WO2021207025A1 - Mesenchymal cell (msc) exosomes increase t cell differentiation towards t regulatory cells - Google Patents

Mesenchymal cell (msc) exosomes increase t cell differentiation towards t regulatory cells Download PDF

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WO2021207025A1
WO2021207025A1 PCT/US2021/025566 US2021025566W WO2021207025A1 WO 2021207025 A1 WO2021207025 A1 WO 2021207025A1 US 2021025566 W US2021025566 W US 2021025566W WO 2021207025 A1 WO2021207025 A1 WO 2021207025A1
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cells
evs
extracellular vesicles
population
disease
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Seth H. ANDREWS
Steven L. Stice
Ross Alexander MARKLEIN
Timothy Scott MAUGHON
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University Of Georgia Research Foundation, Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • AC-EVs often MSC-EVs are subjected to various conditions to enhance quality and therapeutic function of these extracellular vesicles.
  • the inventors examined hypoxia (LO2-EV), acidosis (LPH-EV), and inflammatory cytokines (INF-EV) on AC-EV (MSC-EV) biogenesis and secretion as compared to the unprimed, serum-free culture conditions.
  • the present invention is directed to a method to create immune-suppressive Adherent cell (preferably MSC) exosomes as well as pH stimulated adherent cell (preferably, MSC) exosomes.
  • MSCs Mesenchymal stem cells
  • MSCs When used in preclinical models, MSCs have been shown to greatly reduce inflammation and while the mechanism for this reduction may not have been elucidated, it is known that MSCs reduce the levels of many pro-inflammatory cytokines such as TNF- ⁇ , IL- 1 ⁇ , and IL-6 while increasing the levels of cytokines which reduce inflammation like IL-4, IL-5, and IL-10.
  • MSCs promote bacterial clearance both directly by secreting antimicrobial peptides and proteins such as LL-37 and lipocalin as well as indirectly by activating host monocytes, macrophages and neutrophils which then phagocytose the bacteria.
  • MSC secretion of substances such as keratinocyte growth factor (KGF) has been shown to be essential in alveolar fluid clearance and restoration of epithelial permeability. MSCs have been proven to have an immunomodulatory effect through multiple mechanisms. Although initially, it was thought that these cells would promote regeneration of the injured tissue through engraftment and trans-differentiation, now it has become apparent that engraftment plays little to no role in their therapeutic action. However, it is known that these cells modulate host cells through direct cell-to-cell interactions and through the release of factors (paracrine) including active agents such as KGF, indoleamine 2,3-dihydrogenase, and prostaglandin E2.
  • KGF keratinocyte growth factor
  • EVs extracellular vesicles
  • MSC-EVs Cell-free extracellular vesicles secreted by MSC
  • MSC-EVs possess the similar therapeutic function of MSC therapy, but the MSC-EVs do not divide or engraft like MSCs, thus addressing concerns with tumorigenicity and ectopic tissue growth.
  • extracellular/extrasomal vesicles are being actively explored as an alternative to whole-cell therapy.
  • Novel therapies for disease states and/or conditions where there is high mortality and no pharmacological therapeutic approach to providing therapy such as osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders are critically needed.
  • Additional disease states and/or conditions which can be treated using EVs according to the present invention include, eosinophil-associated gastrointestinal diseases, noninfectious posterior uveitis and cancers, especially including myeloma, multiple myeloma, lymphoma and opsoclonus-myoclonus syndrome (OMS).
  • MSC-EVs demonstrate potent protective effects mediated through a variety of mechanisms related to the transfer of the EVs cargo to the recipient cells.
  • MSC-EVs may suppress pro-inflammatory responses
  • biomanufacturing of MSC-EVs remains an emerging field with great potential.
  • the lack of standardized methods may result in final products with functional heterogeneity.
  • Acidic priming is an easy approach to increase the production of MSC-EVs with anti- inflammatory function. Further research into the mechanism of action, biodistribution, standardization, and biomanufacturing is needed to facilitate clinical translation of this new cell therapy to more general therapeutic outcomes.
  • AC-EVs and in particular, MSC-EVs pursuant to the present invention, are useful for treating auto-immune diseases, traumatic injuries and vascular injuries, including osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as enhancing skeletal muscle repair, bone repair, treating cancer, cardiovascular diseases, and other neurological disorders.
  • These methods comprise administering to a patient or subject in need a composition comprising an effective amount of AC-EVs, preferably MSC-EVs, by intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal routes of administration in order to effect therapy in said patient or subject.
  • the present invention is directed to extracellular vesicles prepared from adherent cells (ACEVs) which are exposed to hypoxic conditions to produce low oxygen extracellular vesicles (LO2-EVs), acidic conditions to produce low pH extracellular vesicles (LPH-EVs) and inflammatory conditions (exposure to concentrations of cytokines, including interferons, interleukins and tumor necrosis factors) to produce inflammatory extracellular vesicles (INF-EVs). Once produced, these EVs find use in the treatment of disease states and/or conditions as otherwise described herein.
  • the EVs pursuant to the present invention may be formulated in pharmaceutical compositions comprising an effective amount of EVs in combination with a pharmaceutically acceptable carrier, additive and/or excipient.
  • compositions according to the present invention may also include an effective amount of one or additional bioactive agent which is useful in the treatment of disease.
  • the bioactive agent is an anti-cancer agent.
  • adherent cells including MSCs as otherwise described herein are first grown to 70-95% confluence in media (confluent cells), harvested and replated at 2,000- 10,000 cells/cm 2 (preferably, 5,000 cells/cm2) on plating material.
  • the replated cells are exposed to hypoxic, low pH or inflammatory conditions in a priming step for approximately one minute to 72 hours, often 24-28 hours, rinsed, and subsequently grown in growth medium for another 12 hours to 6 days or more, preferably 24-48 hours, after which the cells are collected and filtered to remove cells and large debris from the extracellular vesicles in the medium.
  • the period of priming for all conditions is that which is sufficient to affect the cell secretome, to maximize EV production with characteristics of the priming conditions.
  • the period of time sufficient for the adherent cells to release EVs after priming is about 12-36+ hours, more often 24-48 hours.
  • confluent adherent cells preferably, MSCs
  • a hypoxic priming step wherein the confluent cells are exposed to gas/atmosphere mixtures in culture containing from 0.5-21% oxygen, more often about 0.75% to 15% oxygen, often about 1.0% to 10% oxygen, about 1.5% to 5.0% oxygen, 2.0-3.5% oxygen, about 0.25% to about 12.5% oxygen, and about 5% oxygen, the remainder of the gas/atmosphere consisting essentially of nitrogen (from 75% to 98%) and a small percentage of carbon dioxide (about 1% to 10%, often about 5%).
  • the adherent cells are primed in the hypoxic atmospheric conditions for a period sufficient to affect the cell secretome. This period may range from one minute up to 72 hours, often 2- 15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 12-48 hours before washing, collecting and freezing the EVs which are released from the cells.
  • growth medium preferably, serum free medium
  • confluent adherent cells are primed under acidic conditions to provide a pH of the priming medium ranging from 6.0-7.35, often 6.9-7.3 for a period ranging from one minute up to 72 hours, often 2-15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells.
  • growth medium preferably, serum free medium
  • confluent adherent cells are primed under conditions with effective concentrations of cytokines (e.g. interferon type I, including IFN- ⁇ , IFN- ⁇ , IFN- ⁇ IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN-v, interleukins 1, 1 ⁇ , 1 ⁇ , and 2-36 and/or tumor necrosis factors, TNF 1-19 and 13B) at effective concentrations ranging from 1 ng/ml to 100 ng/ml or more, often about 5 ng/ml to about 50 ng/ml, about 10 ng/ml to about 30 ng/ml, often about 15 ng/ml to 25ng/ml for a period ranging from one minute up to 72 hours, often 2-15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6
  • cytokines e.g. interferon type I, including IFN- ⁇ ,
  • FIGURE 1 shows EV characterization. The size and concentration of MSC-EVs is affected by priming. Hypoxic and acidic preconditioning increased EV release.
  • A Size distributions of EV groups as measured by NTA
  • B EV diameter across MSC culture conditions as determined by NTA.
  • C EV concentration across MSC culture conditions as determined by NTA.
  • FIGURE 3 shows the differential uptake of MSC-EVs by T cell subsets. LPH-EV treated PBMCs had significantly higher CFSE+ frequency than untreated cells, indicating uptake of EVs.
  • A Frequency of CD4 + cells positive for CFSE.
  • B Frequency of CD8 + cells positive for CFSE.
  • C Frequency of CD4 + /CD25 + cells positive for CFSE.
  • D Frequency of CD8 + /CD25 + cells positive for CFSE.
  • FIGURE 4 shows the inhibition of T cell proliferation 5 days after treatment with EVs or MSCs as measured by CFSE dilution.
  • FIGURE 5 shows the comparative activation of T-cell subsets 5 days after treatment with EVs or MSCs. Overall, MSCs had significant effects on T effector cells frequency, while LPH-EVs had significant effects on Treg frequency.
  • A Frequency of CD4 + /CD25 + /FOXP3- cells.
  • FIGURE 1S shows experimental workflow. MSCs were split into groups that each underwent different priming steps. MSCs were then either co-cultured with PBMCs for 5 days or used for EV isolation. Isolated EVs were used in NTA or MACSPLEX analysis, stained with CFSE and incubated for 24 hours with PBMCs, or incubated for 5 days with PBMCs.
