting 17.9 million deaths from CVD in 2017. Of these deaths, an estimated 7.4 million are attributable to coronary heart disease (CHD) alone (WHO-Cardiovascular Diseases (CVDs), 2017). CHD is characterized by the narrowing of coronary arteries due to the gradual formation and subsequent rupture of plaque within the vessel wall. The blockage of these arteries leads to oxygen and nutrient deprivation in downstream tissues. Consequently, ischemic damage and cardiomyocyte death occur in the affected areas of the heart, a phenomenon known as myocardial infarction (MI) (Thygesen et al., 2019). Therapeutic neovascularization has been proposed as a possible strategy to generate new myocardial vascular networks and reduce the extent of cardiomyocyte damage. Paracrine cell communication plays a key role in controlling this process (Gnecchi et al., 2008). Paracrine cell communication is regulated by multiple mechanisms, including extracellular vesicles (EVs). EVs carry and transfer various bioactive molecules, such as non-coding small RNAs, proteins, and lipids, which modulate signaling pathways in recipient cells (Théry et al., 2018).

[0005] Despite some controversy, preclinical studies have shown that EVs hold great promise in regulating complex processes, such as post-ischemic neovascularization (Kesidou et al., 2020). However, the optimal sources of such EVs, their respective cargoes and functions, and their translation into clinical opportunities are still relatively nascent.

[0006] Endothelial cell (EC) damage and viability are important factors in the cellular release of EVs. Typically, EC-EVs are present at low concentrations under physiological conditions and are released from ECs at higher levels after activation (Koga et al., 2005). Circulating EVs released from ECs have been shown to play a role in activating vascular ECs (Ridger et al., 2017). However, emerging evidence suggests that endothelial-derived EVs may play a multifunctional role in neovascularization, as their effects depend not only on the EV donor cells, but also on the dose or number of EVs to which the recipient cells are exposed (Lacroix et al., 2007). Nevertheless, EC transplantation studies have suggested a potential role for the endothelial secretome in EC activation and neovascularization, showing improved capillary density and blood flow after EC transplantation, although retention in ischemic tissue was limited (Chekanov et al., 2003). We have previously shown that transplantation of human embryonic stem cell-derived EC products (hESC-to-ECP) after left femoral artery occlusion resulted in increased capillary density in mice 21 days after ischemia. Furthermore, we have shown that cardiac function improves after injection of cells into the heart following myocardial ischemia (Ana-MishelSpiroski et al., 2022), demonstrating the broad relevance of these cells as therapeutics. Although these cells are able to increase capillary density, they exhibit poor retention (MacAskill et al., 2018), suggesting that an acute paracrine mechanism of action may be involved. These findings are consistent with previous cell transplantation studies, which have shown that only a very small number of transplanted cells are able to engraft at the site of injury (Tompkins et al., 2018).

[0007] Overview

[0008] Because the increased rate of neovascularization following hESC-ECP transplantation suggests a paracrine mechanism, here we aimed to investigate the role of hESC-ECP-derived EVs (hESC-eEVs) in angiogenesis. Here, we show that very low concentrations of hESC-eEVs promote EC tube formation and wound healing and are enriched for known angiogenic miRNAs and other miRNAs with previously unreported roles in angiogenesis. Furthermore, we demonstrate that hESC-eEVs induce angiogenesis at low doses, likely due to the presence of selected miRNA molecules in hESC-eEVs as well as other components present in the EVs.

[0009] In a first aspect, an isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents are provided, which are obtainable from a pluripotent stem cell-derived endothelial cell product (ECP).

[0010] Also provided is an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents, which can be obtained from pluripotent stem cell-derived ECPs for use in treating a subject in need thereof, e.g., for promoting angiogenesis and / or neovascularization. In one teaching, the isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents are obtained from pluripotent stem cell-derived ECPs.

[0011] Also provided is a method of treatment (e.g., treatment by promoting angiogenesis and / or neovascularization), comprising administering to a subject in need thereof an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents, wherein the isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents are obtained from pluripotent stem cell-derived ECPs.

[0012] 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 existing blood vessels, while neovascularization refers to the process of forming blood vessels de novo or forming new blood vessels from existing blood vessels. The present disclosure may relate to both angiogenesis and / or neovascularization. That is, the present disclosure may relate only to promoting angiogenesis, only to promoting neovascularization, or to promoting both angiogenesis and neovascularization. The remaining descriptions may refer to either term alone, but unless the context indicates otherwise, references to angiogenesis should be understood to extend to neovascularization, and vice versa.

[0013] As used herein, the terms "therapy" or "treatment" are understood to refer to the prevention and / or treatment of a condition or disease.

[0014] As used herein, the phrase "pluripotent stem cells" refers to cells capable of differentiating 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).

[0015] As used herein, the phrase "embryonic stem cells" refers to cells obtained from: embryonic tissue formed after pregnancy (e.g., blastocyst) but before implantation (i.e., pre-implantation 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 reproductive tissue of a fetus at any time during pregnancy (preferably before 10 weeks of gestation).

[0016] According to some embodiments, pluripotent stem cells are embryonic stem cells, for example, from humans or primates (such as monkeys). 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 usually obtained from preimplantation embryos in the human body or from in vitro fertilization (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. In order to separate human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is separated by immunosurgery, wherein the trophectoderm cells are lysed and removed from the complete ICM by gentle pipetting. The ICM is then plated onto a tissue culture flask containing an appropriate culture medium that enables it to grow. After 9 to 15 days, the outgrowth derived from the ICM is dissociated into clumps by mechanical dissociation or by enzymatic degradation, and the cells are then re-plated onto fresh tissue culture medium. Colonies showing undifferentiated morphology are selected individually by micropipette, mechanically dissociated into clumps, and then re-plated. Subsequently, conventional divisions are performed on the resulting ES cells every 4-7 days. For more details on methods for making human ES cells, 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].

[0017] It will also be understood 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.

[0018] As used herein, the phrase "induced pluripotent stem (iPS) cells" (or embryonic-like stem cells) 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 almost unlimited periods of time.

[0019] iPS cells can be conferred pluripotency by genetic manipulation, which reprograms the cells to acquire characteristics of embryonic stem cells. For example, iPS cells of the present invention can be generated from somatic cells by inducing 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 expression of Oct4, Sox2, Nanog, and Lin28, essentially as described in Yu et al., 2007 and Nakagawa et al., 2008. It should be noted that genetic manipulation (reprogramming) of somatic cells can be performed using any known method, such as using plasmid or viral vectors, or derivative methods without any integration into the genome [Yu J, et al., Science. 2009, 324: 797-801].

[0020] 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 dermis and skin tissue [Hanna et al., 2007; Lowry et al., 2008], b-lymphocytes [Hanna et al., 2007], and adult liver and stomach cells [Aoi et al., 2008].

[0021] iPS cell lines can also be obtained through cell banks (e.g., 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].

[0022] According to some embodiments, the iPS cells are human induced pluripotent stem cells.

[0023] As used herein, the term "pluripotent stem cell-derived ECP" should be understood to refer to a cell product derived from differentiated or partially differentiated pluripotent stem cells and comprising at least 40%, 50%, 60%, or 70% endothelial cells. hESC-derived ECPs can be a mixed population comprising 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).

[0024] As used herein, the term "extracellular vesicle population" refers to a population of extracellular vesicles having pro-angiogenic and / or pro-angiogenic characteristics as a result of production by ECPs derived from pluripotent stem cells. The terms "extracellular vesicle population" and "extracellular vesicles" are used interchangeably to refer to a population of extracellular vesicles having pro-angiogenic and / or pro-angiogenic characteristics.

[0025] The term "extracellular vesicle" (abbreviated as EV) as used herein encompasses exosomes. The terms "extracellular vesicle" and "EV" as used herein may refer in some embodiments to membranous particles having a diameter (or maximum dimension when the particle is non-spherical) of about 10 nm to about 5000 nm, more typically 30 nm to 1000 nm, and most typically about 50 nm to 750 nm. Most commonly, EVs will have a size (average diameter) of up to 5% of the size of the donor cell. Therefore, EVs of particular consideration include those shed from cells.

[0026] As used herein, the isolated EV (or EV population) is an EV that is physically separated from its natural environment. The isolated EV can be physically separated in whole or in part from the ECP derived from the pluripotent stem cell that secretes the EV. In some embodiments of the present disclosure, the isolated extracellular vesicle composition may not contain the ECP that produced them. In some embodiments, the EV may be present in a culture medium for culturing the ECP, or may be free of or substantially free of a culture medium for culturing the ECP. In some embodiments, the isolated EV may be provided at a concentration higher than the concentration of EV present in the culture medium from which the EV is derived.

[0027] The extracellular vesicles (EVs) of the present disclosure can be obtained from ECPs derived from pluripotent stem cells. Thus, pluripotent stem cells have been directed to differentiate into endothelial cell products using appropriate techniques known in the art [see, for example (Ana-MishelSpiroski et al., 2022; MacAskill et al., 2018; McCracken et al., 2019; Zhang et al., 2017)].

[0028] Typically, suitable endothelial cells can be characterized by expressing one or more (ideally both) of the cell surface markers CD31 and CD144. Typically, at least 40%, 50%, or 60% of cells derived from pluripotent stem cells co-express CD31 and CD144. In addition, typically less than 10%, 5%, 2.5%, or 1% of ECPs express the pluripotency marker SSEA-3 / TRA-1-60.

[0029] Generally, any suitable method for isolating, purifying and / or enriching EVs can be used, including, for example, magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick TM (Systems Biosciences, CA, USA) and / or chromatography methods. In some embodiments, extracellular vesicles are separated by centrifugation and / or ultracentrifugation. EVs can also be purified by ultracentrifugation of clarified conditioned medium. They can also be purified into a sucrose cushion by ultracentrifugation. This experimental protocol is described in, for example, Thery et al. Current Protocols in Cell Biol. (2006) 3.22, which is incorporated herein by reference (Théry et al., 2006). In some embodiments, extracellular vesicles are separated by step-by-step size exclusion chromatography. This experimental protocol is described in, for example, Boing et al., Journal of Extracellular Vesicles (2014) 3: 23430, which is incorporated herein by reference. 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, for examples of other experimental protocols, see below.

