Stimulation of angiogenesis by fibroblast-derived exosomes

Abstract

Disclosed are methods, means, and compositions useful for stimulating angiogenesis directly by administering membrane vesicles (such as fibroblast-derived exosomes) and / or via the induction of angiogenic cytokines from blood cells that contact fibroblast-derived exosomes. The present invention provides means for treating conditions in which angiogenesis is beneficial by local or systemic administration of exosomes (including exosomes derived from fibroblasts), wherein the fibroblasts are cultured under basal or hypoxic conditions. In other embodiments, fibroblast-derived exosomes are utilized to enhance endogenous regenerative processes (such as hematopoiesis, angiogenesis, and neurogenesis), as well as regenerative processes stimulated by the administration of exogenous therapeutic agents (such as cells, growth factors, or genes). [Selected Figure] Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 487,143, filed April 19, 2017, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] Various embodiments of the present disclosure include at least the fields of cell biology, molecular biology, physiology, and medicine. [Background technology]

[0003] Exosomes are nanoparticles (40-100 nm) in size with highly defined, uniform characteristics [1]. They differ from microvesicles, which are released in a nonspecific manner (Figure 1). Initially thought to be by-products of cellular protein turnover [2], these nanoparticles are increasingly recognized as crucial intercellular communication mechanisms in a variety of domains, ranging from neurotransmission [3] to immunomodulation [2] to infectious diseases [4]. Compared to other secreted vesicles, exosomes have much more clearly defined biophysical and biochemical properties. Specifically, exosomes are 40-100 nm in diameter (with a density of 1.13-1.19 g / ml in sucrose) and can sediment at 100,000 g [1]. Their membranes are rich in cholesterol, sphingomyelin, and ceramide and are known to contain lipid rafts. Exosomes were originally discovered as a vehicle for the export of transferrin receptors during sheep reticulocyte maturation. [5] In recent years, interest in exosomes has exploded, and a wide variety of cells have been reported to secrete these nanoparticles, including T cells [6, 7], B cells [8, 9], dendritic cells [10, 11], tumor cells [12, 13], neurons [14, 15], oligodendrocytes

[16] , and placental cells

[17] .

[0004] At the very least, the present disclosure provides a solution to a long-standing need in the art of exosome-based therapeutics. Summary of the Invention [Means for solving the problem]

[0005] Various embodiments of the present disclosure relate to the unexpected discovery that membrane vesicles (e.g., exosomes) have therapeutic properties. While in some cases the therapeutic properties can be of any type, in specific cases the membrane vesicles include properties for stimulating angiogenesis, hematopoiesis, and / or neurogenesis. While the source of the membrane vesicles can be any source, in specific embodiments, the source of exosomes is derived from mammalian cells. In specific embodiments, the membrane vesicles are derived from one or more types of fibroblasts. In specific embodiments, the membrane vesicles are utilized to deliver one or more therapeutic signals from one or more types of cells. In at least certain cases, membrane vesicles allow for the utilization of the benefits of cell therapy, for example, without the drawbacks of having to store and deliver actual cells.

[0006] Specific embodiments of the present disclosure relate to the field of angiogenesis stimulation, including, for example, as it relates to the use of conditioned medium to stimulate angiogenesis. In specific embodiments, the present disclosure encompasses the use of membrane vesicles derived from fibroblast-conditioned medium for any therapeutic and / or prophylactic application involving at least the stimulation of angiogenesis.

[0007] Examples of various embodiments include a method for stimulating angiogenesis in an individual, comprising administering to the individual an effective amount of fibroblast-derived exosomes or one or more biologically active fractions thereof. Exosomes may be considered to be derived from fibroblasts if they are obtained from a culture of fibroblasts, for example. The culture medium may be specifically engineered for the purpose of producing exosomes with one or more characteristics; for example, the medium may include one or more factors that are mitogenic for fibroblasts.

[0008] In one embodiment, there is a method for stimulating angiogenesis in an individual, the method comprising the steps of: a) obtaining one or more fibroblasts; b) culturing the fibroblasts in culture under conditions that allow the production of exosomes into the culture medium; c) removing exosomes from the culture medium; and d) administering the removed exosomes, or one or more biologically active fractions thereof (i.e., fractions capable of stimulating angiogenesis), to an individual in need of angiogenesis (including therapeutic angiogenesis). The individual in need of angiogenesis may be an individual at risk of limb loss, an individual in need of prevention of limb loss, an individual with or at risk of cardiovascular disease or coronary artery disease, etc. The individual may have one or more underperfused tissues and / or organs. The tissue and / or organ in need of angiogenesis may be of any type, including muscle, skin, blood vessels, cartilage, heart, brain, stomach, duodenum, intestine, pancreas, spleen, uterus, kidney, liver, etc. The individual may have an ischemic disease (e.g., ischemic heart disease or ischemic brain disease (stroke)), including those that develop due to insufficient angiogenesis. The individual may need healing after a stroke. The individual may have a gastrointestinal ulcer (e.g., a duodenal ulcer, etc.). If the individual has coronary artery disease, the coronary artery disease may not be amenable to complete revascularization by medical intervention (e.g., percutaneous transluminal coronary angioplasty and / or coronary artery bypass grafting, etc.). The individual may need any type of wound healing. As an example, the individual may have diabetes, and in some cases, wound healing is poor as part of the diabetes.