  • FIGURE 2S shows an antibody table. All antibodies used in the experimental study, were titrated to optimal concentration prior to experiments.
  • FIGURE 3S shows a fFlow analysis diagram.
  • FIGURE 4S shows the dynamic Light Scattering of EVs. Histograms of EV size distribution confirm the existence of a large-diameter population present in INF-MSC-EV at a high concentration.
  • FIGURE 5S shows the comparative activation of T cell subsets 24 hours after treatment with CFSE-EVs. There were no significant differences between treatment groups.
  • A Frequency of CD4 + cells.
  • B Frequency of CD8 + cells.
  • C Frequency of CD4 + /CD25 + cells.
  • D Frequency of CD8 + /CD25 + cells.
  • E Frequency of CD4 + /CD25 + /FOXP3 + cells.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • effective is used to describe an amount of a composition or component which is included to effect an intended result, including the production of MSC-EVs as otherwise described herein .
  • therapeutically effective refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • the terms “treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a patient at risk for or afflicted by a disease state, condition or deficiency which may be improved using cellular compositions according to the present invention.
  • Treating a condition includes improving the condition through lessening or suppression of at least one symptom, delay in progression of the effects of the disease state or condition, including the prevention or delay in the onset of effects of the disease state or condition, etc.
  • treatment can involve reducing the impact of a spinal cord injury or stroke, including reversing and/or inhibiting the effects of such injury, reversing, improving, inhibiting and/or stabilizing a neurodegenerative disease such that the disease improves and/or does not progress or worsen.
  • prophylactic is used to describe a method which “reduces the likelihood” that a particular result will occur, often the progression and/or worsening of a disease state and/or condition.
  • Standard techniques for growing cells, separating cells, and where relevant, cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.
  • a number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (Ed.) 1993 Meth. Enzymol.218, Part I; Wu (Ed.) 1979 Meth.
  • culture media is used to describe a growth medium in which MSC’s are grown, such media especially including media in which MSCs are grown to produce EVs as otherwise described herein. Culture media describeds media which comprises sufficient components to promote the growth and health of cells.
  • Culture media are well known in the art and comprise at least a minimum essential medium plus one or more optional components such as growth factors, including fibroblast growth factor (FGF), ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine (often at 2mM L-glutamine), penicillin (often, for example at 50U/ml), streptomycin (often 50 ⁇ g/ml), insulin (where indicated and not excluded), transferrin, beta mercaptoethanol, and other agents well known in the art and as otherwise described herein.
  • FGF fibroblast growth factor
  • ascorbic acid glucose
  • non-essential amino acids salts (including trace elements)
  • glutamine often at 2mM L-glutamine
  • penicillin often, for example at 50U/ml
  • streptomycin often 50 ⁇ g/ml
  • insulin where indicated and not excluded
  • transferrin beta mercaptoethanol
  • Culture media may include basal cell media which may contain between 1% and 20% (often 10%)
  • Other components which optionally may be added to culture medium are as otherwise described herein, especially including the experimental section which follows. Each of these components, when included, are included in effective amounts, i.e., in amount sufficient to promote the growth and health of cells.
  • LIF leukemia inhibitory factor
  • BDNF brain-derived neurotrophic factor
  • EGF epidermal growth factor receptor
  • bFGF basic fibroblast growth factor
  • FGF-6 glial-derived neurotrophic factor
  • GDNF glial-derived neurotrophic factor
  • GCSF granulocyte colony-stimulating factor
  • HGF hepatocyte growth factor
  • IGFBP-2 insulin-like growth factor binding protein
  • IGFBP-6 insulin-like growth factor binding protein
  • IGFBP-6 insulin-like growth factor binding protein
  • IGFBP-6 IL-1ra
  • IL-6 IL-8
  • MCP-1 mononuclear phagocyte colony-stimulating factor
  • NT3 tissue inhibitor of metalloproteinases
  • TIMP-2 tumor necrosis factor
  • TNF- ⁇ tumor necrosis factor
  • VEGF vascular endothelial growth factor
  • VEGF-D vascular endothelial growth factor
  • suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco # 10829- 018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non- essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
  • DMEM Dulbecco's modified Eagle's medium
  • KO DMEM Knockout Dulbecco's modified Eagle's medium
  • bFGF basic fibroblast growth factor
  • culture media useful in the present invention are commercially available and can be supplemented with commercially available components, available from Invitrogen Corp. (GIBCO), Cell Applications, Inc., among numerous other commercial sources, including Calbiochem.
  • GEBCO Invitrogen Corp.
  • Cell Applications, Inc. among numerous other commercial sources, including Calbiochem.
  • One of ordinary skill in the art will be able to readily modify the cell media to grow MSCs or to produce any one or more of the target MSC-EVs pursuant to the present invention.
  • human Pluripotent Stem Cells of which ”human Embryonic Stem Cells” (hESCs) and human induced pluripotent stem cells (hiPSCs) are a subset, are derived from pre-embryonic, embryonic, fetal tissue or adult stem cells (in the case of human induced pluripotent stem cells) at any time after fertilization, and have the characteristic of being capable under appropriate conditions of producing progeny of several different cell types, especially including mesenchymal stem cells (MSCs) and related proliferative and non- proliferative cells.
  • MSCs mesenchymal stem cells
  • the term includes both established lines of stem cells of various kinds, and cells obtained from primary tissue that are pluripotent in the manner described.
  • embryonic stem cell refers to pluripotent cells, preferably of primates, including humans, which are isolated from the blastocyst stage embryo.
  • extracellular vesicle producing adherent cells or “EVPAC” is used to describe adherent cells which can be used to produce LO2-EVs, LPH-EVs and INF-EVs “adherent cell extracellular vesicles” or “EC-EVs”, preferably MSC-EVs pursuant to the present invention.
  • adherent cells include primary cells (e.g. mesenchymal stem cells/MSCs, pericytes, fibroblasts, and immortalized cells lines, such as HEK-293T), among others.
  • MSCs are preferred adherent cells for providing LO2- EVs, LPH-EVs and INF-EVs pursuant to the present invention.
  • the term “mesenchymal stem cell” or “MSC” refers to multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes. These cells are multipotent non-hematopoietic, self-renewable cells that are capable of trilineage differentiation into mesoderm, ectoderm, and endoderm cells. These cells also characterized by their pluripotency and immunomodulatory features, and their ability to be cultured over lengthy periods of time.
  • MSCs are used to produce the various extracellular vesicles/extrasomes pursuant to the present invention.
  • MSCs may be purchased from commercial sources (e.g. Lifeline Cell Technologies, Oceanside, California USA, among others).
  • MSCs can be readily produced following literature preparations well-known in the art. US patent no.5,486,359 and Pittenger et al., Science, Vol.284, pg.143 (1999) provide methods for the production of MSCs.
  • MSCs Mesenchymal stem cells
  • extracellular vesicle and“EV” are used herein to refer to a vesicle of about 10nm to 10 ⁇ m in size consisting of fluid, macro-molecules, solutes, and metabolites from a cell contained by a lipid bilayer or micelle.
  • the EV is a cell-derived EV (from adherent cells, preferably MSCs).
  • MSCs adherent cells
  • EV may also include lipid vesicles engineered to contain bioactive molecules found in a cell-derived EVs, such as MSC-EVs. These terms encompass both exosomes and ectosomes. Exosomes are extracellular vesicles released on the exocytosis of multivesicular bodies (MVBs).
  • Ectosomes are vesicles assembled at and released from the plasma membrane.
  • the EV is about 20nm to 10 ⁇ m, 20nm to 1 ⁇ m, 20 nm-500 nm, 30 nm-100nm, 30 nm-160nm, or 80-160 nm in size.
  • the EVs are exosomes that are about 20 to 150 nm in size. Often the exosomal vesicles range in size from 10 nm to 250 nm or more.
  • autologous EV is used to describe a population of EVs which are obtained from MSC cells from a subject or patient to whom the EVs are to be administered.
  • MSC-EV is used to refer to a cell-derived EV produced from mesenchymal stem cells derived in vitro from pluripotent stem cells or progenitor cells.
  • the term also refers to vesicles engineered to contain a sufficient number of the bioactive molecules found in the cell-derived MSC-EVs to have substantially the same bioactivity.
  • LO2-EV is used to describe extracellular vesicles which are prepared using a hypoxic priming step of the adherent cells prior to incubating.
  • AC-EVs Preferably MSC-EVs
  • MSC-EVs LO2-EV Extracellular vesicles produced from adherent cells (preferably MSCs) exposed to hypoxic conditions
  • LPH-EV Extracellular vesicles produced from adherent cells preferably MSCs
  • INF-EV Extracellular vesicles produced from adherent cells preferably MSCs exposed to inflammation cytokines.
  • MSCs are plated at approximately 1,000-10,000, or 2,500-10,000, often 5,000 cells cells/cm 2 on tissue culture flasks in complete medium (Alpha-Minimum Essential Medium (Gibco), 10% defined fetal bovine serum (Hyclone), 2 mM L-glutamine, 50 U/mL penicillin, 50 ⁇ g/mL streptomycin (Gibco)) and allowed to grow to 80% confluence (approximately 20,000–25,000 cells/cm 2 ). The cells were harvested using 0.05% trypsin (Gibco) and replated at 5000 cells/cm 2 . All proliferation cultures were maintained at 37°C and at 5% CO 2 .