[0030] Detailed methods for collecting EVs from ECPs derived from pluripotent stem cells comprise, consist of, or consist essentially of a combination of centrifugation, ultrafiltration, and size exclusion chromatography (SEC). Detailed experimental protocols are provided in the Examples.

[0031] Thus, in another aspect, there is provided a method of isolating EVs from ECPs derived from pluripotent stem cells, the method comprising:

[0032] a) centrifuging and / or ultrafiltration the fluid containing EVs to separate the EVs from larger molecular weight species and obtain a fluid enriched in EVs, wherein the EVs are produced by endothelial cell products derived from pluripotent stem cells; and

[0033] b) The EV-enriched fluid is subjected to size exclusion chromatography to obtain a fluid fraction containing isolated EVs.

[0034] The fluid containing EVs produced by ECPs derived from pluripotent stem cells is conditioned medium from which the ECPs have been removed by centrifugation (conditioned medium is medium that has been exposed to cells for a certain amount of time (e.g., 12-36 hours, e.g., 24 hours. The medium contains factors secreted by the cells, including extracellular vesicles). Centrifugation can be performed to remove cells and cell debris from the medium, but retain the EVs in the medium.

[0035] Ultrafiltration can involve centrifugation through a filter of appropriate molecular weight (e.g., 100 kDa) to separate lower molecular weight species from EVs.

[0036] Size exclusion chromatography (SEC) separates molecules based on the size of the molecules by filtering through a resin. The resin is composed of spherical beads containing holes of a specific size. When molecules of different sizes are contained in or excluded from the holes in the matrix, separation occurs. Small molecules diffuse into the holes, and their flow through the column slows down according to their size, while macromolecules do not enter the holes but are eluted into the void volume of the column. Therefore, molecules are separated based on their size when passing through the chromatographic column and are eluted in the order of decreasing molecular weight (MW). Operating conditions and gel selection depend on application and required resolution. Exemplary SEC media include sephacryl, sephadex, superose, superdex and sepharose.

[0037] As described herein, also provided are isolated populations of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents prepared according to the methods described herein for use in promoting angiogenesis and / or neovascularization.

[0038] Suitable isolated populations of pro-angiogenic EVs can be determined as being positive for the markers CD63 and CD81 and negative for the cell contamination marker calnexin. In addition, isolated populations of pro-angiogenic EVs are desirably free of non-EV protein contaminants.

[0039] The angiogenic activity of EVs can be determined using known techniques (Irvin et al., 2014). For example, as described herein, EVs can be used in angiogenesis and / or wound healing assays. Other suitable assays include in vitro proliferation assays, as well as 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.

[0040] One disadvantage of pro-angiogenic EVs previously known in the art is that in order to provide their angiogenic effects, pro-angiogenic EVs need to be used in high doses. 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 less than 50, 25, 10, 5 EVs / cell, for example, as low as 1 EV / cell). In addition, increasing the EV dose does not increase the angiogenic effect. This shows that the pro-angiogenic and / or pro-angiogenic EVs of the present disclosure are significantly different from the pro-angiogenic and / or pro-angiogenic EVs previously known in the art.

[0041] EV can be used immediately or stored before use, whether short-term or long-term, for example, in a frozen state. Protease inhibitors are typically contained in freezing media because they provide the integrity of extracellular vesicles during long-term storage. Freezing at -20°C is not preferred because it is associated with an increased loss of extracellular vesicle activity. More preferably, it is rapidly frozen at -80°C because it retains activity. See, for example, Kidney International (2006) 69, 1471-1476, which is incorporated herein by reference (Zhou et al., 2006). Additives added to the freezing medium can be used to enhance the retention of extracellular vesicle biological activity. Such additives are similar to those used to cryopreserve intact cells and may include, but are not limited to, DMSO, glycerol, and polyethylene glycol.

[0042] The present disclosure also relates to a pharmaceutical composition comprising an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents that can be obtained from endothelial cell products derived from pluripotent stem cells or obtained from endothelial cell products derived from pluripotent stem cells. Preferably, the pharmaceutical composition comprises an isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents that can be obtained from ECPs derived from pluripotent stem cells or obtained from ECPs derived from pluripotent stem cells, and a pharmaceutically acceptable carrier or excipient. The composition may comprise carriers and excipients well known to those skilled in the art. Such compositions can be used to promote angiogenesis and / or neovascularization.

[0043] The isolated populations of pro-angiogenic and / or pro-angiogenic EVs and / or their contents and compositions described herein can be used to treat, for example, ischemic tissue disorders, cardiovascular diseases (e.g., pulmonary hypertension) and / or wound healing, for example. The term ischemic tissue disorder as used herein includes disorders and complications comprising cardiovascular ischemic disorders, including cardiac damage after myocardial infarction and progression to heart failure and lower limb ischemia, mesenteric ischemic disorders and / or renal vascular ischemic disorders.

[0044] The isolated populations of pro-angiogenic and / or pro-angiogenic EVs and / or their contents and compositions described herein can be used therapeutically, for example, when new / improved blood vessel formation is beneficial for tissue transplantation therapy and / or graft engraftment.

[0045] In other embodiments, the present disclosure extends to medical devices coated with the isolated populations of pro-angiogenic and / or pro-angiogenic EVs described herein and / or their contents or compositions described herein. The medical devices can be stents, sutures, bandages, dressings, prostheses, biomaterials engineered for wound healing, and the like.

[0046] The terms "ischemic" and "cardiovascular" disorders and diseases used herein may include interruptions in the blood supply to an organ or tissue. An ischemic event can typically be caused by a blood clot and is most commonly caused when a thrombus breaks off from an atherosclerotic lesion in patients with atherosclerotic stenosis. The resulting stenosis or narrowing or blockage of an artery or other blood vessel may result in a large number of adverse conditions due to this blockage, many of which can have serious consequences for the subject. Ischemic and cardiovascular disorders / diseases referred to herein include, but are not limited to, stroke / transient ischemic attack or cerebrovascular attack, myocardial infarction, myocardial ischemia (angina pectoris), any cardiomyopathy complicated by myocardial ischemia (e.g., symptomatic aortic stenosis, HOCM), cerebral hemorrhage, peripheral (unstable) angina pectoris, peripheral atherosclerotic arterial disease, and other major intravascular abnormalities. The term "intravascular abnormality" includes reference to coronary and cerebrovascular events and peripheral vascular disease. The term "ischemic cardiovascular or cerebrovascular event" generally refers to the acute phase of a medical condition broadly encompassed by the terms "cardiovascular, cerebrovascular, and peripheral arterial disease" (collectively referred to herein as "cardiovascular disease"). Such diseases include cerebrovascular disease and also peripheral arterial disease.

[0047] As used herein, the term "ischemia" refers to an absolute or relative shortage of blood supply, or insufficient blood flow to an organ, body part, or tissue. Relative shortage refers to the difference between blood supply (oxygen delivery) and blood demand (oxygen consumption of tissue). Limited blood supply (usually due to factors in the blood vessels) is most commonly (but not exclusively) caused by contraction or blockage of blood vessels by thromboembolism (blood clots) or atherosclerosis (lipid-laden plaques blocking the arterial lumen). Ischemia causes tissue damage or dysfunction. Myocardial ischemia causes angina pectoris and is referred to herein as ischemic heart disease.

[0048] The term "cardiovascular disease" (CVD) broadly refers to a number of diseases that affect the heart and circulatory system, including aneurysms; angina pectoris; arrhythmias; atherosclerosis; cardiomyopathy; cerebrovascular accident (stroke); cerebrovascular disease; congenital heart disease; congestive heart failure; coronary heart disease (CHD), also known as coronary artery disease (CAD), 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 encompasses ischemia; arterial damage (damage to the endothelial cell lineage) due to physical injury (endarterectomy, balloon angioplasty) or due to chronic injury (including atherosclerosis); myocardial damage (myocardial necrosis); and myonecrosis. In general, any physiological or pathophysiological condition that elicits an angiogenic or neovascularization response is encompassed by the term "cardiovascular disease" as used herein.

[0049] Unless otherwise stated, "a" or "an" means "one or more".

[0050] Unless specifically defined otherwise, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

[0051] The term "subject" (also referred to herein as "patient") as used herein includes warm-blooded animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In more preferred embodiments, the subject is a human.

[0052] As used herein, the terms "treating," "treat," or "treatment" include alleviating, relieving, or eliminating at least one symptom of a disease or condition.

[0053] As used herein, the term "preventing," "prevent," or "prevention" includes stopping or preventing the appearance or presence of at least one symptom of a disease or condition. Alternatively, the term "prevention" can include stopping or preventing the appearance or presence of at least one symptom of a disease or condition.

[0054] The present disclosure is based at least in part on the discovery of a group of miRNAs present in EVs. Without being bound by theory, the pro-angiogenic and / or pro-angiogenic effects of EVs disclosed herein are at least in part due to the miRNA content of EVs. Each member (or combination thereof) of this group of miRNAs represents a target for regulating cell proliferation (and migration), affecting (or regulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and (for example) disorders, diseases, syndromes, and / or conditions affecting blood vessels and / or the vascular system of the human or animal body.

[0055] In the context of the present disclosure, any disclosed miRNA can be targeted to, for example, regulate the expression level of pro-angiogenesis and / or pro-angiogenesis genes in cells. For example, the expression level of any disclosed miRNA can be increased or decreased as needed. Without being bound by theory, it has been shown that the expression level of any disclosed miRNA is associated with a potentially beneficial therapeutic effect. For example, and also without being bound by theory, by targeting one or more of the disclosed miRNAs, angiogenesis and / or neovascularization can be promoted. This may help to change the architecture / structure of the blood vessel wall after injury. Therefore, the present disclosure provides a group of miRNAs, each member of which can be targeted as a tool for regulating (e.g., promoting) angiogenesis and / or neovascularization and / or for treating or preventing (vascular) complications, (vascular) injuries and / or cardiovascular diseases.