[0009] In particular, fibroblasts are derived from biopsy specimens.Fibroblasts may or may not be derived from individuals.Fibroblasts may be cultured in a medium that allows fibroblasts to grow, for example, in a medium that contains one or more factors that are mitogenic to fibroblasts.Examples of factors that are mitogenic to fibroblasts include one or more factors selected from the group consisting of: a) FGF-1, b) FGF-2, c) FGF-5, d) EGF, e) CNTF, f) KGF-1, g) PDGF, h) platelet-rich plasma, i) TGF-alpha, j) HGF-1, and (k) combinations thereof.

[0010] In a specific embodiment, the fibroblasts are cultured under hypoxia. Exosomes may be collected from the fibroblasts while they are in a proliferative state. In some cases, exosomes are collected from the fibroblasts while they are cultured in a medium that does not contain any growth-inducing factors or in a medium that contains a reduced level (compared to standard levels) of a growth-inducing growth factor. In a specific embodiment, exosomes are collected from the fibroblasts that have been cultured in 2% to 8% oxygen for at least 1 day, such as 1 to 15 days, or 5 to 10 days. In a specific embodiment, the cells are subcultured for at least one passage.

[0011] In certain embodiments, the exosomes are present in a preparation, and the preparation may contain less than 5% polyethylene glycol.The exosomes may be purified using polyethylene glycol and / or may be purified using ultrafiltration.Polyethylene glycol may be added to the exosomes after purification.

[0012] In certain embodiments, the exosomes express a marker selected from the group consisting of (a) CD63, (b) CD9, (c) MHC I, (d) CD56, and (e) a combination thereof. The fibroblasts may be cultured in a medium selected from the group consisting of a) Roswell Park Memorial Institute (RPMI-1640), b) Dulbecco's Modified Essential Medium (DMEM), c) Eagle's Modified Essential Medium (EMEM), d) Optimem, e) Iscove's Medium, and f) a combination thereof. In certain embodiments, during and / or after removal, the exosomes are selected based on one or more markers expressed by the exosomes, including, by way of example, the markers listed above.

[0013] In the method comprising the removing step, the removing step may comprise anion exchange chromatography under high pressure. The support for anion exchange chromatography may be functionalized with quaternary amine. The support for anion exchange chromatography may be, for example, in the form of beads. In a specific case, the removing step comprises gel permeation chromatography, which may be performed before or after anion exchange chromatography. In a specific case, the removing step further comprises an enrichment step for exosomes. Such enrichment step may comprise one or more of centrifugation, clarification, filtration, concentration and / or ultrafiltration. In certain cases, the removing step further comprises non-specific affinity chromatography. The removing step may further comprise filtration.

[0014] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of this invention, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the invention. [Brief explanation of the drawings]

[0015] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0016] [Figure 1] 1 shows stimulation of VEGF from PBMCs by fibroblast-derived exosomes.

[0017] [Figure 2] Stimulation of HUVEC proliferation is shown. DETAILED DESCRIPTION OF THE INVENTION

[0018] I. Definition As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more. Still further, the terms "having," "including," "containing," and "comprising" are interchangeable, and those of skill in the art will recognize that these terms are open-ended. Some embodiments of the present invention may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0019] The term "exosome," as used herein, refers to small membrane vesicles of endocytic origin that are secreted by cells in culture (such as, for example, fibroblasts). II. General Embodiments of the Disclosure

[0020] The present disclosure encompasses, for example, treatment for an individual suffering from or at risk of a medical condition by providing an effective amount of exosomes that improves at least one symptom of the medical condition. In specific embodiments, the medical condition is one for which stimulating angiogenesis would be therapeutic. Thus, in at least certain cases, the present disclosure provides a means for stimulating angiogenesis using exosomes. The exosomes may be from any source, including exosomes derived from fibroblasts. Fibroblasts may or may not be present in tissue culture.

[0021] In one embodiment, the present disclosure encompasses a method for extracting exosomes from a culture of fibroblasts, which may also include concentrating the exosomes and administering the exosomes, including for the purpose of stimulating angiogenesis. Without being bound by theory, exosomes produced by tissue culture may stimulate angiogenesis by acting directly as mitogens on endothelial cells and / or by inducing the production of pro-angiogenic cytokines in bodily cells, for example, in blood cells. In certain embodiments, such a mechanism is not involved.