  • Confluent MSCs (as grown above to about 70-95% confluence, preferably 80% confluence and 80-100%, often 90-95% or more viability) are then exposed (primed) to hypoxic, acidosis or inflammatory conditions for between one minute up to 72 hours, often 2- 15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for a time sufficient to release EVs into culture media, often between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells.
  • growth medium preferably, serum free medium
  • the primed cells can be placed in co-culture with human Peripheral Blood Mononuclear Cells (PBMCs). Extracellular Vesicle/Exosome Isolation After priming, MSCs are rinsed twice with PBS before adding fresh serum free medium (Alpha-Minimum Essential Medium (Gibco), 2 mM L-glutamine, 50 U/mL penicillin, 50 ⁇ g/mL streptomycin (all from Gibco/Invitrogen) and incubating cultures for 24- 48 hours. The resulting conditioned media containing cells were collected and filtererd (e.g. 0.22 ⁇ m filters) to remove cells and large debris.
  • PBMCs Peripheral Blood Mononuclear Cells
  • the filtered media were subjected to ultrafiltration (with, for example a 100kDa MWCO (Amicon, Millipore-Sigma) at 4000g for 10 minutes as previously published (26).
  • the EVs remained on top of the filter and were then washed twice with PBS +/+ (Thermo Fisher Scientific, Waltham, MA) at 2000g for 10 minutes.
  • the EVs in PBS+/+ were then collected, aliquoted, and frozen at -20°C.
  • Nanoparticle tracking analysis was performed using a Nanosight NS3200 (Nanosight, Salisbury UK) according to the manufacturer’s recommendations. Briefly, aliquots of EV suspensions were thawed at room temperature and diluted to 10 7 -10 9 particles/mL with the same lot of PBS +/+ the EVs were isolated in. A minimum of three samples and five one-minute videos were recorded for each exosome isolation. All videos were captured at the same camera level and analyzed with the same detection threshold.
  • vesicles were further verified via Dynamic Light Scattering using a Malvern Zetasizer Nano ZS Analyzer (Malvern Instruments, Malvern, UK). Samples were diluted to a total vesicle concentration of approximately 2x10 8 vesicles/ml in 0.22 ⁇ m filtered Phosphate Buffered Saline containing Calcium and Magnesium, pH 7.4 prior to measurements. Disposable polystyrene cuvettes were rinsed with 1mL of filtered PBS +/+ prior to adding sample. Measurements were taken using cuvette with 800uL of prepared sample.
  • EV surface marker characterization was performed using the MACSPLEX Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s directions. Briefly, an equal number of exosomes as determined by NTA were analyzed from each isolation in triplicate. Flow cytometry analysis was performed using a CytoFLEX S (Beckman Coulter, Hialeah, Florida) alongside bead only controls, with FlowJo being used for data analysis. Data was processed with background subtraction and normalized to the median of the average value of CD9, CD63, and CD81 for each sample (27). The data was transformed to be a percentage of the difference between the maximum and minimum relative expression of a marker.
  • PCA Principal component analysis
  • the MSCs are primed using a hypoxic gas/atmospheric mixture as described above for a period ranging from about 1 minute up to 1 hour or much longer in certain instances, more often 2- 15 minutes, more often 5-10 minutes before growing the MSCs in serum free medium for a period between 12 hours and 6 days or more, more often 24 hours to 72 hours, 36 to 60 hours, often about 48 hours or as otherwise described herein.
  • Acidosis/Acidic Conditions - confluent adherent cells (MSCs) in media under acidic conditions (use of protic acid such as HCl, phosphoric acid, organic acid or other acid to reduce pH of priming media to about 6.0 to 7.35, often 6.9 to about 7.3, often 7.1 + 0.05).
  • Inflammation Conditions prime confluent adherent cells (preferably, MSCs) are primed under conditions with effective concentrations of cytokines (e.g.
  • interferon type I including IFN- ⁇ , IFN- ⁇ , IFN- ⁇ IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ , interleukins 1, 1 ⁇ , 1 ⁇ , and 2-36 and/or tumor necrosis factors, TNF 1-19 and 13B) at effective concentrations ranging from 1 ng/ml to 100 ng/ml or more, often about 5 ng/ml to about 50 ng/ml, about 10 ng/ml to about 30 ng/ml, often about 15 ng/ml to 25ng/ml for a period ranging from one minute up to 72 hours, often 2-15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells.
  • growth medium preferably, serum free medium
  • AC-EVs preferably MSC-EVs (e.g. extracellular vesicles or exosomes) and methods of using these EVs in the treatment of disease states and conditions, including osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders.
  • MSC-EVs mesenchymal stem cells
  • pluripotent stem cells e.g.
  • hESCs human embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • LO2-EV hypoxia
  • LPH-EV acidosis
  • INF-EV inflammatory cytokines
  • the disclosed EVs can be obtained in some embodiments by culturing MSCs, derived directly or indirectly from pluripotent stem cells in cell culture medium under conditions and for a time sufficient to produce EVs, and isolating said EVs from the culture medium.
  • MSCs may be purchased from commercial sources or produced by known methods, long known in the art.
  • MSCs are cultured in hypoxic, acidic (acidosis) or in the presence of inflammatory cytokines conditions for a sufficient period (generally about 12 hours to 72 hours or more (about 1 week), often 24-60 hours, more often about 48 hours (about 2 days) to produce MSC-EVs in the media and the resulting media is subjected to filtration to remove cellular debris to isolate the MSC-EVs which are further subjected to ultrafiltration to isolate the MSC-EVs.
  • Pluripotent stem cells used to produce the EV-producing MSCs include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
  • Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1.
  • SSEA stage-specific embryonic antigens
  • Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.
  • the types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • Non- limiting examples are established ethical lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines WA01, WA07, and WA099 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells.
  • mutant human embryonic stem cell lines such as, for example, BG01v (BresaGen, Athens, Ga.), as well as normal human embryonic stem cell lines such as WA01, WA07, WA09 (WiCell) and BG01, BG02 (BresaGen, Athens, Ga.).
  • Human embryonic stem cells hESCs
  • hESCs may be prepared by methods which are described in the in the art as described for example, by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995).
  • Epiblast stem cells EpiScs
  • iPSCs induced pluripotent stem cells isolated from early post-implantation stage embryos. They express Oct4 and are pluripotent. iPSCs are made by dedifferentiating adult somatic cells back to a pluripotent state by retroviral transduction of four genes (c-myc, Klf4, Sox2, Oct4).
  • EpiScs Epiblast stem cells
  • iPSCs induced pluripotent stem cells isolated from early post-implantation stage embryos. They express Oct4 and are pluripotent. iPSCs are made by dedifferentiating adult somatic cells back to a pluripotent state by retroviral transduction of four genes (c-myc, Klf4, Sox2, Oct4).
  • [e]xosomes produced from cells can be collected from the culture medium and/or cell tissue by any suitable method.
  • a preparation of EVs can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of
  • EVs can be prepared by differential centrifugation, that is low speed ( ⁇ 2,000g) centrifugation to pellet larger particles followed by high speed (>100,000 g) centrifugation to pellet EVs, size filtration with appropriate filters (for example, 0.22 ⁇ m filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods.”
  • the MSCs are cultured over a sufficient period (12 hours to a week or more as otherwise described herein) to produce EVs and the EVs are isolated by filtration (to remove cellular debris), followed by ultrafiltration to separate EVs from remaining fluid.
  • the EVs After washing the EVs may be collected and aliquoted and used directly or frozen (at preferably -10oC to -30oC) for subsequent use after thawing. It is noted that the contents of EVs, i.e., EVs in which the lipid bilayer has been removed or eliminated and the contents obtained may also be used to engineer artificial EVs. Further, as described in U.S. Patent Application Document No.20140356382, exogenous protein and/or peptide and other cargo can be introduced into the EVs by a number of different techniques including electroporation or the use of a transfection reagent. Electroporation conditions may vary depending on the charge and size of the biotherapeutic cargo.
  • Typical voltages are in the range of 20V/cm to 1,000V/cm, such as 20V/cm to 100V/cm with capacitance typically between 25 ⁇ F and 250 ⁇ F, such as between 25 ⁇ F and 125 ⁇ F.
  • a voltage in the range of 150 mV to 250 mV, particularly a voltage of 200 mV is preferred for loading EVs with an antibody.
  • the EVs may be loaded with exogenous protein and/or peptide using a transfection reagent.
  • conventional transfection agents may be used for transfection of EVs with protein and/or peptide.
  • EVs may also be loaded by transforming or transfecting a host cell with a nucleic acid construct which expresses therapeutic protein or peptide of interest, such that the therapeutic protein or peptide is taken up into the EVs as the EVs are produced from the cell.
  • the EV-producing MSCs disclosed herein are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or for as long as about 1, 2, 3, 4, 5, 6, 7, 8 weeks or about 1, 2, 3, 4, 5, or 6 months, depending on the cell and its ability to produce EVs.
  • the MSCs are cultured for between about 12 hours and one week, often 24-72 hours, more often about 24-60 hours, even more often about 48 hours to produce the MSC-EVs according to the present invention.