[0056] The present disclosure provides compounds and compositions for various therapeutic uses, uses of the compounds and compositions for the manufacture of therapeutically effective medicaments, and methods for treating various diseases, disorders, and conditions. In the context of the present disclosure, such compounds and compositions encompass EVs and / or their contents comprising one or more miRNAs disclosed herein.

[0057] One particular application of the compounds / compositions, medicaments, uses and methods described herein may be in the treatment or prevention of, for example, cardiovascular diseases, such as coronary heart disease, ischemic tissue disorders and pulmonary hypertension.

[0058] In the context of the present invention, the terms "comprising" and "including" encompass embodiments wherein the invention "consists essentially of" or "consists of" the relevant features.

[0059] In one teaching, isolated EVs or isolated populations of EVs and / or their contents are provided for use in therapy, wherein the EVs comprise one or more pro-angiogenic and / or pro-angiogenic miRNAs, for example, including any one or more miRNAs disclosed herein. The isolated EVs or isolated populations of EVs of the present disclosure can be used to treat or prevent cardiovascular disease in a subject in need thereof.

[0060] The disclosed EVs or isolated EVs (or isolated populations of EVs) and / or their contents may contain 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 may contain at least one, two or more of the miRNAs disclosed herein.

[0061] Those skilled in the art are familiar with the term "microRNA" (or "miR"). MicroRNAs are small, non-coding RNA molecules that affect the regulation of gene expression. They are produced by gene sequences or intron / exon sequences; many are encoded by intergenic sequences.

[0062] In the context of the present disclosure, the term microRNA or "miR" may encompass one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0063] (i) miR-126-3p;

[0064] (ii) miR-21-5p;

[0065] (iii) miR-92a-5p;

[0066] (iv) miR-92a-3p;

[0067] (v) miR-7-1-5p;

[0068] (vi) miR-196b-5p;

[0069] (vii) miR-302b-3p;

[0070] (viii) miR-222-3p;

[0071] (ix) miR-6087;

[0072] (x)miR-10a-5p;

[0073] (xi) miR-99b-5p;

[0074] (xii) miR-184;

[0075] (xiii)miR-302a-3p;

[0076] (xiv)miR-423-3p;

[0077] (xv)miR-3184-5p;

[0078] (xvi) miR-7-2-5p;

[0079] (xvii)miR-3529-3p;

[0080] (xviii)miR-92b-3p;

[0081] (xix)miR-342-3p;

[0082] (xx)miR-23a-3p;

[0083] (xxi)miR-302d-3p;

[0084] (xxii) miR-483-3p;

[0085] (xxiii)miR-302c-3p;

[0086] (xxiv)miR-151a-5p;

[0087] (xxv)miR-26a-5p;

[0088] (xxvi)miR-149-5p;

[0089] (xxvii) miR-miR-27b-3p;

[0090] (xxviii)miR-302a-5p.

[0091] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0092] (i) miR-126-3p;

[0093] (ii) miR-21-5p;

[0094] (iii) miR-92a-3p

[0095] (iv) miR-7-1-5p;

[0096] (v) miR-196b-5p;

[0097] (vi) miR-302b-3p;

[0098] (vii) miR-222-3p;

[0099] (viii) miR-6087;

[0100] (ix) miR-10a-5p;

[0101] (x)miR-99b-5p;

[0102] (xi) miR-184;

[0103] (xii) miR-302a-3p;

[0104] (xiii) miR-423-3p;

[0105] (xiv)miR-3184-5p;

[0106] (xv)miR-7-2-5p;

[0107] (xvi)miR-3529-3p;

[0108] (xvii) miR-92b-3p;

[0109] (xviii)miR-342-3p;

[0110] (xix)miR-23a-3p;

[0111] (xx)miR-302d-3p.

[0112] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0113] (i) miR-92a-3p;

[0114] (ii) miR-21-5p;

[0115] (iii) miR-302b-3p;

[0116] (iv) miR-126-3p;

[0117] (v) miR-222-3p;

[0118] (vi) miR-92b-3p;

[0119] (vii) miR-196-5p;

[0120] (viii) miR-10a-5p;

[0121] (viv)miR-483-3p;

[0122] (x)miR-302a-3p;

[0123] (xi) miR-302c-3p;

[0124] (xii) miR-7-1-5p;

[0125] (xiii) miR-99b-5p;

[0126] (xiv)miR-151a-5p;

[0127] (xv)miR-26a-5p;

[0128] (xvi) miR-149-5p;

[0129] (xvii) miR-23a-3p;

[0130] (xviii) miR-27b-3p;

[0131] (xiv)miR-302d-3p;

[0132] (xx)miR-302a-5p.

[0133] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0134] (i) miR-126-3p;

[0135] (ii) miR-21-5p;

[0136] (iii) miR-92a-3p;

[0137] (iv) miR-7-1-5p;

[0138] (v) miR-196b-5p;

[0139] (vi) miR-302b-3p;

[0140] (vii) miR-222-3p;

[0141] (viii) miR-10a-5p;

[0142] (viv)miR-99b-5p;

[0143] (x)miR-302a-3p;

[0144] (xi) miR-92b-3p;

[0145] (xii) miR-23a-3p;

[0146] (xiii)miR-302d-3p.

[0147] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0148] (i) miR-126-3p;

[0149] (ii) miR-21-5p;

[0150] (iii) miR-92a-3p

[0151] (iv) miR-7-1-5p;

[0152] (v) miR-196b-5p;

[0153] (vi) miR-302b-3p;

[0154] (vii) miR-126-3p;

[0155] (viii)miR-222-3p.

[0156] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0157] (i) miR-126-3p;

[0158] (ii) miR-21-5p;

[0159] (iii) miR-92a-3p;

[0160] (iv) miR-7-1-5p; and

[0161] (v)miR-196b-5p.

[0162] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0163] (i) miR-92a-3p;

[0164] (ii) miR-21-5p;

[0165] (iii) miR-302b-3p;

[0166] (iv) miR-126-3-p; and

[0167] (v)miR-222-5p.

[0168] In one teaching, the term "miR" can encompass any one or more miRs (or combinations thereof) selected from the group consisting of or consisting essentially of:

[0169] (i) miR-126-3p;

[0170] (ii) miR-21-5p; and

[0171] (iii)miR-92a-3p.

[0172] The miRs described herein may each individually have pro-angiogenic and / or pro-angiogenic effects on target cells or tissues. A combination of any two or more of the miRs described herein may produce additive or synergistic effects on target cells or tissues, thereby promoting angiogenesis and / or neovascularization.

[0173] Without being bound by theory, a combination of 2, 3, 4, 5, 10, 15 or 20 of the miRs detailed herein may comprise at least 50%, 60%, 65%, 70% or 75% of the total miR content of a disclosed EV or isolated EV (or isolated population of EVs).

[0174] In one teaching, the miR content of the disclosed EVs or isolated EVs (or isolated populations of EVs) may comprise a combination of two, three, four, or five or more of the miRs disclosed herein. Without being bound by theory, it is contemplated that EVs or isolated EVs (or isolated populations of EVs) of the present disclosure comprising a combination of two or more of the miRs disclosed herein can produce enhanced pro-angiogenic and / or pro-angiogenic effects on target cells or tissues.

[0175] Among the miRs listed above, the following specific combinations of miRs or EVs / isolated EVs containing them have been shown to be associated with angiogenesis and / or neovascularization:

[0176] (i) miR-126-3p;

[0177] (ii) miR-21-5p;

[0178] (iii) miR-92a-3p;

[0179] (iv) miR-7-1-5p; and

[0180] (v) miR-196b-5p;

[0181] or

[0182] (i) miR-92a-3p;

[0183] (ii) miR-21-5p;

[0184] (iii) miR-302b-3p;

[0185] (iv) miR-126-3-p; and

[0186] (v) miR-222-5p;

[0187] or

[0188] (i) miR-126-3p;

[0189] (ii) miR-21-5p; and

[0190] (iii)miR-92a-3p.

[0191] Therefore, by targeting one or more or a combination of these miRs, or using EVs / isolated EVs containing the same, it may be possible to treat or prevent conditions or diseases in which enhanced pro-angiogenic and / or pro-angiogenic effects on target cells or tissues may be desired or beneficial.

[0192] The present disclosure further provides modulators of any of the miRs described herein.

[0193] A miR modulator of the present disclosure can be any molecule or compound that is capable of increasing or inhibiting (decreasing) the expression of a particular miR, such as one or more of the miRs described herein.

[0194] Thus, a "miR modulator" 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 miRs listed in (i)-(xxviii) above (and any combination thereof).

[0195] Thus, the present invention provides miR modulators (e.g., modulators that increase 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 in (i)-(xxviii) above (and any combination thereof), for use in any one or more of the therapeutic applications described herein, including:

[0196] used in medicine or as a drug;

[0197] Treat or prevent cardiovascular disease;

[0198] Treat or prevent coronary heart disease;

[0199] treating or preventing ischemic tissue disorders;

[0200] Treat or prevent pulmonary hypertension;

[0201] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0202] Regulate or promote wound healing.

[0203] In one aspect, the present disclosure provides a miR modulator for use in:

[0204] medicine or as a drug;

[0205] Treat or prevent cardiovascular disease;

[0206] Treat or prevent coronary heart disease;

[0207] treating or preventing ischemic tissue disorders;

[0208] Treat or prevent pulmonary hypertension;

[0209] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0210] Regulate or promote wound healing.

[0211] In another aspect, the present disclosure provides a method for:

[0212] Treat or prevent cardiovascular disease;

[0213] Treat or prevent coronary heart disease;

[0214] treating or preventing ischemic tissue disorders;

[0215] Treat or prevent pulmonary hypertension;

[0216] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0217] Regulate or promote wound healing;

[0218] The method comprises administering a miR modulator to a subject in need thereof. The modulator can be administered in a therapeutically effective amount or a modulating amount. The subject in need thereof can be a human or animal subject.