[0022] In one embodiment, fibroblasts are cultured to preserve their viability and proliferation. The present disclosure may be applied to both individualized autologous exosome preparations and exosome preparations obtained from one or more other individuals, including, for example, established cell lines for experimental, biological, or therapeutic use. In one embodiment, the present disclosure encompasses the use of chromatographic separation methods to prepare membrane vesicles, particularly to separate the membrane vesicles from potential biological contaminants, where the microvesicles may be exosomes and the cells used to generate the exosomes are fibroblasts. The present disclosure encompasses methods for preparing exosomes from fibroblasts for therapeutic use.

[0023] As shown herein, membrane vesicles (particularly exosomes) having the ability to stimulate angiogenesis can be purified.In one embodiment, strong anion exchange or weak anion exchange can be performed, and in a specific embodiment, the anion exchange is strong anion exchange.In addition, in a specific embodiment, chromatography is performed under pressure.Therefore, more specifically, the chromatography can include high performance liquid chromatography (HPLC).

[0024] When anion exchange chromatography is employed, various types of supports can be used to perform the anion exchange chromatography. In certain embodiments, these include cellulose, poly(styrene-divinylbenzene), agarose, dextran, acrylamide, silica, ethylene glycol-methacrylate copolymer, or mixtures thereof (e.g., agarose-dextran mixtures). In some cases, specific chromatography devices composed of supports and, in particular, the following gels can be utilized: SOURCE.POROS®, SEPHAROSE®, SEPHADEX®, TRISACRYL®, TSK-GEL SW OR PW®, SUPERDEX®, TOYOPEARL HW, and SEPHACRYL®. Thus, in a specific embodiment, the present disclosure relates to a method for preparing membrane vesicles (in particular exosomes) from a biological sample, such as from a tissue culture containing fibroblasts, comprising at least one step in which the biological sample is treated by anion exchange chromatography on a given support, including (for example) a support selected from cellulose, poly(styrene-divinylbenzene), silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate copolymer, alone or in mixture, and optionally the support is functionalized.

[0025] In addition, to improve chromatographic resolution, it is useful within the scope of the present disclosure to use a support in the form of beads in specific embodiments. In certain embodiments, these beads have a uniform and calibrated diameter with a porosity large enough to allow penetration of the chromatographic entity (i.e., exosomes). In this manner, assuming the diameter of exosomes (generally between 50 nm and 100 nm), highly porous gels, particularly those between 10 nm and 5 μm, such as those between approximately 20 nm and approximately 2 μm (e.g., between about 100 nm and about 1 μm), may be used to apply the teachings of the present disclosure. For anion exchange chromatography, the support used may be functionalized with a group capable of interacting with anionic molecules. Generally, this group is composed of amines, which may be ternary or quaternary, thereby defining weak or strong anion exchangers, respectively. It is useful within the scope of the present disclosure to use strong anion exchangers. In this manner, according to the present disclosure, a chromatography support such as that described above, e.g., a chromatography support functionalized with a quaternary amine, is used. Thus, according to more specific embodiments of the present invention, anion exchange chromatography is performed on a support functionalized with a quaternary amine. In at least some cases, the support is selected from poly(styrene-divinylbenzene), acrylamide, agarose, dextran, and silica, alone or in combination, and functionalized with a quaternary amine. Examples of supports functionalized with a quaternary amine include SOURCEQ.MONO Q, Q SEPHAROSE®, POROS® HQ, and POROS® QE gels, FRACTOGEL® TMAE-type gels, and TOYOPEARL SUPER® Q gel.

[0026] In certain embodiments, the support for performing anion exchange chromatography comprises poly(styrene-divinylbenzene). One example of this type of gel that can be used within the scope of the present disclosure is SOURCE Q gel, particularly SOURCE 15 Q (Pharmacia). This support offers the advantage of having very large internal pores, thus providing low resistance to the circulation of liquid through the gel, while allowing rapid diffusion of exosomes to functional groups, which are useful parameters for exosomes given their size. Biological compounds retained on the column can be eluted in one or more ways, particularly using a salt gradient of increasing concentration (e.g., from 0 M to 2 M). Sodium chloride solutions can be particularly used, for example, at concentrations ranging from 0 M to 2 M. The various fractions purified in this way are detected by measuring their optical density (OD) at the column outlet using successive spectrophotometric readings. As a guideline, under the conditions used in the examples, fractions containing membrane vesicles were eluted at ionic strengths comprised between approximately 350 mM and 700 mM, depending on the type of vesicle.

[0027] Various types of columns can be used to perform this chromatography step, depending on the requirements and throughput. For example, depending on the preparation, columns ranging from approximately 100 μl to 10 ml or more can be used. In this manner, available carriers have capacities that can reach, for example, 25 mg of protein per ml. Thus, a 100 μl column has a protein capacity of approximately 2.5 mg, allowing the processing of approximately 2 L of culture supernatant (e.g., corresponding to a volume of 100 ml to 200 ml per preparation after 10- to 20-fold concentration), given the sample in question. It is understood that larger volumes can also be processed, for example, by increasing the column volume. In addition, to perform at least certain methods of the present disclosure, it is also possible to combine an anion exchange chromatography step with a gel permeation chromatography step. In this manner, according to specific embodiments of the present disclosure, a gel permeation chromatography step is added either before or after the anion exchange chromatography step. In certain cases, in this embodiment, the gel permeation chromatography step is performed after the anion exchange step. Additionally, in a specific variation, the anion exchange chromatography step is replaced by a gel permeation chromatography step. The present disclosure reveals that membrane vesicles can also be purified using gel permeation liquid chromatography, as described in detail below, particularly when this step is combined with an anion exchange chromatography step or other treatment step of the biological sample.