  • the EV-producing cells may be cultured in suitable media and grown under conditions that are readily determined by one of ordinary skill in the art. Cell culture conditions may vary with cell type and the examples presented hereinafter illustrate suitable media and conditions as otherwise described herein.
  • CMRL 1066 medium from Invitrogen
  • fetal bovine serum e.g., at 10%
  • glutamine or glutamine-containing mixtures and antibiotics could be used.
  • Cells can be grown on a surface (feeder cells) in some embodiments, e.g.
  • EV producing adherent cells may include primary cells (mesenchymal stem cells, pericytes, fibroblasts, immortalized cell lines (HEK-293T).
  • the density of the cells (which are primed) sufficient to produce EVs include a range of 1000-10000 cells/cm 2 on plating material (often 2D culture, tissue culture plastic, although other plating material may be used).
  • the media which is used to promote the growth and health of adherent cells often includes a minimum essential medium, which may include alpha-Minimum Essential Medium (Gibco), 10% defined fetal bovine serum (Hyclone), 2 mM L-glutamine, 50 U/ml penicillin, 50 ⁇ g/ml streptomycin (Gibco) among other antibiotics.
  • a minimum essential medium which may include alpha-Minimum Essential Medium (Gibco), 10% defined fetal bovine serum (Hyclone), 2 mM L-glutamine, 50 U/ml penicillin, 50 ⁇ g/ml streptomycin (Gibco) among other antibiotics.
  • the adherent cells are generally grown at body temperate.
  • cell growth media are well known in the art and comprise at least a minimum essential medium plus one or more optional components such as growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (where indicated and not excluded), Activin A, transferrin, beta mercaptoethanol, and other agents well known in the art and as otherwise described herein.
  • a preferred media is a low protein, serum-free based growth medium that supports neural cells.
  • the growth factor used can be fibroblast growth factor 2 (FGF2), alone or preferably in combination with leukemia inhibitor factor (LIF). Depending on the MSCs to be grown in the growth media, the inclusion of may be preferred.
  • FGF2 fibroblast growth factor 2
  • LIF leukemia inhibitor factor
  • Additional media includes basal cell media which may contain serum, for example, between about 0.1% and 20% (preferably, about 2-10%) fetal calf serum, or for defined medium, an absence of fetal calf serum and KSR, and optionally including bovine serum albumin (about 1-5%, preferably about 2%).
  • Preferred medium is defined and is serum-free and low protein.
  • the components of the growth media depends on the type of MSC to be grown, all of which are well known in the art. Particularly preferred media is media and supplement is identified in the experimental section hereinbelow. The medium and supplement are engineered for versatility to meet all mesenchymal stem cell culture needs.
  • agents which optionally may be added to the medium include, depending on the cell type grown in the media, for example, any one or more of nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, Activin A, nodal, Bone Morphogen Proteins (BMP 2 to 7) serum albumin, members of the fibroblast growth factor (FGF) family, platelet- derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II, LR-IGF), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin,
  • suitable media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco # 10829- 018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non- essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
  • DMEM Dulbecco's modified Eagle's medium
  • KO DMEM Knockout Dulbecco's modified Eagle's medium
  • bFGF basic fibroblast growth factor
  • MSC-EV-producing cells may be cultured on a layer of feeder cells that support the cells in various ways. Approaches for culturing cells on a layer of feeder cells are well known in the art.
  • the cells may be grown on a cellular support or matrix, as adherent monolayers, rather than as embryoid bodies or in suspension.
  • cellular supports preferably comprise at least one substrate protein.
  • Substrate proteins include, for example, an extracellular matrix protein, which is a protein found in the extracellular matrix, such as laminin, tenascin, thrombospondin, and mixtures thereof, which exhibit growth promoting and contain domains with homology to epidermal growth factor (EGF) and exhibit growth promoting activity.
  • Other substrate proteins which may be used include for example, collagen, fibronectin, vibronectin, polylysine, polyornithine and mixtures thereof.
  • gels and other materials such as methylcellulose of other gels which contain effective concentrations of one or more of these embryonic stem cell differentiation proteins may also be used.
  • Exemplary differentiation proteins or materials which include these differentiation proteins include, for example, laminin, BD Cell-TakTM Cell and Tissue Adhesive, BDTM FIBROGEN Human Recombinant Collagen I, BDTM FIBROGEN Human Recombinant Collagen III, BD MatrigelTM Basement Membrane Matrix, BD MatrigelTM Basement Membrane Matrix High Concentration (HC), BDTM PuraMatrixTM Peptide Hydrogel, Collagen I, Collagen I High Concentration (HC), Collagen II (Bovine), Collagen III, Collagen IV, Collagen V, and Collagen VI, among others.
  • these MSCs may be cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of the cells to produce EVs.
  • the growth of cells in feeder-free culture can be supported using a medium conditioned by culturing previously with another cell type.
  • the growth of EV-producing cells in feeder-free culture without differentiation can be supported using a chemically defined medium.
  • the cells are grown in feeder cell free medium.
  • EVs can be harvested at various time intervals (e.g. at about 12 hours, 1, 2, 4, 6, 8 or 3, 6, 9, 12 day or longer intervals, depending upon the rate of production of EVs).
  • Exemplary yields of EVs can range from at least about 1 ng EVs/1 million cells, at least about 10 ng EVs/1 million cells, at least about 50 ng EVs/1 million cells, at least about 100 ng EVs/1 million cells, at least about 500 ng EVs/1 million cells, at least about 750 ng EVs/1 million cells, at least about 800 ng EVs/1 million cells, at least about 900 ng EVs/1 million cells, at least about 1.0 ⁇ g EVs/1 million cells, at least about 1.5 ⁇ g EVs/1 million cells, at least about 2.0 ⁇ g EVs/1 million cells, at least about 2.5 ⁇ g EVs/1 million cells, at least e.g.
  • EVs are harvested and collected by filtration, ultrafiltration, ultracentrifugation or differential centrifugation or any combination thereof, pelleted EVs are collected, and, optionally, collected pelleted EVs are washed with a suitable medium.
  • a preparation of EVs can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods.
  • the EVs can be prepared by differential centrifugation, that is low speed ( ⁇ 2,0000 g) centrifugation to pellet larger particles followed by high speed (>100,000 g) centrifugation to pellet EVs, size filtration with appropriate filters (for example, 0.22 ⁇ m filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods.
  • EVs may be purified by differential centrifugation, micro and ultra-filtration, polymeric precipitation, microfluidic separation, immunocapture and size-exclusion chromatography.
  • the EVs pursuant to the present invention are isolated by filtration (e.g.0.22 ⁇ M), followed by ultrafiltration.
  • the EVs which remain (often on top of the filter) are washed for several minutes collected, aliquoted and used directly or frozen for later use.
  • Pharmaceutical compositions Disclosed are pharmaceutical compositions containing therapeutically effective amounts of one or more of the disclosed EVs and a pharmaceutically acceptable carrier.
  • Formulations containing the disclosed EVs may take the form of liquid, solid, semi-solid or lyophilized powder forms, such as, for example, solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • Pharmaceutical compositions typically include a conventional pharmaceutical carrier and/or excipient and may additionally include other medicinal agents, carriers, adjuvants, additives and the like.
  • the weight percentage ratio of the EVs to the one or more excipients can be between about 20:1 to about 1:60, or between about 15:1 to about 1:45, or between about 10:1 to about 1:40, or between about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1 to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, and preferably is about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1 or 5:1.
  • the disclosed composition comprises between about 0.1 ⁇ g to about 1 g or more of total EVs, about 1 ⁇ g to aobut 750 mg, 500 ⁇ g about 500 mg, about 1 mg to about 500 mg of total EVs, about 5 to about 500 mg, about 10 to about 500 mg, about 25 to about 500 mg, about 50 mg to about 350 mg, about 75 mg to about 450 mg, about 50 mg to about 450 mg, or about 75 mg to about 325 mg or about 100 mg to about 650 mg of total EVs and may optionally contain one or more suitable pharmaceutical carriers, additives and/or excipients.
  • An injectable composition for parenteral administration e.g.
  • intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal, among other routes) will typically contain the EVs and optionally additional components in a suitable i.v. solution, such as sterile physiological salt solution.
  • a suitable i.v. solution such as sterile physiological salt solution.
  • the composition may also be formulated as a suspension in an aqueous emulsion.
  • Liquid compositions can be prepared by dissolving or dispersing the pharmaceutical composition comprising the EVs, and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension.
  • the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.
  • the compositions may be provided as liquid composition which can be sprayed into the nose, trachea and/or lungs.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.
  • auxiliary substances such as wetting agents, emulsifying agents, or buffers.
  • the preparations may be tablets, granules, powders, capsules or the like.
  • the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.
  • additives e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.
  • Methods for preparing such dosage forms are known or are apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub
  • Intravenous formulations can comprise the EVs described herein, an isotonic medium and one or more substances preventing aggregation of the EVs.
  • Exemplary intravenous/ intrathecal/ intracerebrospinal fluid formulations may contain saline solutions (e.g. normal saline (NS); about 0.91% w/v of NaCl, about 300 mOsm/L) and/or dextrose 4% in 0.18% saline, and optionally 1%, 2% or 3% human serum albumin.