[0219] In another aspect, the present disclosure provides the use of a miR modulator in the manufacture of a medicament for:

[0220] Treat or prevent cardiovascular disease;

[0221] Treat or prevent coronary heart disease;

[0222] treating or preventing ischemic tissue disorders;

[0223] Treat or prevent pulmonary hypertension;

[0224] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0225] Regulate or promote wound healing.

[0226] Thus, the present disclosure provides modulators (e.g., modulators that increase expression) of one or more of:

[0227] (i) miR-126-3p;

[0228] (ii) miR-21-5p;

[0229] (iii) miR-92a-3p

[0230] (iv) miR-7-1-5p;

[0231] (v) miR-196b-5p;

[0232] (vi) miR-302b-3p;

[0233] (vii) miR-126-3p;

[0234] (viii) miR-222-3p;

[0235] or one or more of the following:

[0236] (i) miR-126-3p;

[0237] (ii) miR-21-5p;

[0238] (iii) miR-92a-3p;

[0239] (iv) miR-7-1-5p; and

[0240] (v) miR-196b-5p;

[0241] or one or more of the following:

[0242] (i) miR-92a-3p;

[0243] (ii) miR-21-5p;

[0244] (iii) miR-302b-3p;

[0245] (iv) miR-126-3-p; and

[0246] (v) miR-222-5p;

[0247] or one or more of the following:

[0248] (i) miR-126-3p;

[0249] (ii) miR-21-5p; and

[0250] (iii)miR-92a-3p.

[0251] The regulator is used for:

[0252] Treat or prevent cardiovascular disease;

[0253] Treat or prevent coronary heart disease;

[0254] treating or preventing ischemic tissue disorders;

[0255] Treat or prevent pulmonary hypertension;

[0256] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0257] Regulate or promote wound healing.

[0258] The extent of regulation exerted by the miR modulators of the invention can be assessed relative to "normal" or "control" levels of miR expression and / or miR target gene expression that may be present in healthy / normal tissue that does not exhibit pathology associated with a cardiovascular disease or condition.

[0259] MiR modulators for any use or method described herein can take the form of inhibitors of one or more miRs described herein. The term "miR inhibitor" can include compounds or molecules that inhibit or reduce the expression, function and / or activity of a miR, including, for example, one or more miRs described herein.

[0260] Modulators of the present disclosure may include miR promoters, molecules that increase the expression of the relevant miR (in a cell). The term "miR promoter" may include compounds or molecules that increase the expression, function and / or activity of a miR (including, for example, one or more miRs described herein).

[0261] The miR promoter may include, for example, a miR mimic, i.e., a nucleic acid encoding a related miR for expression in a cell. It will be understood by those skilled in the art that when a nucleic acid encoding a particular miR is introduced into a cell, the nucleic acid will provide additional copies of the miR (the additional copies supplementing any natural copies (or copies) expressed by the cell) such that the end result is overexpression of the miR in the cell.

[0262] Suitable miR mimics can comprise double-stranded RNA molecules that mimic the mature miR duplex.

[0263] The nucleic acid encoding the relevant miR may comprise a stem-loop miRNA.

[0264] The nucleic acid can encode any miR described herein (including any miR listed in (i)-(xxviii) herein).

[0265] A miR-encoding nucleic acid for expression may also comprise (or be operably linked to) a promoter element and a polyA element. The promoter element and the polyA element may "flank" the miR-encoding nucleic acid sequence.

[0266] The nucleic acid can be provided in the form of a vector for delivery to the cell.

[0267] The vector may include a viral vector.

[0268] The vector may comprise an adenoviral vector, such as HAdV5 or an adeno-associated virus (such as AAV1, AAV2, AAV3, AAV4 or AAV5), or a lentivirus.

[0269] The miR promoter, eg, nucleic acid encoding the miR, can be packaged or contained within a viral, adenoviral, or AAV5 vector.

[0270] MiR inhibitors suitable 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 peptidyl nucleic acids, including PNA), carbohydrates, lipids, antibodies (including antigen-binding fragments thereof), and the like.

[0271] Any miR modulator of the present disclosure can be administered directly to the vessel wall to be treated (e.g., a vessel wall that has been surgically repaired and / or shows signs of disease and / or injury or damage). The miR modulator of the present disclosure can be administered packaged in a vector (e.g., a viral (adenoviral) vector).

[0272] The present disclosure provides an adenoviral vector comprising sequences for expressing one or more of the following miRs in a cell:

[0273] (i) miR-126-3p;

[0274] (ii) miR-21-5p;

[0275] (iii) miR-92a-5p;

[0276] (iv) miR-92a-3p;

[0277] (v) miR-7-1-5p;

[0278] (vi) miR-196b-5p;

[0279] (vii) miR-302b-3p;

[0280] (viii) miR-222-3p;

[0281] (ix) miR-6087;

[0282] (x)miR-10a-5p;

[0283] (xi) miR-99b-5p;

[0284] (xii) miR-184;

[0285] (xiii)miR-302a-3p;

[0286] (xiv)miR-423-3p;

[0287] (xv)miR-3184-5p;

[0288] (xvi) miR-7-2-5p;

[0289] (xvii)miR-3529-3p;

[0290] (xviii)miR-92b-3p;

[0291] (xix)miR-342-3p;

[0292] (xx)miR-23a-3p;

[0293] (xxi)miR-302d-3p;

[0294] (xxii) miR-483-3p;

[0295] (xxiii)miR-302c-3p;

[0296] (xxiv)miR-151a-5p;

[0297] (xxv)miR-26a-5p;

[0298] (xxvi)miR-149-5p;

[0299] (xxvii) miR-miR-27b-3p;

[0300] (xxviii)miR-302a-5p.

[0301] The present disclosure provides a composition comprising a miR modulator of the present disclosure and one or more excipients.

[0302] The present disclosure also provides a pharmaceutical composition comprising a miR modulator of the present disclosure and one or more pharmaceutically acceptable excipients.

[0303] A composition or pharmaceutical composition of the present disclosure may comprise a miR modulator that is a mimetic of one or more of the following miRs:

[0304] (i) miR-126-3p;

[0305] (ii) miR-21-5p;

[0306] (iii) miR-92a-5p;

[0307] (iv) miR-92a-3p;

[0308] (v) miR-7-1-5p;

[0309] (vi) miR-196b-5p;

[0310] (vii) miR-302b-3p;

[0311] (viii) miR-222-3p;

[0312] (ix) miR-6087;

[0313] (x)miR-10a-5p;

[0314] (xi) miR-99b-5p;

[0315] (xii) miR-184;

[0316] (xiii)miR-302a-3p;

[0317] (xiv)miR-423-3p;

[0318] (xv)miR-3184-5p;

[0319] (xvi) miR-7-2-5p;

[0320] (xvii)miR-3529-3p;

[0321] (xviii)miR-92b-3p;

[0322] (xix)miR-342-3p;

[0323] (xx)miR-23a-3p;

[0324] (xxi)miR-302d-3p;

[0325] (xxii) miR-483-3p;

[0326] (xxiii)miR-302c-3p;

[0327] (xxiv)miR-151a-5p;

[0328] (xxv)miR-26a-5p;

[0329] (xxvi)miR-149-5p;

[0330] (xxvii) miR-miR-27b-3p;

[0331] (xxviii)miR-302a-5p.

[0332] The composition or pharmaceutical composition can be used (i) for the following or (ii) for use in the following methods:

[0333] Treat or prevent cardiovascular disease;

[0334] Treat or prevent coronary heart disease;

[0335] treating or preventing ischemic tissue disorders;

[0336] Treat or prevent pulmonary hypertension;

[0337] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0338] Regulate or promote wound healing.

[0339] When the composition or pharmaceutical composition is for use in a method of treatment, the composition or pharmaceutical composition may be administered to a subject in need thereof (as defined herein).

[0340] Furthermore, the composition or pharmaceutical composition can be used in the manufacture of a medicament for:

[0341] Treat or prevent cardiovascular disease;

[0342] Treat or prevent coronary heart disease;

[0343] treating or preventing ischemic tissue disorders;

[0344] Treat or prevent pulmonary hypertension;

[0345] Regulating or promoting angiogenesis and / or neovascularization; and / or

[0346] Regulate or promote wound healing.

[0347] In another aspect, the present disclosure further provides a method for identifying pro-angiogenic or pro-angiogenic EVs or their contents, wherein the method comprises detecting the presence of 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.

[0348] In preferred embodiments, the method comprises detecting the presence and / or measuring the expression level of any one or more miRNAs selected from the group consisting of or essentially consisting of:

[0349] (i) miR-126-3p;

[0350] (ii) miR-21-5p;

[0351] (iii) miR-92a-5p;

[0352] (iv) miR-92a-3p;

[0353] (v) miR-7-1-5p;

[0354] (vi) miR-196b-5p;

[0355] (vii) miR-302b-3p;

[0356] (viii) miR-222-3p;

[0357] (ix) miR-6087;

[0358] (x)miR-10a-5p;

[0359] (xi) miR-99b-5p;

[0360] (xii) miR-184;

[0361] (xiii)miR-302a-3p;

[0362] (xiv)miR-423-3p;

[0363] (xv)miR-3184-5p;

[0364] (xvi) miR-7-2-5p;

[0365] (xvii)miR-3529-3p;

[0366] (xviii)miR-92b-3p;

[0367] (xix)miR-342-3p;

[0368] (xx)miR-23a-3p;

[0369] (xxi)miR-302d-3p;

[0370] (xxii) miR-483-3p;

[0371] (xxiii)miR-302c-3p;

[0372] (xxiv)miR-151a-5p;

[0373] (xxv)miR-26a-5p;

[0374] (xxvi)miR-149-5p;

[0375] (xxvii) miR-miR-27b-3p;

[0376] (xxviii)miR-302a-5p;

[0377] or:

[0378] (i) miR-126-3p;

[0379] (ii) miR-21-5p;

[0380] (iii) miR-92a-3p

[0381] (iv) miR-7-1-5p;

[0382] (v) miR-196b-5p;

[0383] (vi) miR-302b-3p;

[0384] (vii) miR-126-3p;

[0385] (viii) miR-222-3p;

[0386] or:

[0387] (i) miR-126-3p;

[0388] (ii) miR-21-5p;

[0389] (iii) miR-92a-3p;

[0390] (iv) miR-7-1-5p; and

[0391] (v) miR-196b-5p;

[0392] or:

[0393] (i) miR-92a-3p;

[0394] (ii) miR-21-5p;

[0395] (iii) miR-302b-3p;

[0396] (iv) miR-126-3-p; and

[0397] (v) miR-222-5p;

[0398] or:

[0399] (i) miR-126-3p;

[0400] (ii) miR-21-5p; and

[0401] (iii)miR-92a-3p.