[0028] To perform the gel permeation chromatography step, a support selected from silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate copolymer, or a mixture thereof (e.g., an agarose-dextran mixture) may be used. As an example, a support such as SUPERDEX.RTM.200HR (Pharmacia), TSK G6000 (TosoHaas), or SEPHACRYL® S (Pharmacia) may be used for gel permeation chromatography.

[0029] The processes according to the present disclosure may be applied to one or more different biological samples, which may include, in particular, biological fluids from a subject (such as bone marrow, peripheral blood, etc.), culture supernatants, cell lysates, pre-purified solutions, or any other compositions containing membrane vesicles.

[0030] In this regard, in a specific embodiment of the present disclosure, the biological sample is a culture supernatant of membrane vesicle-producing fibroblasts.

[0031] Additionally, according to certain embodiments of the present invention, the biological sample is treated prior to the chromatography step, for example, to enrich for membrane vesicles (enrichment step). In this manner, in a specific embodiment, the present disclosure relates to a method for preparing membrane vesicles from a biological sample, characterized in that the method comprises at least a) an enrichment step for preparing a sample enriched in membrane vesicles, and b) a step in which the sample is treated by anion exchange chromatography and / or gel permeation chromatography.

[0032] In one embodiment, biological sample is the culture supernatant that is processed to enrich membrane vesicles.Particularly, biological sample may be composed of the pre-purified solution obtained from the culture supernatant of a population of membrane vesicle-producing cells or from biological fluid by various processes (for example, centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography, etc.), particularly by using clarification and / or ultrafiltration and / or affinity chromatography.Therefore, a specific method for preparing membrane vesicles according to the present disclosure may include the following steps: a) culturing a population of membrane vesicles (for example, exosomes) producing cells under conditions that allow vesicle release; b) enriching the sample in membrane vesicles; and c) anion exchange chromatography and / or gel permeation chromatography of the sample.

[0033] As noted above, sample (e.g., supernatant) enrichment steps can include one or more of the following: centrifugation, clarification, ultrafiltration, nanofiltration, and / or affinity chromatography steps on the supernatant. In a first specific embodiment, the enrichment step includes (i) removal of cells and / or cell debris (clarification), optionally followed by (ii) a concentration and / or affinity chromatography step. In another specific embodiment, the enrichment step includes an affinity chromatography step, optionally preceded by a step of cell and / or cell debris removal (clarification). Particular enrichment steps according to the present disclosure include (i) removal of cells and / or cell debris (clarification), (ii) concentration, and (iii) affinity chromatography. Cells and / or cell debris can be removed by centrifugation of the sample, for example, at a low speed, preferably below 1000 g, e.g., between 100 g and 700 g. Preferred centrifugation conditions during this step are, for example, approximately 300 g or 600 g for a period between 1 and 15 minutes.

[0034] Cells and / or cell debris may also be removed by filtration of the sample, optionally combined with the centrifugation described above. Filtration may in particular be carried out by successive filtration using filters of decreasing porosity. For this purpose, filters with a porosity greater than 0.2 μm, for example between 0.2 μm and 10 μm, may be used. It is particularly possible to use successive filters with porosities of 10 μm, 1 μm, 0.5 μm, followed by 0.22 μm.

[0035] A concentration step may also be performed, for example, to reduce the volume of the sample to be processed during the chromatography step. In this manner, concentration may be achieved by centrifuging the sample at high speed, for example, between 10,000 g and 100,000 g, to cause sedimentation of membrane vesicles. This may involve a series of differential centrifugations, the final centrifugation being performed at approximately 70,000 g. The membrane vesicles in the resulting pellet may be carried out in a smaller volume and in an appropriate buffer for subsequent steps of the process. A concentration step may also be performed by ultrafiltration. In fact, this ultrafiltration allows both concentrating the supernatant and performing an initial purification of the vesicles. According to certain embodiments, the biological sample (e.g., supernatant) is subjected to ultrafiltration, preferably tangential ultrafiltration. Tangential ultrafiltration consists of concentrating and fractionating a solution between two compartments (filtrate and retentate) separated by a membrane with a defined cutoff threshold. Separation is achieved by applying a flow through the retentate compartment and applying transmembrane pressure between this compartment and the filtrate compartment. Various systems can be used to perform ultrafiltration, such as spiral wound membranes (Millipore, Amicon), flat membranes, or hollow fibers (Amicon, Millipore, Sartorius, Pall, GF, Sepracor), etc. While within the scope of the present disclosure, it is advantageous to use membranes with a cutoff threshold of less than 1000 kDa, or that may be between 300 kDa and 1000 kDa, or even that may be between 300 kDa and 500 kDa.