  • formulations of the invention may comprise about 50 ng EVs/ml intravenous/intrathecal/intracerebrospinal fluid medium, including about 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1.0 ⁇ g, 1.5 ⁇ g, 2.0 ⁇ g, 2.5 ⁇ g, 3.0 ⁇ g, 5.0 ⁇ g, 10.0, 15.0 ⁇ g, 20.0 ⁇ g, 100 ⁇ g, or more EVs/ml intravenous/intrathecal/intracerebrospinal fluid medium for use in treating spinal cord injury, stroke, traumatic brain injury and/or neurodegenerative diseases.
  • intravenous formulations may comprise about 0.1 ⁇ g EVs/ml medium, about 0.2 ⁇ g EVs/ml intravenous medium, about 0.3 ⁇ g EVs/ml intravenous medium, about 0.4 ⁇ g EVs/ml intravenous medium, about 0.5 ⁇ g EVs/ml intravenous medium, about 0.6 ⁇ g EVs/ml intravenous medium, about 0.7 ⁇ g EVs/ml intravenous medium, about 0.8 ⁇ g EVs/ml intravenous medium, about 0.9 ⁇ g EVs/ml intravenous medium, about 1.0 ⁇ g EVs/ml intravenous medium, about 1.5 ⁇ g EVs/ml intravenous medium, about 2.0 ⁇ g EVs/ml intravenous medium, about 2.5 ⁇ g EVs/ml intravenous medium, such as at least e.g.
  • EVs/ml intravenous medium such as e.g. at least about 5.0 ⁇ g EVs/ml intravenous medium, about 10.0 ⁇ g EVs/ml intravenous medium, 15.0 ⁇ g EVs/ml intravenous medium or about 20.0 ⁇ g or more EVs/ml intravenous medium.
  • the pharmaceutical composition is in a dosage form comprising at least 25 mg of EVs, at least 50 mg of EVs, at least 60 mg of EVs, at least 75 mg of EVs, at least 100 mg of EVs, at least 150 mg of EVs, at least 200 mg of EVs, at least 250 mg of EVs, at least 300 mg of EVs, about 350 mg of EVs, about 400 mg of EVs, about 500 mg of EVs, about 750 mg of EVs, about 1g (1,000mg) or more of EVs, alone or in combination with a therapeutically effective amount of at least one additional bioactive agent, which agent may be useful in the treatment of osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders.
  • ARDS acute respiratory distress syndrome
  • Parkinson’s disease as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders.
  • the pharmaceutical composition comprises between about 10 mg to about 750 mg, about 25 mg to about 650 mg, or between about 30 mg to about 500 mg, or about 35 mg to about 450 mg, most often about 50 to about 500 mg of EVs.
  • an intravenous formulation comprises the EVs described herein, an isotonic medium, and one or more substances preventing aggregation of the EVs. Intravenous formulations may therefore contain saline solutions (e.g. normal saline (NS); about 0.91% w/v of NaCl, about 300 mOsm/L) and/or dextrose 4% in 0.18% saline, and optionally 1%, 2% or 3% human serum albumin.
  • saline solutions e.g. normal saline (NS); about 0.91% w/v of NaCl, about 300 mOsm/L
  • dextrose 4% in 0.18% saline
  • optionally 1%, 2% or 3% human serum albumin
  • the composition comprising the disclosed EVs further comprises one more neurotrophic agents.
  • the composition can further comprises one or more agents selected from the group consisting of leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte colony- stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN- ⁇ , insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factors (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF- ⁇ ), vascular endothelial growth factor (VEGF), VEGF-D, urokinas
  • the disclosed EVs are contained in or on a biocompatible scaffold, such as a hydrogel.
  • a biocompatible scaffold such as a hydrogel.
  • Suitable hydrogels include temperature dependent hydrogels that solidify or set at body temperature, e.g., PLURONICSTM; hydrogels crosslinked by ions, e.g., sodium alginate; hydrogels set by exposure to either visible or ultraviolet light, e.g., polyethylene glycol polylactic acid copolymers with acrylate end groups; and hydrogels that are set or solidified upon a change in pH, e.g., TETRONICSTM.
  • Examples of materials that can be used to form these different hydrogels include polysaccharides such as alginate, polyphosphazenes, and polyacrylates, which are cross-linked ionically, or block copolymers such as PLURONICSTM (also known as POLOXAMERSTM), which are poly(oxyethylene)- poly(oxypropylene) block polymers solidified by changes in temperature, or TETRONICSTM (also known as POLOXAMINESTM), which are poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine solidified by changes in pH.
  • Suitable hydrogels also include undefined extracellular matrix derived hydrogels that originated from tissues including but not limited to bladder intestine, blood and brain.
  • the disclosed EVs are contained in or on a biocompatible scaffold comprising collagen, fibrin, silk, agarose, alginate, hyaluronan, chitosan, a biodegradable polyester such as polylactic-co-glycolic acid, polylacic acid, or polyglycolic acid, polyethylene glycol, polyvinylpyrrolidone, polyethersulfone, a peptide-based biomaterial, glycose amino glycan, fibronectin, laminin, or any combination thereof.
  • the hydrogel is produced by cross-linking the anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with ions, such as calcium cations.
  • the strength of the hydrogel increases with either increasing concentrations of calcium ions or alginate.
  • U.S. Pat. No.4,352,883 describes the ionic cross-linking of alginate with divalent cations, in water, at room temperature, to form a hydrogel matrix.
  • EVs are mixed with an alginate solution, the solution is delivered to an already implanted support structure and then solidifies in a short time due to the presence in vivo of physiological concentrations of calcium ions.
  • the solution is delivered to the support structure prior to implantation and solidified in an external solution containing calcium ions.
  • these polymers are at least partially soluble in aqueous solutions, e.g., water, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof.
  • aqueous solutions e.g., water, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof.
  • polymers with acidic side groups that can be reacted with cations e.g., poly(phosphazenes), poly(acrylic acids), and poly(methacrylic acids).
  • acidic groups include carboxylic acid groups, sulfonic acid groups, and halogenated (preferably fluorinated) alcohol groups.
  • polymers with basic side groups that can react with anions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinyl imidazole).
  • Polyphosphazenes are polymers with backbones consisting of nitrogen and phosphorous atoms separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two side chains. Polyphosphazenes that can be used have a majority of side chains that are acidic and capable of forming salt bridges with di- or trivalent cations. Examples of acidic side chains are carboxylic acid groups and sulfonic acid groups.
  • Bioerodible polyphosphazenes have at least two differing types of side chains, acidic side groups capable of forming salt bridges with multivalent cations, and side groups that hydrolyze under in vivo conditions, e.g., imidazole groups, amino acid esters, glycerol, and glucosyl.
  • Bioerodible or biodegradable polymers i.e., polymers that dissolve or degrade within a period that is acceptable in the desired application (usually in vivo therapy), will degrade in less than about five years and most preferably in less than about one year, once exposed to a physiological solution of pH 6-8 having a temperature of between about 25° C. and 38° C. Hydrolysis of the side chain results in erosion of the polymer.
  • hydrolyzing side chains are unsubstituted and substituted imidazoles and amino acid esters in which the side chain is bonded to the phosphorous atom through an amino linkage.
  • Methods for synthesis and the analysis of various types of polyphosphazenes are described in U.S. Pat. Nos.4,440,921, 4,495,174, and 4,880,622.
  • Methods for the synthesis of the other polymers described above are known to those skilled in the art. See, for example Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz, editor (John Wiley and Sons, New York, N.Y., 1990).
  • Many polymers, such as poly(acrylic acid), alginates, and PLURONICSTM are commercially available.
  • Water soluble polymers with charged side groups are cross-linked by reacting the polymer with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups, or multivalent anions if the polymer has basic side groups.
  • Cations for cross-linking the polymers with acidic side groups to form a hydrogel include divalent and trivalent cations such as copper, calcium, aluminum, magnesium, and strontium. Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels.
  • Anions for cross-linking the polymers to form a hydrogel include divalent and trivalent anions such as low molecular weight dicarboxylate ions, terepthalate ions, sulfate ions, and carbonate ions.
  • Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels, as described with respect to cations.
  • a useful polymer size in the hydrogel is in the range of between 10,000 D and 18,500 D.
  • Temperature-dependent, or thermosensitive, hydrogels have so-called “reverse gelation” properties, i.e., they are liquids at or below room temperature, and gel when warmed to higher temperatures, e.g., body temperature.
  • these hydrogels can be easily applied at or below room temperature as a liquid and automatically form a semi-solid gel when warmed to body temperature.
  • these gels are especially useful when the support structure is first implanted into a patient, and then filled with the hydrogel-EV composition.
  • temperature-dependent hydrogels examples include PLURONICSTM (BASF-Wyandotte), such as polyoxyethylene-polyoxypropylene F-108, F-68, and F-127, poly(N-isopropylacrylamide), and N-isopropylacrylamide copolymers.
  • PLURONICSTM BASF-Wyandotte
  • these copolymers can be manipulated by standard techniques to affect their physical properties such as porosity, rate of degradation, transition temperature and degree of rigidity.
  • the addition of low molecular weight saccharides in the presence and absence of salts affects the lower critical solution temperature (LCST) of typical thermosensitive polymers.