[0402] Without being 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:

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412] Each one or more of the disclosed genes may represent a target for regulating cell proliferation (and migration), affecting (or regulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and (for example) disorders, diseases, syndromes, and / or conditions that affect blood vessels and / or the vascular system of the human or animal body. Thus, the expression, function, and / or activity of any one or more of the genes described may be modulated, alone or in combination, to regulate cell proliferation (and migration), affecting (or regulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and (for example) to treat or prevent disorders, diseases, syndromes, and / or conditions that affect blood vessels and / or the vascular system of the human or animal body.

[0413] Detailed description

[0414] The present disclosure will now be further described by way of example and with reference to the accompanying drawings, which show:

[0415] Figure 1. Isolation and characterization of extracellular vesicles (EVs) from human embryonic stem cell-derived endothelial cell product (hESC-ECP) conditioned medium. A. Schematic diagram of human embryonic stem cell-derived endothelial cell product (hESC-ECP) differentiation at the mesoderm (day 4) and endothelial cell-rich (day 8) stages and secretion of extracellular vesicles (EVs) from cells. B. Workflow for separation of particles from hESC-ECP conditioned medium by a combination of ultrafiltration and size exclusion chromatography (SEC). Created using BioRender.com. C. Representative graphs of nanoparticle tracking analysis (NTA) showing the size distribution of EVs in mixed fractions 5 and 6. D. Characterization of EV surface markers in mixed fractions 5 and 6 by Western blotting. Cell lysates were used as positive controls. Detection of calnexin appeared as a band at 90 KDa, detection of CD63 appeared at 30-65 KDa, and detection of CD81 appeared at 22-26 KDa. E. Transmission electron microscopy (TEM) imaging of particles in mixed fractions 5 and 6. Arrows indicate EVs. Scale bar = 100 nm (left panel), scale bar = 1 μm (right panel). The scatter plot (bottom panel) shows the EV size distribution and circularity ratio (scale: 0-1) determined from a total of 40 TEM images using the "TEM Exosome Analyzer" tool. Each dot (pink) corresponds to a single EV. Error bars represent the median with interquartile range.

[0416] Figure 2. Extracellular vesicles from human embryonic stem cell-derived endothelial cell products (hESC-eEVs) induce endothelial cell (EC) tube formation at low concentrations. A. Hypoxic human umbilical vein endothelial cells (HUVECs) were treated with increasing concentrations of EVs (dose range: 1-10 5Figure 3 Tube formation assay of human cardiac microvascular endothelial cells (HCMEC) using 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 served as positive controls (GF+). The figure (right) shows the number of grids formed and the total length of the tubes at 4 hours. Images were analyzed using the "Angiogenesis Analyzer" tool on ImageJ. Control samples (GF- and GF+) are depicted as black circles, and HUVEC hypoxic EV-treated samples (HUVEC hyp EVs) are depicted as purple triangles. Statistical significance (expressed as p-values) was determined by one-way ANOVA using Dunnett's multiple comparison test. Error bars represent SD. Scale bar = 1 mm. B. hESC-eEVs were cultured using increasing concentrations (dose range: 1-10 5 EV / cell) (n=4) were quantified for tube formation assay on HCMEC. Cells cultured in basal medium and treated with sterile 0.1 μm filtered PBS (vehicle control) served as negative control (GF-). 5 HUVEC hypoxic EV / cell treated cells served as a positive control. The graph shows the number of meshes formed and the total length of the tubes at 4 hours. The data were analyzed using the "Angiogenesis Analyzer" tool. Control samples (GF- and GF+) are depicted as black circles, HUVEC hypoxic EV treated samples (HUVEC hyp EV) are depicted as purple triangles, and hESC-eEV treated samples are depicted as green circles. Statistical significance (expressed as p-value) was determined by one-way ANOVA using Dunnett's multiple comparison test. Error bars represent SD. C. Using increasing concentrations of hESC-eEV (dose range: 1-10 5 HCMECs were stained with Calcein AM for tube formation assay (EV / cell, n=4). Cells cultured in basal medium and treated with sterile 0.1 μm filtered PBS (vehicle control) served as negative controls (GF-). 5 HUVEC hypoxic EV / cell treated cells served as positive control. Scale bar = 500 μm. D. Human embryonic stem cell-derived mesodermal cell products extracellular vesicles (hESC-mEV) (dose range: 1-10 5HCMECs treated with 100 EVs / cell, n=3) were subjected to tube formation assays. Cells cultured in basal medium and treated with sterile-filtered PBS (vehicle control) served as negative controls (GF-). Cells treated in fully supplemented medium (GF+) or with 1 hESC-eEV / cell served as positive controls. Images were analyzed using the "Angiogenesis Analyzer" tool on ImageJ. Control samples (GF- and GF+) are depicted as black circles, hESC-eEV-treated samples are depicted as green circles, and hESC-mEV-treated samples are depicted as red triangles. Statistical significance (expressed as p-value) was determined by single-factor ANOVA using Dunnett's multiple comparison test. The figure (right) shows the number of grids formed and the total length of the tube at 4 hours. Error bars represent SD. Scale bar = 1 mm. E. Schematic diagram of samples collected at different stages of separating particles from hESC-EC cell culture medium. Created using BioRender.com. F. Tube formation assays performed using samples collected at different stages of separating particles from hESC-EC cell culture medium. Cells cultured in basal medium and treated with PBS (vehicle control) served as negative controls (GF-). Cells cultured in fully supplemented medium (GF+) or treated with human recombinant VEGFA 165 Cells treated with or cultured with basal medium and treated with one hESC-eEV / cell served as positive controls.

[0417] Figure 3 Extracellular vesicles from human embryonic stem cell-derived endothelial cell products promote endothelial cell wound healing at low concentrations. 2 EV / cell) or 10 5 Wound healing assay of human cardiac microvascular endothelial cells (HCMEC) treated with hypoxic EV / cells of human umbilical vein endothelial cells (HUVEC) (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. The figure (below) shows the percentage of wound closure 6 hours, 12 hours, and 24 hours after wound induction. The data were analyzed using the "MRI wound healing" tool. Control samples (GF+ and GF-) are depicted as black circles, HUVEC hypoxic EV-treated samples (HUVEC hyp EV) are depicted as purple triangles, and hESC-eEV-treated samples are depicted as green circles. Statistical significance (expressed as p-value) was determined by one-way ANOVA using Dunnett's multiple comparison test. Error bars represent SD. Scale bar = 100 μm.

[0418] Figure 4. Extracellular vesicles from human embryonic stem cell-derived endothelial cell products are enriched for pro-angiogenic miRNAs. A. Graph shows the normalization of small RNA biotypes to the total number of small RNA reads (RPMM) in EV samples and human embryonic stem cell-derived endothelial cell products (hESC-ECPs) (n=3). 总 ) per million mapped reads. B. Graph showing the percentage of reads accounted for by the top 20 miRNAs for each EV and cellular RNA sample. Across all datasets, the top 20 miRNAs corresponded to 68% to 86% of the total miRNA reads. The top 5 miRNAs in each group are highlighted. In the stacked bar chart, each miRNA is represented by a different color to provide an understanding of the differences and similarities between the top 20 miRNAs in each group. C. Graph showing the RPMM of the top 20 miRNAs in each group (corresponding to 68% to 86% of the total RPMM) and their role in angiogenesis. The top 5 miRNAs in each group are separated by a vertical red dashed line. Angiogenic miRNAs are highlighted in green, and anti-angiogenic miRNAs are highlighted in red. A literature search was performed on PubMed in July 2022.

[0419] Supplementary Figure 1. Representative flow cytometric analysis of hESC-ECPs. Cells were stained for endothelial cells (CD31 and CD144) (right panel), pluripotency markers (TRA-1-60 and SSEA-3) (left panel), and their corresponding isotype controls.

[0420] Supplementary Figure 2. Combination of ultrafiltration and SEC allows for good separation of fractions containing particles and proteins. This figure shows the particle and protein concentrations of hESC-ECP conditioned medium fractions generated after SEC, as determined by NTA and spectrophotometry, respectively. Particles eluted in fractions 5 and 6, while proteins eluted in fractions 12 and 13.

[0421] Replenish Figure 3 Small RNA sequencing revealed a diverse composition of small RNA classes in EV samples. A Venn diagram shows the presence of unique and common small RNA molecules in different EV samples. Average RPMM was filtered out from the analysis. 总 Numerator <10.

[0422] Supplementary Figure 4. Principal component analysis (PCA) of the total reads of the small RNA sequencing dataset.

[0423] Replenish Figure 5 .Small RNA sequencing read characteristics. A. RNA input reads for alignment after filtering out UniVec / rRNA / low-quality reads (n=3). B. Stacked bar chart showing the read composition of each sample. Reads have been aligned to the total number of reads (RPMM 总C. Correlation between the percentage of unmapped reads and the number of particles used in small RNA sequencing of EV samples.