[0036] The affinity chromatography step can be performed in various ways using various chromatographic supports and materials. In certain embodiments, the chromatography is nonspecific affinity chromatography, which aims to retain (i.e., bind) certain contaminants present in the solution without retaining the target substance (i.e., exosomes). Therefore, this is negative selection. In some embodiments, affinity chromatography for dyes is used, which allows for the removal (i.e., retention) of contaminants (e.g., proteins and enzymes, such as albumin, kinases, dehydrogenases, clotting factors, interferons, lipoproteins, or cofactors, etc.). In certain cases, the support used for this chromatography step is a support functionalized with a dye, such as that used for ion exchange chromatography. As a specific example, the dye may be selected from Blue SEPHAROSE® (Pharmacia), YELLOW 86, GREEN 5, and BROWN 10 (Sigma). The support is more preferably agarose. It should be understood that any other carrier and / or dye or reactive group that allows for the retention (binding) of contaminants from the processed biological sample can be used in the present disclosure.

[0037] In one embodiment, a membrane vesicle preparation process within the scope of the present disclosure comprises the following steps: a) culturing a population of membrane vesicle (e.g., exosome)-producing cells under conditions that allow the release of vesicles; b) treating the culture supernatant with at least one ultrafiltration or affinity chromatography step to produce a biological sample enriched in membrane vesicles (e.g., exosomes); and c) treating the biological sample with anion exchange chromatography and / or gel permeation chromatography. In a specific embodiment, the above step b) comprises filtration of the culture supernatant, followed by ultrafiltration, preferably tangential ultrafiltration. In another embodiment, the above step b) comprises clarification of the culture supernatant, followed by affinity chromatography for a dye, preferably affinity chromatography with Blue SEPHAROSE®.

[0038] In addition, after step c), the collected material may be subjected, if applicable, to one or more further processing steps and / or a filtration step d), in particular for sterilization purposes. For this filtration step, filters having a diameter of 0.3 μm or less are preferentially used, or even more preferentially filters having a diameter of 0.25 μm or less. Such filters have, for example, a diameter of 0.22 μm.

[0039] After step d), the obtained material is dispensed in a suitable storage medium, for example, into a suitable device (e.g., bottle, tube, bag, syringe, etc.). The purified vesicles thus obtained may be refrigerated, frozen, or used immediately. Thus, a specific preparation process within the scope of the present invention comprises at least the following steps: c) anion exchange chromatography and / or gel permeation chromatography of the biological sample, and d) a filtration step (particularly sterile filtration) of the material collected after step c). In a first variant, the process according to the present disclosure comprises c) anion exchange chromatography of the biological sample, and d) a filtration step (particularly sterile filtration) of the material collected after step c).

[0040] In another variant, the process according to the present disclosure comprises c) a gel permeation chromatography treatment of the biological sample, and d) a filtration step (in particular a sterile filtration) of the material collected after step c).According to a third variant, the process according to the invention comprises c) an anion exchange treatment of the biological sample, which is carried out before or after the gel permeation chromatography, and d) a filtration step (in particular a sterile filtration) of the material collected after step c).

[0041] Further embodiments include methods for optimizing the production of angiogenesis-stimulating therapeutic factors from fibroblast cultures by using filters to separate compositions based on charge, size, or elution capacity from an adsorbent. Numerous techniques are known in the art for purifying therapeutic factors and concentrating the agents. For some specific uses, fibroblast-derived compounds may be sufficient for use as the culture supernatant of the cells in culture media. Currently, media useful for this purpose include Roswell Park Memorial Institute (RPMI-1640), Dulbecco's Modified Essential Medium (DMEM), Eagle's Modified Essential Medium (EMEM), Optimem, and Iscove's Medium.