  • LCST critical solution temperature
  • Nos.4,474,751, '752, '753, and 4,478,822 describe drug delivery systems which utilize thermosetting polyoxyalkylene gels; with these systems, both the gel transition temperature and/or the rigidity of the gel can be modified by adjustment of the pH and/or the ionic strength, as well as by the concentration of the polymer.
  • pH-dependent hydrogels are liquids at, below, or above specific pH values, and gel when exposed to specific pHs, e.g., 7.35 to 7.45, the normal pH range of extracellular fluids within the human body.
  • these hydrogels can be easily delivered to an implanted support structure as a liquid and automatically form a semi-solid gel when exposed to body pH.
  • pH-dependent hydrogels examples include TETRONICSTM (BASF-Wyandotte) polyoxyethylene-polyoxypropylene polymers of ethylene diamine, poly(diethyl aminoethyl methacrylate-g-ethylene glycol), and poly(2-hydroxymethyl methacrylate). These copolymers can be manipulated by standard techniques to affect their physical properties. Hydrogels that are solidified by either visible or ultraviolet light can be made of macromers including a water soluble region, a biodegradable region, and at least two polymerizable regions as described in U.S. Pat. No.5,410,016.
  • the hydrogel can begin with a biodegradable, polymerizable macromer including a core, an extension on each end of the core, and an end cap on each extension.
  • the core is a hydrophilic polymer
  • the extensions are biodegradable polymers
  • the end caps are oligomers capable of cross- linking the macromers upon exposure to visible or ultraviolet light, e.g., long wavelength ultraviolet light.
  • Examples of such light solidified hydrogels include polyethylene oxide block copolymers, polyethylene glycol polylactic acid copolymers with acrylate end groups and 10K polyethylene glycol-glycolide copolymer capped by an acrylate at both ends.
  • the copolymers comprising these hydrogels can be manipulated by standard techniques to modify their physical properties such as rate of degradation, differences in crystallinity, and degree of rigidity.
  • Methods of Treatment Also disclosed is a method of treating a subject with any one of a number of disease states and/or conditions as described herein including osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis, Parkinson’s disease, skeletal muscle or bone in need of repair, cancer, cardiovascular disease, including stroke and other neurological disorders comprising administering to the subject in need an effective amount of a composition containing a population of neural EVs disclosed herein, optionally in combination with at least one additional bioactive agent.
  • ARDS acute respiratory distress syndrome
  • Parkinson’s disease skeletal muscle or bone in need of repair
  • cancer cardiovascular disease
  • stroke and other neurological disorders comprising administering to the subject in need an effective amount of a composition containing a population of neural EVs disclosed herein, optionally in combination with at least one additional bioactive agent.
  • the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, a Parkinson’s-related disorder, Huntington’s disease, prion disease, motor neuron disease (MND), spinocerebellar ataxia (SCA) or spinal muscular atrophy (SMA).
  • the term “stroke” is used to describe a cerebrovascular accident (CVA), cerebrovascular insult (CVI), or brain attack, occurs when poor blood flow to the brain results in cell death.
  • CVA cerebrovascular accident
  • CVI cerebrovascular insult
  • brain attack occurs when poor blood flow to the brain results in cell death.
  • Signs and symptoms of a stroke may include an inability to move or feel on one side of the body, problems understanding or speaking, a sense of spinning, or loss of vision to one side, among others. Signs and symptoms often appear soon after the stroke has occurred. If symptoms last less than one or two hours it is known as a transient ischemic attack. Hemorrhagic strokes may also be associated with a severe headache. The symptoms of a stroke can be permanent. Long term complications of stroke may include pneumonia or loss of bladder control.
  • the main risk factor for stroke is high blood pressure. Other risk factors include tobacco smoking, obesity, high blood cholesterol, diabetes mellitus, previous transient ischemic attack (TIA), and atrial fibrillation, among others.
  • An ischemic stroke is typically caused by blockage of a blood vessel.
  • a hemorrhagic stroke is caused by bleeding either directly into the brain or into the space surrounding the brain. Bleeding may occur due to a brain aneurysm. Both ischemic and hemorrhagic stroke are treated pursuant to the present invention.
  • the term “neurodegenerative disease” is used throughout the specification to describe a disease which is caused by damage to the central nervous system and which damage can be reduced and/or alleviated through transplantation of neural cells according to the present invention to damaged areas of the brain and/or spinal cord of the patient.
  • Exemplary neurodegenerative diseases which may be treated using the neural cells and methods according to the present invention include for example, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, lysosomal storage disease (“white matter disease” or glial/demyelination disease, as described, for example by Folkerth, J. Neuropath. Exp. Neuro., 58, 9, Sep., 1999), Tay Sachs disease (beta hexosamimidase deficiency), other genetic diseases, multiple sclerosis, brain injury or trauma caused by ischemia, accidents, environmental insult, etc., spinal cord damage, ataxia and alcoholism.
  • Parkinson's disease Huntington's disease
  • amyotrophic lateral sclerosis Lou Gehrig's disease
  • Alzheimer's disease lysosomal storage disease (“white matter disease” or glial/demyelination disease, as described, for example by Folkerth, J. Neuro
  • the present invention may be used to reduce and/or eliminate the effects on the central nervous system of a stroke or a heart attack in a patient, which is otherwise caused by lack of blood flow or ischemia to a site in the brain of said patient or which has occurred from physical injury to the brain and/or spinal cord.
  • neurodegenerative diseases also includes neurodevelopmental disorders including for example, autism and related neurological diseases such as schizophrenia, among numerous others.
  • osteoarthritis is used to describe a type of joint disease that results from the breakdown of joint cartilage and underlying bone. Osteoarthritis (OA) is caused by aging joints, injury, and obesity. OA symptoms include joint pain and stiffness.
  • ARDS acute respiratory distress syndrome
  • fluid leaks from small blood vessels and collects in tiny air sacs in the lungs so the air sacs can’t fill with enough air. Because of this, the blood can’t pick up the oxygen it needs to carry to the rest of the body. Organs such as the kidneys and brain often become dysfunctional or may shut down. ARDS is sometimes life-threatening and can get worse quickly.
  • ARDS is usually triggered by another health problem, so most people who have it are already in the hospital for something else.
  • causes of ARDS include sepsis, accidents and breathing in toxins such as dense smoke or chemical fumes.
  • the term “multiple sclerosis” or “MS” is used to describe a potentially disabling disease of the brain and spinal cord (central nervous system).
  • MS the immune system attacks the protective sheath (myelin) that covers nerve fibers and causes communication problems between the brain and the rest of the body. Eventually, the disease can cause permanent damage or deterioration of the nerves.
  • auto-immune disease is used to describe disease in which a subject’s own immune system produces an abnormal immune response to a normal organ or tissue of a subject.
  • Exemplary auto-immune diseases which can be treated using the extracellular vesicles (EVs) pursuant to the present invention include rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticarial, Sjogren’s disease, autoimmune-related Type 1 diabetes, rheumatoid arthritis (RA), psoriasis/psoriatic arthritis, multiple sclerosis, inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, Addison’s disease, Grave’s disease, Hashimoto’s thyroiditis, Myasthenia gravis, autoimmune vasculitis, pernicious anemia and celiac disease.
  • compositions including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • Methods of treating subjects involve administration of a pharmaceutical composition comprising an effective amount of EVs described herein and optionally at least one additional bioactive (e.g. an agent which is useful in the treatment of osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis, Parkinson’s disease, skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders, among others) agent and otherwise as described herein.
  • additional bioactive e.g. an agent which is useful in the treatment of osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis, Parkinson’s disease, skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders, among others
  • compositions could be formulated so that a therapeutically effective dosage of between about 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg of patient/day or in some embodiments, greater than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/kg of the disclosed EVs can be administered to a patient receiving these compositions.
  • the dose of EVs administered to a subject can be less than 10 ⁇ g, less than 25 ⁇ g, less than 50 ⁇ g, less than 75 ⁇ g, less than 0.10 mg, less than 0.25 mg, less than 0.5 mg, less than 1 mg, less than 2.5 mg, less than 5 mg, less than 10 mg, less than 15 mg, less than 20 mg, less than 50 mg, less than 75 mg, less than 100 mg, less than 500 mg, less than 750 mg, less than 1 g or more than 1 g.
  • Administration may be by numerous routes of administration, but intravenous, intrathecal, intranasal and/or intracerebrospinal fluid are often used as preferred routes of administration.
  • the disclosed EVs are administered within 24 hours after a stroke or trauma.
  • the EVs are administered at least 1, 2, 3, or 4 weeks after a stroke or trauma, including a heart attack (myocardial infarction).
  • the disclosed EVs are administered in multiple doses 1, 2, 3, or more days apart.
  • the EVs are administered continuously (e.g., once every 1, 2, 3, or 4 weeks or more often) over the course of the disease.
  • EVs may be loaded with small molecules, antisense oligonucleotides, siRNAs, peptides, proteins or antibodies that target, peptides or peptide translation products which are involved in therapeutic, including neurodegenerative processes.
  • the disclosed EVs are loaded with additional bioactive agents or are co-administered with additional bioactive agents, especially agents which are useful in the treatment of cancer and/or neurodegenerative diseases.
  • coadministered”, “coadministration” or “combination therapy” is used to describe a therapy in which at least two active compounds/compositions in effective amounts are used to treat neural injury and/or a neurodegenerative disease.