[0424] Figure 5 Extracellular vesicles from RC11 human embryonic stem cell-derived endothelial cell products (RC11-eEVs) induce endothelial cell (EC) tube formation at low concentrations. 5 Tube formation assay (n=3) was performed on human cardiac microvascular endothelial cells (HCMEC) treated with 1% EV / cell (n=3). Cells cultured in basal medium and treated with sterile-filtered PBS (vehicle control) served as negative controls (GF-). Cells treated in fully supplemented medium (GF+) or with human embryonic stem cell-derived endothelial cell products (hESC-eEV) served as positive controls. The number of grids formed and the total length of the tubes at 4 hours were quantified. Images were analyzed using the “Angiogenesis Analyzer” tool on ImageJ. Control samples (GF+ and GF-) are depicted as black circles, hESC-eEV-treated samples are depicted as green circles, and RC11-eEV-treated samples are depicted as blue circles. Statistical significance (expressed as p-values) was determined by one-way ANOVA using Dunnett’s multiple comparison test. Error bars represent SD. Scale bar = 1 mm.

[0425] Figure 6 :Extracellular vesicles (RC11eEV) from RC11 human embryonic stem cell-derived endothelial cell products promote endothelial cell (EC) wound healing at low concentrations. Human cardiac microvascular endothelial cells (HCMEC) treated with EVs were subjected to wound healing assays (n=3). Cells cultured in basal medium and treated with PBS (vehicle control) served as negative controls (GF-). Cells treated in fully supplemented medium (GF+) or with human embryonic stem cell-derived endothelial cell products (hESC-eEV) served as positive controls. The percentage of wound closure was quantified 6 hours, 12 hours, and 24 hours after wound induction. Control samples (GF+ and GF-) are depicted as black circles, hESC-eEV-treated samples are depicted as green circles, and RC11-eEV-treated samples are depicted as blue circles. Statistical significance (expressed as p-values) was determined by one-way ANOVA using Dunnett's multiple comparison test. Error bars represent SD. Scale bar = 100 μm.

[0426] Figure 7 : The top 20 microRNA cargos of RC11-eEV and hESC-eEV showed 65% overlap.

[0427] Materials and Methods

[0428] hESC-ECP differentiation

[0429] 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 the guidelines of the UK Stem Cell Bank Steering Committee (project approval numbers SCS11-51 and SCSC17-26). Briefly, hESCs were plated on a fibronectin matrix on day 0 (d0). On d1, lateral mesoderm was induced using a GSK3 inhibitor (CHIR99021) (7 μM) and BMP4 (25 ng / mL) added to N2B27 / Neurobasal / DMEM:F12 medium. Endothelial induction was then performed on d4 using forskolin (2 μM) and vascular endothelial growth factor (VEGF) (200 ng / mL) in StemPro34 medium. In the final step, the cells were replated and cultured without matrix until d8. For EV isolation, the medium was replaced with EV-free medium on d7, and the conditioned medium was collected 24 hours later. To prepare EV-free culture medium, human serum was centrifuged at 3,000 × g for 55 min (4°C), ultrafiltered using an Amicon-Ultra15 centrifugal filter unit (Merck Millipore) (molecular weight cutoff, 100 kDa), and supplemented with the cell culture medium.

[0430] hESC-ECP flow cytometry

[0431] Cells were stained with antibodies on day 8 to determine the proportion of multipotent cells (SSEA-4+ / TRA-181+) and endothelial cells (CD31+ / CD144+) in the cell population. Flow cytometry was performed using the BD LDR Fortessa system (Becton) or the Attune NxT system (Thermo Fisher Scientific), and data were analyzed using FlowJo software (FlowJo LLC, Ashland, USA).

[0432] EV isolation

[0433] To isolate EV, conditioned medium was obtained from the cells at 70-90% confluence and centrifuged at 3,000 x g for 15 minutes (4 ° C) to remove cell debris. The supernatant was collected and concentrated to 1 ml by centrifugation at 4,000 x g for 15 minutes (4 ° C) using an Amicon-Ultra 15 centrifugal filter unit (Merck Millipore) (molecular weight cutoff of 100 KDa). 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 entered the column matrix, 0.1 μm filtered DPBS (Gibco) supplemented with 1% penicillin / streptomycin (Gibco) was continuously added to ensure that the sample was completely discharged. The eluate was collected in 15 consecutive 1 ml fractions by gravity. For each fraction, the amount of protein was determined spectrophotometrically (absorbance 280 nm, Nanodrop, Thermo Fisher Scientific).

[0434] Nanoparticle Tracking Analysis (NTA)

[0435] Particle concentration and size distribution 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 2*10 8 -1*10 9 The concentration of particles / ml was calculated. The camera screen gain was set to 2 and the camera level was set to 16. The settings remained unchanged between samples, and each video was analyzed to obtain the mean, mode, median, and estimated concentration for each particle size. Analysis was performed using NTA software (version 3.3) using 60 seconds of video capture for each sample (5 replicates per sample). For video processing, the screen gain was set to 14 and the detection threshold was set to 3.

[0436] Western blotting

[0437] The EV-rich fraction was concentrated by centrifugation at 4,000 x g for 15 minutes (4 ° C) using Amicon-Ultra 15 centrifugal filter units (Merck Millipore) with a molecular weight cutoff of 100 KDa. For the 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 minutes at 4 ° C. The supernatant was collected and the protein concentration was determined using a PierceTM BCA protein assay kit (ThermoFisher Scientific). By NuPAGE TM 4-12% Bis-Tris polyacrylamide gel (ThermoFisher Scientific) was used to separate 5*10 10 Each particle and 15 mg of protein were separated and transferred to a nitrocellulose membrane (Invitrogen) by electrophoresis. The membrane was probed for CD63 (Santa Cruz Biotechnology), CD81 (Santa Cruz Biotechnology), and calnexin (Abcam).

[0438] Transmission electron microscopy (TEM)

[0439] The isolated particles were diluted 100-fold in 0.1 μm-filtered DPBS and mixed with 4% methanol-free formaldehyde solution (ThermoFisher Scientific) in a 1:1 ratio. A drop of this solution was placed on a Petri dish, and a Formvar-coated 200-mesh gold grid (Taab, Aldermaston, UK) was floated on it for 20 minutes. The grid was then transferred to PBS and washed twice for 5 minutes. EVs were then re-fixed on the grid with 1% glutaraldehyde solution for 5 minutes and washed twice again in PBS. Finally, the grid was transferred to a drop of 0.5% uranyl acetate-2% 25 centipoise methylcellulose (Sigma-Aldrich) solution. After staining for 5 minutes, excess liquid was removed, the grid was allowed to air dry, and then examined on a JEOL JEM-1400 series 120 kV transmission electron microscope. TEM images were analyzed using the "TEM Exosome Analyzer" (Kotrbová et al., 2019).

[0440] Endothelial cell culture

[0441] HCMEC and HUVEC were obtained from PromoCell, UK. HCMEC were cultured at 37°C in a humidified atmosphere containing 5% CO2 and 95% O2 in a solution supplemented with 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 R3 IGF), 0.5 ng / ml VEGF 165 HUVECs were cultured in MV2 medium (Promocell) supplemented with 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 R3 IGF), 0.5 ng / ml VEGF 165 (recombinant human), 1 μg / ml ascorbic acid, 22.5 μg / ml heparin, 0.2 μg / ml hydrocortisone in EBM2 medium (Promocell). To isolate EVs from hypoxic HUVECs, when 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 unit (Merck Millipore) (molecular weight cutoff of 100 KDa) after centrifugation at 3,000 x g for 55 minutes (4°C) and supplemented into the cell culture medium.

[0442] Tube formation assay

[0443] The angiogenic effect of hESC-eEV, hESC-mEV and HUVEC hypoxic EV on HCMEC was evaluated by tube formation assay. After incubation at 37°C for 30 minutes, the cells were stained with 2 μM / ml calcein AM (Invitrogen). ECM gel (Sigma) with reduced growth factors was thawed on ice at 4°C overnight and then plated onto 96-well angiogenesis μ-plates (Ibidi, UK) at 10 μl per well. After gelation at 37°C for 30 minutes, 1.5×10 4 HCMEC were seeded into each well. HCMEC were cultured with fully supplemented MV2 medium (Promocell) or basal MV2 medium supplemented with DPBS+P / S (Promocell, UK) or EV containing increasing concentrations (1-10 5Cells were cultured in basal MV2 medium (100 EVs / cell). 4× bright-field micrographs were acquired using the EVOS XL Imaging System (Invitrogen). The number of lattices and branches, as well as the total length of the tubes, were analyzed using the “Angiogenesis Analyzer” plugin (Gilles Carpentier) in ImageJ software (Carpentier et al., 2020).

[0444] Wound healing assay

[0445] The ability of hESC-eEVs to promote wound healing in vitro was assessed by a scratch assay. HCMECs were seeded in 6-well plates to form a confluent monolayer. When the cells reached 70-90% confluence, the cell monolayer was scratched with a sterile P200 pipette tip and the culture medium was replaced with fully supplemented MV2 medium (Promocell), or basal MV2 medium supplemented with DPBS+P / S (Promocell, UK), or containing increasing concentrations of hESC-eEVs (1-10 μg / ml). 2 EVs / cell) or 10 5 HUVECs were cultured in basal medium containing hypoxic EVs / cells for 24 hours. Wound closure of HCMECs was captured using bright-field microscopy at 0, 6, 12, and 24 hours after wounding. Percent wound coverage was calculated using the MRI Wound Healing Tool (Montpellier Resources) on ImageJ software, with the 0-hour value being 0%.

[0446] RNA extraction

[0447] 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 quantity and quality were further analyzed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Inc.) using a total RNA Pico Series II chip.