[0042] In one embodiment, a therapeutic factor for stimulating angiogenesis is derived from a tissue culture that may contain exosomes, or may not contain exosomes but may contain factors capable of stimulating angiogenesis. In such an embodiment, the culture conditioned medium may be concentrated by filtration / desalting means known in the art, including the use of an Amicon filter with a specific molecular weight cutoff, which may be selected for molecular weights greater than 1 kDa to 50 kDa. Alternatively, the supernatant may be concentrated using means known in the art, such as solid-phase extraction using a C18 cartridge (Mini-Speed ​​C18-14%, SPELimited, Concord, ON). The cartridge is prepared by washing with methanol followed by deionized distilled water. Up to 100 ml of fibroblast-conditioned medium supernatant may be passed through each of these specific cartridges before elution. Those skilled in the art will understand that larger cartridges may be used. After washing the cartridge, the adsorbate is eluted with 3 ml of methanol, evaporated under a stream of nitrogen, redissolved in a small volume of methanol, and stored at 4°C. Before testing the eluate for activity in vitro, the methanol is evaporated under nitrogen and replaced with culture medium. C18 cartridges are used to adsorb small hydrophobic molecules from fibroblast-conditioned supernatants, allowing for the removal of salts and other polar contaminants. However, it may be desirable to use other adsorption means to purify certain compounds from the supernatant. The concentrated supernatant may be directly evaluated for biological activity useful for practicing the present invention or may be further purified. Further purification may be performed using, for example, gel filtration using a Bio-Gel P-2 column (Bio-Rad, Richmond, CA) with a nominal exclusion limit of 1800 Da. The column may be washed and pre-swollen in 20 mM Tris-HCl buffer (pH 7.2) (Sigma) and degassed by gentle swirling under vacuum.Bio-Gel P-2 material is packed into a 1.5 x 54 cm glass column and equilibrated with three column volumes of the same buffer. The fibroblast supernatant concentrate removed by the C18 cartridge may be dissolved in 0.5 ml of 20 mM Tris buffer (pH 7.2) and passed through the column. Fractions may be collected from the column and analyzed for biological activity. Other purification, fractionation, and identification means are known to those skilled in the art and include anion exchange chromatography, gas chromatography, high-performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. Administration of the active fraction of the supernatant may be performed locally or systemically.

[0043] The present disclosure includes a method for treating an individual at risk of limb loss, ischemic heart disease, ischemic brain disease, gastrointestinal ulcer, and / or one or more wounds by providing the individual with an effective amount of fibroblast-derived exosomes or one or more biologically active fractions thereof.Fractions can be determined to be biologically active using routine methods in the art, such as determining fractions of a starting material and testing each fraction for specific active substances.In a specific case of the present disclosure, the activity tested can be the production of one or more specific compounds (e.g., one or more factors, such as VEGF).In additional or alternative cases, fractions can be tested in an in vivo model, such as an in vivo mouse model for limb loss. [Example]

[0044] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples below represent techniques found by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results, without departing from the spirit and scope of the invention.

[0045] Example 1 Fibroblast exosomes stimulate VEGF production In one embodiment of the present invention, exosomes derived from fibroblasts are used for "angiogenic therapy." Angiogenic therapy has previously been described as "biological bypass," with the idea being that a more natural type of "bypass" can be achieved by administering agents capable of inducing collateral formation. Indeed, it has been recognized that ischemic muscles secrete angiogenic factors in response to hypoxia, and that natural angiogenesis, to some extent, does occur in animal models of critical limb ischemia (CLI) and in humans (15, 16). Therefore, researchers have attempted to prevent amputation by enhancing these natural processes. One angiogenic factor that has gained attention in many ischemic conditions, including cardiac ischemia, stroke, and CLI, is vascular endothelial growth factor (VEGF) (17-19). In 1994, limb salvage and increased angiogenesis were reported in a rabbit CLI model after a single bolus intra-arterial administration of VEGF-165 (20). Other experiments using the same model demonstrated the absence of calf muscle atrophy and distal limb necrosis after VEGF administration, compared with 85.7% of control rabbits (21, 22). Various studies have replicated these findings in other models of CLI (23-25). Unfortunately, this has not been successfully replicated in clinical practice. Studies using VEGF protein (26) or DNA plasmids have failed to demonstrate significant benefit in reducing limb amputations in double-blind settings (25, 27). In one embodiment of the present invention, fibroblast-derived exosomes are utilized to stimulate VEGF production from patient cells.

[0046] Another approach involved the use of the cytokine fibroblast growth factor-1 (FGF-1). Assuming that FGF-1 is "upstream" of VEGF, it was thought that FGF-1 stimulates numerous angiogenic processes, resulting in the creation of more mature blood vessels (28). Like VEGF, FGF is part of the natural tissue response to hypoxia, as demonstrated in both animal models (29) and clinical trials (30). The crucial role of FGF in endogenous angiogenesis was conclusively demonstrated in FGF-conditional knockout mice, which exhibited impaired post-wound healing and angiogenesis (31). Although FGF-1 gene therapy has been used clinically in CLI patients with some improvement in ABI and perfusion, results have been modest (32). Attempts to recapitulate the in vivo angiogenic cascade using a combination of cytokines have yielded more promising results. Cao et al. demonstrated a synergistic effect between PDGF-BB and FGF-2 administration in enhancing angiogenesis and function in a femoral artery ligation model in rats and rabbits (33). Similarly, in cancer angiogenesis, several tumor-derived angiogenic factors are known to cooperate to promote angiogenesis (34). Researchers have attempted to activate upstream mediators of several angiogenic signals by transfection of genes encoding transcription factors (e.g., HIF-1 alpha) (35). In fact, this approach has been shown to be superior to VEGF gene administration in terms of new capillary sprouting. In a phase I dose-escalation study, transfection of HIF-1 alpha in patients with CLI was found to be tolerable, with some evidence of efficacy (36). In conclusion, although administration of angiogenic factors to patients with CLI does indeed induce some benefit in early studies, data from randomized trials to date do not support widespread use. Transfection of upstream transcription factors (e.g., HIF-1alpha) is a promising approach because it mimics natural angiogenesis in that multiple growth factors are induced after transfection (35, 37).In another embodiment of the present invention, the use of fibroblast-derived exosomes to enhance cytokines and other angiogenic therapies is disclosed.