  • co- administration preferably includes the administration of EVs and at least one additional active compound to the subject at the same time, it is not necessary that the compounds/compositions be administered to the patient simultaneously, only that effective amounts of the individual compounds/compositions be present in the patient at the same time.
  • co-administration includes an administration in which the EVs and the bioactive agent(s) are administered at approximately the same time (contemporaneously), or from about one to several minutes to about eight hours, about 30 minutes to about 6 hours, about an hour to about 4 hours, or even much earlier than the other compound/composition as otherwise described herein including up to a day or substantially more.
  • Agents which may be loaded or coadministered along with EVs may include, for example aricept, namenda, donepezil, excelon, razadyne, glantamine, rivastigmine, memantine, ergoloid, namzaric and mixtures thereof for Alzheimer’s disease, biperiden, apomorphine, trihexyphenidyl, carbidopa/levodopa, rasagline, belladona, levodopa, benztropine, entacapone, selegiline, rivastigmine, pramipexole, rotigotine, bromocriptine, pergolide, ropinirole, carbidopa/entacapone/levodopa, amantadine, tolcopone, trihexiphenidyl and mixtures thereof, for Parkinson’s disease, tetrabenazine, haloperidol, chlorpromazine, o
  • Agents for the treatment of stroke include salicylates, such as aspirin, a thrombolytic agent (alteplase) and a platelet aggregation inhibitor (clopidogrel), among others.
  • Anticancer agents may also be used in conjunction with EVs according to the present invention.
  • the term “anti-cancer compound”, “anti-cancer drug” or “anti-cancer agent” is used to describe any compound (including its derivatives) which may be used to treat cancer.
  • the “anti-cancer compound”, “anti-cancer drug” or “anti-cancer agent” can be an anticancer agent.
  • the co-administration of another anti-cancer compound according to the present invention results in a synergistic anti-cancer effect.
  • anti-metabolites agents which are broadly characterized as antimetabolites, inhibitors of topoisomerase I and II, alkylating agents and microtubule inhibitors (e.g., taxol), as well as tyrosine kinase inhibitors (e.g., surafenib), EGF kinase inhibitors (e.g., tarc
  • Anti-cancer compounds for co-administration include, for example, agent(s) which may be co-administered with EVs according to the present invention in the treatment of cancer.
  • agents include chemotherapeutic agents and include one or more members selected from the group consisting of everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101 , pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor
  • the activities of EVs described herein can be evaluated by methods known in the art.
  • the amount of EVs required for use in treatment can vary not only with the particular cell from which the EVs are prepared, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and can be ultimately at the discretion of the attendant physician or clinician. In general, however, a dose can be in the range of from about 0.01 mg/kg to about 10 mg/kg of body weight per day.
  • Identifying EVs useful in the present methods for treating a spinal cord injury, stroke, traumatic brain injury and/or a neurodegenerative disease which occurs by modulating the activity and expression of a disease-related protein and biologically active fragments thereof can be made by screening EV activity in any of a variety of screening techniques.
  • the screening can be made for whole EVs or their contents. Fragments employed in such screening tests may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the blocking or reduction of biological activity or the formation of binding complexes between the disease-related protein, the EVs and/or one or more components of the EVs may be measured by methods available in the art.
  • a number of embodiments of the invention are described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the claims which follow.
  • MSCs-based therapy is considered as a promising approach for a number of disease states and conditions because of their ability to target major aspects of the pathophysiology associated with these disease states.
  • MSC-EVs are expected to act similarly by way of effectiveness but with fewer of the side effects of MSC therapy, such as tumorogenicity.
  • Experimental Overview Mesenchymal stromal cells (MSCs) are being explored as an immunomodulatory therapy to treat immune diseases such as osteoarthritis, multiple sclerosis, and Parkinson’s disease (1, 2); however, cell therapies face challenges associated with manufacturing consistent, high quality products.
  • cryopreserved and thawed MSCs have diminished efficacy and require a recovery period at point of care when compared to fresh, non-thawed MSCs (3).
  • Transplantation of any cell therapy, even MSCs can also raise safety concerns in the event of uncontrolled differentiation into undesired tissue or promotion of tumor growth following engraftment (4- 6).
  • Cell-free extracellular vesicle (EV) preparations from MSC cultures that possess similar MSC therapeutic function are a potential alternative to MSC therapy. Secreted factors from MSCs are responsible for much of their regenerative and immunomodulatory functions (7).
  • MSC-derived extracellular vesicles are secreted nanoscale vesicles that participate in intercellular signaling through transference of bioactive molecules including RNA, proteins, and lipids (11). MSC-EVs do not divide, engraft, or dynamically respond to their environment like MSCs, thus addressing concerns with tumorigenicity and ectopic tissue growth (5, 6).
  • MSC-EVs have demonstrated preliminary evidence of regenerative effects ranging from recovery from myocardial ischemia and reperfusion injury (12), stroke (13), gentamicin induced acute kidney injury (14), and allogeneic skin grafts (15) and MSC-EV clinical trials are being conducted for a debilitating skin disease
  • the injury microenvironment is often characterized by inflammation, involving local hypoxia, acidosis, as well as the presence of cytokines such as Tumor Necrosis Factor alpha (TNF- ⁇ ) and Interferon gamma (IFN- ⁇ ) (16-18).
  • TNF- ⁇ Tumor Necrosis Factor alpha
  • IFN- ⁇ Interferon gamma
  • regenerative and anti-inflammatory factors such as Vascular Endothelial Growth Factor, Indolamine 2,3-Dioxygenase, Transforming Growth Factor Beta, and Prostaglandin E2 (19). More recently, inflammatory and hypoxic priming have been shown to increase the potency of MSC-EV immunomodulation (20-22). EVs derived from MSCs primed with hypoxia were more effective than EVs from non- primed MSCs in inducing macrophage proliferation and type 2 macrophage polarization (20). TGF- ⁇ and IFN- ⁇ primed MSCs produced EVs that were more effective in inducing Treg formation than those from resting MSCs (22).
  • MSC-EVs derived from MSCs primed with acidosis induced the formation of Tregs while other MSC-EV groups had no significant effect on T cell activation. Therefore, precise control and monitoring of the pH during MSC-EV manufacturing should be further explored as a means to enhance MSC-EV immunomodulatory function.
  • a hypoxic environment was created by placing the cell culture vessels in a hypoxia incubator chamber (STEMCELL Technologies, Cambridge MA), which was then filled with a gas mixture containing 2% O 2 , 5% CO 2 , and 93% N2 (Airgas) for 5 minutes at 2 psi according to the manufacturer’s recommendation (24, 25).
  • the MSCs undergoing this priming were termed LO2-MSCs, and their EVs were LO2-EV.
  • An inflammatory environment was created by adding the cytokines TNF- ⁇ and IFN- ⁇ (Sigma) to the medium at 15 and 20 ng/mL, respectively. These MSCs were termed INF-MSCs, with their EVs being INF-EV.
  • PBMCs Peripheral Blood Mononuclear Cells
  • MSCs were rinsed twice with PBS before adding fresh serum free medium (Alpha-Minimum Essential Medium (Gibco), 2 mM L-glutamine, 50 U/mL penicillin, 50 ⁇ g/mL streptomycin (all from Gibco/Invitrogen)) and incubating cultures for 24 hours.
  • serum free medium Alpha-Minimum Essential Medium (Gibco)
  • 2 mM L-glutamine 50 U/mL penicillin, 50 ⁇ g/mL streptomycin (all from Gibco/Invitrogen)
  • the resulting conditioned media were collected and passed through 0.22 ⁇ m filters to remove cells and large debris.
  • the media were subjected to ultrafiltration with a 100kDa MWCO (Amicon, Millipore-Sigma) at 4000g for 10 minutes as we previously published (26).
  • the EVs remained on top of the filter and were then washed twice with PBS +/+ (Thermo Fisher Scientific, Waltham, MA) at 2000g for 10 minutes. The EVs in PBS+/+ were then collected, aliquoted, and frozen at -20°C.
  • nanoparticle tracking analysis was performed using a Nanosight NS3200 (Nanosight, Salisbury UK) according to the manufacturer’s recommendations. Briefly, aliquots of EV suspensions were thawed at room temperature and diluted to 10 7 -10 9 particles/mL with the same lot of PBS +/+ the EVs were isolated in. A minimum of three samples and five one-minute videos were recorded for each exosome isolation.
  • EV surface marker characterization was performed using the MACSPLEX Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s directions. Briefly, an equal number of exosomes as determined by NTA were analyzed from each isolation in triplicate. Flow cytometry analysis was performed using a CytoFLEX S (Beckman Coulter, Hialeah, Florida) alongside bead only controls, with FlowJo being used for data analysis. Data was processed with background subtraction and normalized to the median of the average value of CD9, CD63, and CD81 for each sample (27). The data was transformed to be a percentage of the difference between the maximum and minimum relative expression of a marker.
  • PCA Principal component analysis
  • PBMCs were harvested and stained for flow cytometry using Pacific Blue anti-CD4, APC anti-CD8, Brilliant Violet 711 anti-CD25, and PE anti- FOXP3. Antibodies and clones are listed in Table S2.