[0448] Small RNA Sequencing

[0449] EV isolation by a combination of ultrafiltration and SEC yields highly pure EV preparations, but yields are lower than other methods (Théry et al., 2018). To compensate for the low input of our libraries, we used the highest possible input for sequencing each EV library, with a minimum of 1 ng of total RNA (n = 3). First, small RNAs ranging from 18 to 30 nt were selected by 15% urea-PAGE gel electrophoresis and gel extraction. After gel purification, adenylated 3′ adapters were ligated to the small RNA fragments. A barcoded reverse transcription (RT) primer was used to anneal the 3′ adenylated adapters to combine with excess unligated 3′ adenylated adapters. Then, 5′ adapter ligation and RT reactions were performed. After first-strand cDNA synthesis, the products were amplified through 15 cycles. A second size selection of 103-115 bp fragments was performed from the gel. This step was performed to purify the PCR products and remove any nonspecific products. After gel purification, PCR yield was quantified using Qubit (Invitrogen, catalog number Q33216). The samples were pooled together to prepare single-stranded DNA circles (ssDNA circles) to obtain the final small RNA library. DNA nanoballs (DNBs) were generated using ssDNA circles by rolling circle replication (RCR) to amplify the fluorescent signal during sequencing. DNBs were loaded into patterned nanoarrays and 50 bp single-end reads were read through on the BGISEQ-500 platform for subsequent data analysis (sequencing depth: ≥2 × 10 per sample). 7 reads).

[0450] Sequencing read mapping and small RNA annotation

[0451] Raw data from BGI-SEQ500 were in fastq format. We used two mapping and quantification methods to ensure robust analysis of EV miRNA profiles, the first of which used the exceRpt small RNAseq processing pipeline (Rozowsky et al., 2019). Files were exported to the exceRpt small RNA-seq pipeline (version 4.6.2) in Genboree Workbench for quality control and mapping of reads to the human genome and a default small RNA library. This allowed for single mismatches down to 18 nucleotides. Reads that were not successfully sheared (0%), reads that did not pass quality filtering (<1.2%), UniVec contaminants (<0.6%), and rRNA (5–51.4%) were filtered out of the analysis. After these initial filters, the average number of reads per group used for alignment ranged from 2.47 to 2.83 × 10 7 Reads (Supplementary Figure 5A). A high percentage of reads failed to map to either the genomic or small RNA libraries, likely due to background nonspecific amplification in the lower input libraries (particularly in hESC-eEVs and HUVEC hypoxic EVs, where 30.4–78% of reads mapped were unmapped) (Supplementary Figure 5 B). We found a negative correlation between the percentage of unmapped reads and the number of particles used for RNA extraction (rho -0.7, p < 0.05, Spearman rank), suggesting that low initial input for a particular sample may result in higher background amplification (Supplementary Figure 5 C). We consider this low input in the sequencing library an acceptable trade-off for the high purity of our EV preparations. Unmapped reads were discarded from all further analyses. Principal component analysis (PCA) of the remaining reads for each sample revealed distinct small RNA profiles (Supplementary Figure 4).

[0452] Reads were aligned and quantified on the recently released reference human genome (GRCh38) using the Shortstack alignment tool (Axtell, 2013; Rozowsky et al., 2019), mapped only to miRbase. ShortStack provides the ability to provide counts for the same miRNA in multiple genomic loci. Both methods provided nearly identical percentages of miRNA reads that mapped to miRbase v21. We then compared the normalization of miRNA counts relative to the total counts assigned to the miRNA by Shortstack (RPMM) with the default values ​​provided in the exceRpt pipeline (which normalizes miRNA counts to the total number of reads mapped to all small RNA libraries or genomes) (RPMM 总 -exceRpt default). Therefore, these measurements quantify individual miRNAs relative to the total pool of miRNAs or small RNAs present in EVs, respectively. We found that RPMM and RPMM 总 There was an overall strong positive correlation between the values ​​(rho 0.76-0.93, p < 0.0001, Spearman rank). However, for RPMM 总, showing high variability between replicates, particularly for the most highly expressed miRNAs (RPMM > 100). Therefore, our analyses used RPMM values ​​unless otherwise stated. ShortStack-derived counts processed with DESeq2 (v1.34.0) (Love et al., 2014) were used to determine miRNAs enriched in hESC-eEVs relative to other conditions. We also used the ashr package (Stephens, 2017) in DESeq2 to mitigate overestimation of LFCs due to noise and high variability in miRNA counts. Due to the large variability between replicates observed by PCA (which could overly affect the normalization of differential expression analyses), comparisons with hESC-mEVs were treated separately from other comparisons. Alternative differential expression analyses were also performed using the edgeR tool with the “classical” approach (Robinson et al., 2010), again separating the hESC-mEV comparisons from the other comparisons.

[0453] Statistical analysis

[0454] All biological replicates using human primary cells correspond to independent experiments from different amplification and passage numbers. As detailed in the legend, all figures (except for the ratio) are shown as data points, which include the mean ± standard deviation (SD) of biological or technical replicates. As described by Livak and Schmittgen (Livak & Schmittgen, 2001), the qRT-PCR data in the figure are shown as relative expression. Statistical analysis was performed using GraphPad Prism 9.0.0 (GraphPad Software, La Jolla, CA, USA). The test method used to assess significance is detailed in each legend, and the exact p value of the significant change is indicated on the figure. A p value of <0.05 is the nominal significance level. For in vitro experiments, since each experimental data set is the average of a large number of cultured cells, we assumed that the data were normally distributed based on the central limit theorem. A single-factor ANOVA using Dunnett's multiple comparison test was used to determine the significant differences between samples (>2 groups) in our in vitro experiments.

[0455] result

[0456] 1. EVs isolated from hESC-ECP conditioned medium express EV markers and exhibit phenotypic characteristics of exosomes

[0457] To isolate hESC-eEVs, pluripotent hESCs were subjected to an established 8-day differentiation protocol through the mesoderm stage as described previously ( Figure 1AWe confirmed that, as shown by flow cytometry, at day 8, two-thirds (65.7%) of the cells expressed the EC cell surface markers CD31 and CD144, while less than 0.05% of the cells co-expressed the pluripotency markers SSEA-3 / TRA-1-60 (Supplementary Fig. 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 in 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 (Supplementary Figure 2), which contained significantly higher numbers of particles and lower protein concentrations, were pooled and used for further analysis to confirm the isolation of EVs.

[0458] The enrichment of EVs in the isolated fractions was assessed by characterizing their size distribution, surface markers, and morphology. NTA showed that the size of particles in the combined fractions 5 and 6 ranged from 30 to 200 nm, with an average size of 84 ± 7.3 ( Figure 1C Western blot analysis confirmed that these particles were positive for the EV markers CD63 and CD81, but negative for the endoplasmic reticulum marker calnexin ( Figure 1D The characteristic size and shape of EVs were identified by transmission electron microscopy (TEM). Quantitative analysis of TEM images showed that the average EV size was 100.42 ± 38.36 and the circularity ratio was 0.96 ± 0.06 ( Figure 1E ). Therefore, EVs were prepared from hESC-ECP conditioned medium by a combination of ultrafiltration and SEC, allowing the isolation of particles with the characteristic size, morphology, and markers of EVs (CD63 and CD81), while also being free of protein contamination. These high-purity EV preparations are henceforth referred to as hESC-eEVs.

[0459] 2. hESC-eEVs were internalized by ECs and promoted tube formation and wound closure at low concentrations (<100 EVs / cell).

[0460] Since increased EC tube formation and wound closure are considered hallmarks of angiogenesis (S. Guo et al., 2014), and previous reports have demonstrated that the role of EC-derived EVs in angiogenesis depends on the dose used (Lacroix et al., 2007), to evaluate the angiogenic potency 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 / migratory cells, and cells cultured in medium without growth factors and treated with an equal volume of sterile 0.1 μm-filtered PBS (EV-suspension solution) served as a negative control. Since 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 demonstrated that these EVs were ineffective in inducing EC tube formation at low doses (<100 EVs / cell), whereas high doses (10 5 EV / cell) showed the highest effect ( Figure 2A In contrast, treatment with approximately 1 hESC-eEV per cell resulted in a significant increase in HCMEC tube-forming capacity compared to the negative control (p<0.0001) ( Figure 2B ). Therefore, only a relatively low dose of hESC-eEV (1 EV / cell) is required to compare with HUVEC hypoxic EV (10 5 We also showed that, in contrast to HUVEC hypoxic EVs, treatment with high hESC-eEV concentrations (≥100 EVs / cell) failed to replicate this effect. Staining with Calcein AM confirmed cell viability in all conditions in the tube formation assay ( Figure 2C ).

[0461] To understand whether the effect on angiogenesis is specific to EVs derived from the endodermal stage of the hESC-ECP differentiation system, we investigated the effects of EVs derived from the mesodermal stage of the differentiation protocol (hESC-mEVs) on the tube-forming capacity of HCMECs (dose range: 1-10 5 EV / cell). 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 treatment with hESC-mEVs did not induce tube formation at any dose tested, compared to treatment with 1 hESC-eEV / cell ( Figure 2D). Thus, the ability to promote angiogenesis is unique to EVs derived from the endothelial stage of the ESC-ECP differentiation system (hESC-eEVs) and absent in EVs derived from the mesodermal stage (hESC-mEVs). To confirm that the angiogenic effects observed after hESC-eEV treatment were specific to EVs and not to other soluble, non-EV-associated secreted factors, we subjected complete culture medium (not exposed to cells) to the EV purification process and collected samples at different stages of purification ( Figure 2E These samples were tested for their ability to induce HCMEC tube formation ( Figure 2F 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. 165 , and VEGFA is a key driver of angiogenesis (Apte et al., 2019), so this experiment will also use human recombinant VEGFA 165 Treated cells served as a positive control. Our results showed that treatment with complete culture medium that had undergone an EV purification process did not enhance 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 angiogenic effects observed after hESC-eEV treatment.

[0462] Since we observed that hESC-eEVs induced HCMEC tube formation at low concentrations, to evaluate the effect of hESC-eEVs on EC wound healing, we performed a scratch assay using increasing concentrations of hESC-eEVs, with the maximum dose being 100 hESC-eEVs / cell ( Figure 3 ). Our results show that 6-24 hours after wound induction, HCMEC treated with low hESC-eEV doses (<100 EV / cells) migrate significantly faster than cells cultured in basal medium and treated with PBS. Cells treated with high hESC-eEV concentrations (100 EV / cells) or with HUVEC hypoxic EVs did not show significantly improved migration ability compared to the negative control. In short, these results indicate that hESC-eEVs stimulate the effectiveness of angiogenesis at low concentrations (<100 EV / cells). It also appears that this angiogenic effect disappears at higher hESC-eEV concentrations, indicating the presence of a dose-specific effect. In addition, our data show that the ability to induce angiogenesis is unique to EVs derived from the endothelial stage of hESC-ECP differentiation rather than the mesoderm stage.