[0047] Exosomes were prepared from cell culture supernatants of day 4 foreskin fibroblast cultures by differential centrifugation. Briefly, the collected culture supernatants were subjected to three consecutive centrifugations at 300g (5 min), 1,200g (20 min), and 10,000g (30 min) to remove cells and debris, followed by centrifugation at 100,000g for 1 h. To remove excess serum proteins, the exosome pellets were washed with a large volume of PBS, centrifuged at 100,000g for 1 h, and finally resuspended in 120 μl of PBS for further study. Exosomes were quantified by a microvolume Bradford protein assay (Bio-Rad). Each batch was normalized by protein content.

[0048] Peripheral blood mononuclear cells (PBMCs) were isolated from 5 ml of blood using a Ficoll density gradient (Sigma-Aldrich). Cells were washed twice with phosphate-buffered saline (PBS) and plated in a round-bottom 96-well plate (Nunc). In each well, 10,000, 20,000, or 100,000 PBMCs were added to a total volume of 200 μL in RPMI medium containing 10% fetal bovine serum (Life Technologies). Exosomes were added at concentrations of 0.1 μg / ml, 0.2 μg / ml, and 0.4 μg / ml. Cells were cultured for 48 hours, and VEGF concentrations were analyzed by ELISA (R&D Systems). Concentrations are expressed as pg / ml in Figure 1.

[0049] Example 2 Fibroblast exosomes stimulate HUVEC proliferation Fibroblast-derived exosomes were obtained as described in Example 1 and added to cultures of human umbilical vein endothelial cells (HUVECs). The cells were incubated for 48 hours, and proliferation was assessed by thymidine incorporation assay. In Figure 2, proliferation is expressed as counts per minute (CPM).

[0050] Example 3 Prevention of Limb Loss in a Murine Model of Critical Limb Ischemia BALB / c mice were treated with femoral artery ligation and local nerve injury in a previously published model (Meng et al., 2007, J. Trans. Med. 5:57).

[0051] Exosomes were prepared from cell culture supernatants of day 4 foreskin fibroblast cultures by differential centrifugation. Briefly, the collected culture supernatants were subjected to three consecutive centrifugations at 300g (5 min), 1,200g (20 min), and 10,000g (30 min) to remove cells and debris, followed by centrifugation at 100,000g for 1 h. To remove excess serum proteins, the exosome pellets were washed with a large volume of PBS, centrifuged at 100,000g for 1 h, and finally resuspended in 120 μl of PBS for further study. Exosomes were quantified by a microvolume Bradford protein assay (Bio-Rad). Each batch was normalized by protein content.

[0052] Mice were administered 5 micrograms of fibroblast exosomes in a volume of 100 microliters three days after femoral artery ligation (treated). Controls were administered 5 micrograms of fetal bovine serum-derived exosomes (untreated). Limb loss was present in all untreated mice (7 / 7), whereas limb loss was observed in only 1 / 7 treated mice. Administration of mesenchymal stem cell exosomes did not result in limb salvage by day 35.

[0053] [Table 1]

[0054] While the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the invention, as defined by the appended claims. Moreover, the scope of this application is not intended to be limited to the particular embodiments of the processes, apparatus, manufacture, compositions of matter, means, methods, and steps described herein. As those skilled in the art will readily appreciate from this disclosure, any processes, apparatus, manufacture, compositions of matter, means, methods, and steps, whether currently existing or later developed, that perform substantially the same function as, or achieve substantially the same results as, the corresponding embodiments described herein may be utilized in accordance with the present invention. Accordingly, the appended claims are intended to include within their scope such processes, apparatus, manufacture, compositions of matter, means, methods, or steps. (References) All patents and publications cited herein are hereby incorporated by reference in their entirety. 1. Thery, C., M. Ostrowski, and E. Segura, Membrane vesicles as conveyors of immune responses. Nature reviews. Immunology, 2009. 9(8): p. 581-93. 2. Ludwig, AK and B. Giebel, Exosomes: Small vesicles participating in intercellular communication. The international journal of biochemistry & cell biology, 2011. 3. Alvarez-Erviti, L., et al., Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiology of disease, 2011. 42(3): p. 360-7. 4. Silverman, J.M. and N.E. Reiner, Exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes. Cellular microbiology, 2011. 13(1): p. 1-9. 5. Pan, B.T. and R.M. Johnstone, Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell, 1983. 33(3): p. 967-78. 6. Alonso, R., et al., Diacylglycerol kinase alpha regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of Fas ligand-containing exosomes in T lymphocytes. Cell death and differentiation, 2011. 18(7): p. 1161-73. 7. Zhang, H., et al., CD4(+) T cell-released exosomes inhibit CD8(+) cytotoxic T-lymphocyte responses and antitumor immunity. Cellular & molecular immunology, 2011. 8(1): p. 23-30. 8. Mathews, J.A., et al., CD23 Sheddase A disintegrin and metalloproteinase 10 (ADAM10) is also required for CD23 sorting into B cell-derived exosomes. The Journal of biological chemistry, 2010. 285(48): p. 37531-41. 9. Buschow, S.I., et al., MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis. Immunology and cell biology, 2010. 88(8): p. 851-6. 10. Hwang, I. and D. Ki, Receptor-mediated T cell absorption of antigen presenting cell-derived molecules. Frontiers in bioscience : a journal and virtual library, 2011. 16: p. 411-21. 11. Viaud, S., et al., Updated technology to produce highly immunogenic dendritic cell-derived exosomes of clinical grade: a critical role of interferon-gamma. Journal of immunotherapy, 2011. 34(1): p. 65-75. 12. Clayton, A., et al., Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production. Journal of immunology, 2011. 187(2): p. 676-83. 13. Battke, C., et al., Tumour exosomes inhibit binding of tumour-reactive antibodies to tumour cells and reduce ADCC. Cancer immunology, immunotherapy : CII, 2011. 60(5): p. 639-48. 14. Lachenal, G., et al., Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity. Molecular and cellular neurosciences, 2011. 46(2): p. 409-18. 15. Faure, J., et al., Exosomes are released by cultured cortical neurones. Molecular and cellular neurosciences, 2006. 31(4): p. 642-8. 16. Fitzner, D., et al., Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis. Journal of cell science, 2011. 124(Pt 3): p. 447-58. 17. Mincheva-Nilsson, L. and V. Baranov, The role of placental exosomes in reproduction. American journal of reproductive immunology, 2010. 63(6): p. 520-33.

Claims

1. 1. A composition for stimulating angiogenesis in an individual, comprising an effective amount of fibroblast-derived exosomes, A composition for stimulating angiogenesis in an individual, wherein said individual is at risk for limb loss.

2. A composition described in claim 1 for an individual in need of angiogenesis.

3. The composition of claim 1 or 2, wherein the fibroblasts are derived from a biopsy.

4. The composition of claim 1 , 2 or 3, wherein the fibroblasts are obtained from the individual.

5. The composition of any one of claims 1 to 4, wherein the fibroblasts are not obtained from the individual.

6. The composition of any one of claims 1 to 5, wherein the fibroblasts are cultured in a medium that allows proliferation of the fibroblasts.

7. The composition of claim 6, wherein the medium allowing for the proliferation of fibroblasts comprises one or more factors that are mitogenic for fibroblasts.

8. 8. The composition of claim 7, wherein the factor that is mitogenic for fibroblasts comprises one or more factors selected from the group consisting of: a) FGF-1, b) FGF-2, c) FGF-5, d) EGF, e) CNTF, f) KGF-1, g) PDGF, h) platelet-rich plasma, i) TGF-alpha, j) HGF-1, and (k) combinations thereof.

9. The composition of any one of claims 1 to 8, wherein the fibroblasts are cultured under hypoxia.

10. 10. The composition of any one of claims 1 to 9, wherein the exosomes are collected from fibroblasts while the fibroblasts are in a proliferative state.

11. The composition of any one of claims 1 to 10, wherein the exosomes are collected from fibroblasts while the fibroblasts are cultured in a medium that does not contain any proliferation-inducing factor, or in a medium that contains a reduced level of the proliferation-inducing growth factor compared to a standard level.

12. 12. The composition of any one of claims 1 to 11, wherein the exosomes are recovered from the fibroblasts cultured at 2% to 8% oxygen for at least 1 day.

13. The composition of claim 12, wherein the cells are cultured for 1 to 15 days.

14. The composition of claim 12, wherein the cells are cultured for 5 to 10 days.

15. The composition of any one of claims 1 to 14, wherein the cells are subcultured for at least one passage.

16. A composition described in any one of claims 1 to 15, wherein the composition contains less than 5% polyethylene glycol.

17. 17. The composition of any one of claims 1-16, wherein the exosomes express a marker selected from the group consisting of: (a) CD63, (b) CD9, (c) MHC I, (d) CD56, and (e) combinations thereof.

18. 18. The composition of any one of claims 1 to 17, wherein the fibroblasts are cultured in a medium selected from the group consisting of: a) Roswell Park Memorial Institute (RPMI-1640), b) Dulbecco's Modified Essential Medium (DMEM), c) Eagle's Modified Essential Medium (EMEM), d) Optimem, e) Iscove's Medium, and f) combinations thereof.

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