[0463] 3. hESC-eEVs are enriched for angiogenic miRNAs and other miRNAs with potential roles in angiogenesis

[0464] Since the effects of EVs are largely attributed to their small RNA cargo (O'Brien et al., 2020), we aimed to identify the RNA molecules responsible for the angiogenic activity of hESC-eEVs. Small RNAs were extracted from EVs from the following samples: hESC-eEVs, hESC-mEVs, and HUVEC hypoxic EVs (n=3 independent stem cell differentiations), and we also obtained RNA from hESC-ECPs to identify EV-enriched miRNAs. RNA size analysis showed an enrichment of small RNAs (25-200nt) in EVs, while ribosomal RNAs and longer RNAs were absent. Analysis of cellular RNA samples 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 positive-sense miRNAs, while EV RNA samples showed a more diverse composition of small RNA classes, with the majority mapping to tRNAs, miRNAs, promiscuous RNAs (misc_RNAs), and protein-coding molecules ( Figure 4A To understand whether EVs obtained from different conditions harbor distinct RNA molecules, we compared the lists of unique miRNAs, piRNAs, tRNAs, and other RNAs between EV types (Supplementary Figure 3 tRNA and piwiRNA showed less variation, while miRNAs and other small RNAs (including promiscuous RNAs, snRNAs, yRNAs, retained introns, protein-coding molecules, lincRNAs, etc.) were the most heterogeneous groups in EV samples. Overall, a total of 72 miRNAs and 172 other small RNAs were shared across different EV samples. 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) (Kesidou et al., 2020).

[0465] Next, we assessed 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 high abundance in hESC-eEVs and HUVEC hypoxic EVs, but not in hESC-mEVs. Consistent with previous studies on cellular RNA composition, which reported that the top 3-5 abundant miRNAs accounted for more than 50% of the total miRNA pool for any human cell type (de Rie et al., 2017), we found that the top 3 miRNAs in hESC-ECPs corresponded to more than 50% of the total miRNA reads. A similar situation was observed in EV samples, with the top 5 EV miRNAs corresponding to approximately 50% of the total miRNA reads ( Figure 4B A literature search of the 20 most abundant miRNAs per sample (corresponding to 68-86% of total reads) revealed the presence of several miRNAs in hESC-eEVs with previously unreported roles in angiogenesis. The top 5 miRNAs in hESC-eEVs contained molecules that have been extensively studied for their roles in driving angiogenesis, whereas the top 5 miRNAs in hESC-mEVs had 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 demonstrate that hESC-eEVs are enriched for pro-angiogenic miRNAs as well as miRNAs with previously unreported roles in angiogenesis.

[0466] discuss

[0467] Because our previous data suggested that the improvements observed after hESC-ECP transplantation could be attributed to paracrine mechanisms (MacAskill et al., 2018), we aimed here to investigate the role of hESC-eEVs in angiogenesis. This study provides several novel findings in the EV field. We developed a reproducible protocol for isolating pure and bioactive EVs from hESC-ECP cultures and demonstrated that hESC-eEVs induce angiogenesis at low concentrations compared to other EC-derived EVs. We also showed that hESC-eEVs are enriched for pro-angiogenic miRNAs as well as other miRNAs with previously unreported roles in angiogenesis.

[0468] In this study, EVs were isolated by a combination of ultrafiltration and SEC, resulting in the isolation of a highly pure EV population (Théry et al., 2018). Although EV yields are lower compared to other methods (Théry 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 method, we confirmed EVs with intact ultrastructure, positive for the EV markers CD63 and CD81, negative for the cellular marker calnexin (Kozlov & Gehring, 2020), and free of protein contamination. To investigate the impact of hESC-eEVs on angiogenesis, we performed tube formation and wound wound assays on ECs. We also compared our EVs with EVs isolated from the mesodermal stage of hESC-ECP differentiation (hESC-mEVs) and with mature ECs. As a mature EC population, we selected HUVEC-hypoxic EVs because hypoxia induces the release of angiogenic EVs (Bister et al., 2020). Our results showed that low concentrations of hESC-eEVs (<100 EVs / cell) significantly enhanced the angiogenic capacity of ECs, whereas a much higher dose of HUVEC hypoxic EVs (10 5 EVs / cell). We also show that treatment with high hESC-eEV concentrations (≥100 EVs / cell) does not affect EC angiogenic capacity. This may indicate that hESC-eEV-mediated angiogenic effects require precise dosing. 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 comparison between individual EV studies challenging. Another important consideration is that NTA, as a particle quantification technique, has several drawbacks, including the potential for underestimation of particle concentration (Bachurski et al., 2019). Therefore, accurate estimation of the EV dose used in individual experiments remains a key obstacle in the field. In contrast to EC-derived EVs, we found that treatment with EVs derived from the mesodermal stage of hESC-ECP differentiation (hESC-mEVs) did not affect EC angiogenic capacity, suggesting that the ability to induce angiogenesis is unique to EVs isolated from more mature stages of hESC-ECP differentiation. Overall, the finding that only very low doses of hESC-eEVs are required to produce improved effects may be promising for the clinical application of hESC-eEVs for therapeutic angiogenesis.

[0469] One of the most useful tools for studying EV miRNA cargo is small RNA sequencing, as it can provide objective information on EV-miRNA content. To identify key miRNA candidates that may drive the angiogenic potential of hESC-eEVs, we performed small RNA sequencing and compared the small RNA cargo of hESC-eEVs with that of hESC-mEVs, HUVEC hypoxic EVs, and EV donor cells. EV small RNA sequencing was performed by DNBSEQ. TM We performed our method using a NGS technology platform. A potential limitation of our approach is the low RNA input in the EV samples, primarily due to two reasons: the hESC-eEV source from a differentiation system and the low yield of EV isolation by a combination of ultrafiltration and SEC (Théry et al., 2018), which we found to be associated with a high percentage of unmapped reads in our libraries. Our high-purity, low-depth approach to EV isolation and sequencing may miss comprehensive detection of all EV miRNAs; however, we provided sufficient coverage to analyze the most abundant miRNAs, which are most likely to influence angiogenesis, at the low concentrations required in hESC-eEVs. Our results indicate 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 positive-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 groups across different EV samples, but focused on miRNAs in the rest of our analysis because their roles in angiogenesis are better understood (Kesidou et al., 2020).

[0470] Our small RNA sequencing analysis revealed for the first time that, consistent with previous studies of cellular RNA composition reporting that the top 3–5 miRNAs account for over 50% of the total miRNA pool for any human cell type (de Rie et al., 2017), the top 5 EV miRNAs also account for approximately 50% of the total miRNA reads. Our finding that the most abundant miRNAs in hESC-eEVs are molecules with established roles in inducing angiogenesis, whereas the most abundant miRNAs in hESC-mEVs have anti-angiogenic effects, suggests why low doses of hESC-eEVs are required to induce angiogenesis and may explain why hESC-mEVs were ineffective in inducing angiogenesis in our in vitro experiments. Our results also indicate 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 angiogenic effects as hESC-eEVs. Differential expression analysis was performed to assess whether the differences in EV angiogenic effects could be explained by their miRNA cargo. Our results showed that several miRNAs were differentially expressed between angiogenic hESC-eEVs and non-angiogenic hESC-mEVs. The most abundant miRNA in hESC-eEVs compared to hESC-mEVs was the EC-specific (de Rie et al., 2017) miRNA miR-126-3p, which has been extensively studied in driving angiogenesis (Qu et al., 2019).

[0471] Overall, this study is the first to investigate the role of hESC-eEVs in angiogenesis, providing a comprehensive understanding of the miRNA content of hESC-eEVs and comparing the miRNA cargo of hESC-eEVs with that of hESC-mEVs and mature EC-EVs. Our data highlight several factors that will contribute to the field of EVs and CVD: (1) hESC-eEVs induce angiogenesis at low concentrations compared to other EC-derived EVs, whereas EVs from more immature stages of the hESC-ECP differentiation protocol lack angiogenic function; (2) hESC-eEVs are enriched for angiogenic miRNAs, while hESC-mEVs are enriched for anti-angiogenic miRNAs, and the most abundant miRNAs in HUVEC hypoxic EVs are molecules with multifunctional roles in driving angiogenesis.

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Claims

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

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

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

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

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

6. An isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents, pharmaceutical compositions or medical devices according to claim 5 for the treatment of ischemic tissue disorders, cardiovascular diseases such as pulmonary hypertension and / or wound healing.

7. An isolated population of pro-angiogenic and / or pro-angiogenic EVs and / or their contents or pharmaceutical compositions according to claim 4, which are used in an amount less than or equal to 50, 25, 10, 5, 2 or 1 EV / cell.

8. A method for isolating EVs from endothelial cells derived from pluripotent stem cells, the method comprising: (a) ultrafiltration of a fluid containing EVs to separate EVs from larger molecular weight substances and obtain a fluid enriched in EVs, wherein the EVs are produced by endothelial cells derived from pluripotent stem cells; as well as (b) The EV-enriched fluid is subjected to size exclusion chromatography (SEC) to obtain fluid fractions containing isolated EVs.

9. The method of claim 8, wherein the ultrafiltration comprises using a filter with a molecular weight cut-off of 100 KDa.

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

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 8-10, for use in promoting angiogenesis and / or neovascularization.

12. An 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 disorders, cardiovascular diseases 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, wherein the isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents comprise one or more miRNAs.

14. The isolated population of pro-angiogenic and / or pro-angiogenic extracellular vesicles (EVs) and / or their contents according to claim 13, wherein the one or more miRNAs are 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; 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.

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

16. The miR modulator for use according to claim 15, wherein the miR modulator increases the expression of one or more miRs selected from the group consisting 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; 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.

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