  • PBMCs were first washed, then stained with Zombie Yellow viability dye, blocked with 2% FBS and Fc receptors blocked with Trustain FcX. The PBMCs were then stained for CD4, CD8, and CD25 as appropriate at room temperature in the dark for 30 minutes and fixed in FOXP3 TrueNuclear fix before storing overnight in the dark at 4C.
  • PBMCs were permeabilized with TrueNuclear permeabilization buffer and stained with PE anti-FOXP3 according to the manufacturer’s directions.
  • Immunomodulation assay MSCs were plated at 20,000 cells/cm 2 in 48 well plates in complete medium and allowed to adhere for 24 hours. The cells were then subjected to priming as previously described, followed by two PBS -/- washes. PBMCs were labeled with CFSE (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions and 500,000 PBMCs were added to each well.
  • CFSE Thermo Fisher Scientific, Waltham, MA
  • PBMCs peripheral blood mononuclear cells
  • stimulating anti-CD3/CD28 Dynabeads were added at 500,000 each per well, and 10 9 EVs were added to the appropriate wells.
  • the assays took place in complete RPMI medium formulated as above, but with EV- depleted FBS. EVs were depleted by centrifuging FBS at 100,000g for 1 hour at 4°C (Sorvall WX Ultra 80, Thermo Fisher Scientific, Waltham, MA) and using the supernatant (Li 2017). The cultures incubated for 5 days at 37°C, 5% CO 2 for the duration of the experiments.
  • Panel 1 was composed of Pacific Blue anti- CD4, APC anti-CD8, Brilliant Violet 711 anti-CD25, and PE anti-FOXP3.
  • Panel 2 included Pacific Blue anti-CD4, APC anti-CD8, PE anti-IFN- ⁇ , and Brilliant Violet 711 anti-TNF- ⁇ .
  • Antibodies and clones are listed in Table S2. PBMCs were first washed, then stained with Zombie Yellow viability dye, blocked with 2% FBS and FC receptors blocked with Trustain FcX.
  • the PBMCs were then stained for CD4, CD8, and CD25 as appropriate at room temperature in the dark for 30 minutes and fixed in 4% PFA for Panel 2 or FOXP3 TrueNuclear fix for Panel 1 before storing overnight in the dark at 4°C.
  • Panel 1 was then permeabilized with TrueNuclear permeabilization buffer and stained for FOXP3 according to the manufacturer’s directions.
  • Panel 2 was permeabilized with Permwash (BD Biosciences) and stained for IFN- ⁇ and TNF- ⁇ . Samples were resuspended in 2% FBS at 4°C in the dark for up to 2 days before flow analysis. All antibodies and reagents were from Biolegend (San Diego, CA) unless otherwise specified and were used at previously titrated optimal concentrations.
  • T cell proliferation was calculated according to the formula below, where MI is the median fluorescence intensity of CFSE stained samples, and PS is the proliferation score (31): Statistics All data is expressed as mean +/- SEM, with all experiments performed in triplicate. All statistical tests were one-way ANOVA against controls unless stated otherwise with Dunnett’s post-hoc test using Prism (Graphpad, San Diego CA).
  • NC-EV, LO2-EV, and LPH-EV had unimodal size distributions centered at approximately 100nm (FIGURE 1A-C).
  • the size distribution of INF-EV was bimodal, with one population centered at 100 nm as well as a larger diameter population between 150 and 400 nm (FIGURE 1D).
  • INF-EVs had a significantly higher mean diameter than NC-EVs (p ⁇ 0.0001), while LPH-EV and LO2-EV did not differ in size from the control (FIGURE 1E).
  • DLS Dynamic Light Scattering
  • S4 bimodal distribution
  • hypoxic and acidosis priming increased EV release, while only inflammatory priming affected EV size.
  • Inflammatory priming affects MSC-EV surface marker expression
  • Surface marker characterization via MACSPLEX analysis revealed significant changes in the relative expression of EV surface markers in the preconditioned groups compared to the NC-EV group (FIGURE 2A).
  • CD9, CD63, and CD81 exosome markers were present in all EV groups, although to varying degrees. INF-EV had elevated expression of CD63 (p ⁇ 0.0001), while expression of CD9 (p ⁇ 0.0001) and CD81 (p ⁇ 0.0001) were decreased.
  • INF-EV had greatly decreased expression of CD29 (Integrin beta-1, p ⁇ 0.0001), CD44 (p ⁇ 0.0001), CD49e (Integrin alpha-5, p ⁇ 0.0001), CD105 (Endoglin, p ⁇ 0.0001), and melanoma-associated chondroitin sulfate proteoglycan (MCSP, p ⁇ 0.0001).
  • LPH-EV did not have significantly different expression of any surface marker compared to NC-EV.
  • PCA enabled visualization of the high dimensional MACSPLEX data to better discriminate differences in the overall expression of surface markers with priming (FIGURE 2B).
  • the first and second principal components (PC1, PC2) were responsible for 28% and 10% of the variance in the data set, respectively.
  • INF-EV separated from the other groups along PC1, and INF-EV’s mean value of PC1 was significantly lower than that of NC-EV (p ⁇ 0.0001, FIGURE 2C).
  • the largest contributors to PC1 were cell adhesion markers, while PC2 was largely made up of cell membrane and immune signaling proteins (Table S5).
  • CFSE-labeled exosome uptake was determined by %CFSE + cells from T cell sub- groups at 24 hours post-treatment. LPH-EV had the highest uptake of all groups (FIGURE 3). Treatment with LPH-EV led to significantly greater percent CFSE + cells than the untreated controls for helper T cells (p ⁇ 0.05, FIGURE 3A), cytotoxic T cells (p ⁇ 0.05, FIGURE 3B), activated helper T cells, (p ⁇ 0.05, FIGURE 3C), activated cytotoxic T cells (p ⁇ 0.01, FIGURE 3D), CD4 + Tregs (FIGURE 3E, p ⁇ 0.05), and CD8 + Tregs (FIGURE 3F, p ⁇ 0.01).
  • helper T cells p ⁇ 0.05, FIGURE 3A
  • cytotoxic T cells p ⁇ 0.05, FIGURE 3B
  • activated helper T cells p ⁇ 0.05, FIGURE 3C
  • activated cytotoxic T cells p ⁇ 0.01,
  • CD25 expression in helper T cells was significantly reduced by INF-MSC (p ⁇ 0.0001), LO2-MSC (p ⁇ 0.001), LPH-MSC (p ⁇ 0.01), and NC-MSC (p ⁇ 0.001) (FIGURE 5A).
  • CD25 expression was similarly reduced in cytotoxic T cells exposed to INF-MSC (p ⁇ 0.05), LO2- MSC (p ⁇ 0.05), LPH-MSC (p ⁇ 0.05), and NC-MSC (p ⁇ 0.05) (FIGURE 5B).
  • MSC-EV immunomodulation through acidic priming.
  • Tumors are known to create an immunosuppressive microenvironment, and it is hypothesized that MSCs might be involved in this phenomenon (32-34).
  • MSCs cultured in an acidic environment enhanced in vivo melanoma growth, partly through their increased expression of TGF- ⁇ (35).
  • TGF- ⁇ is a potent growth factor and has been shown to induce the maturation of T regs (36).
  • MSC-EV have been shown to associate with TGF- ⁇ , and EVs may bind TGF- ⁇ on their surface (37, 38).
  • Acidosis primed MSCs upregulated osteosarcoma expression of CXCL5 and CCL5 (39). These chemokines have also been implicated in the formation and recruitment of T regs , respectively (40-42).
  • MSC-EV may play a role in the MSC driven immunosuppressive role since we observed increased Tregs in PBMC cultures treated with acidic preconditioned MSC-EVs. EV biogenesis and release is known to take place through several mechanisms, including sphingomyelinases (43). Sphingomyelinase activity has been shown to be increased in an acidic environment (44, 45).
  • INF-EVs exhibited a biomodal size distribution that included a population with size range similar to microvesicles, which bud from the plasma membrane and are between 100nm and 1 ⁇ m in diameter (43, 46). EVs from both IFN- ⁇ and TNF- ⁇ /IFN- ⁇ preconditioned MSCs have previously been observed with larger size distributions tending towards the microvesicle size range (21, 47).
  • the biogenesis of microvesicles differs from that of exosomes, and their immunomodulatory potency has been shown to be less than that of exosomes (48). Additionally, we observed decreases in adhesion-related markers of INF-EV compared to NC-EV.
  • INF-EV contain multiple heterogeneous vesicle populations as indicated here, this could dilute the overall potency, possibly explaining the lack of immunomodulation by INF-EVs compared to other studies.
  • MSC-EVs delivered to PBMCs consistently associated with T cells compared to macrophages or NK cells (50).
  • NK cells NK cells
  • MSCs are more effective than their EVs alone at inhibiting T cell proliferation (21, 48, 50, 51).
  • inhibition of EV release impairs the suppression of T cell proliferation by MSC co-culture, so EVs likely play some role in this process (21).
  • Isolated MSC-EVs also induce T reg formation, while their source cells do not (48, 50), which occurred for the LPH-EV group in this study.
  • MSC-EVs can contain a wide range of micro RNAs (miRNAs), many of which are associated with angiogenesis and tissue remodeling (52). Certain miRNAs have been shown to have increased frequency in EVs derived from MSCs primed with inflammation-relevant signals.
  • miRNA- 155 When MSCs were primed with TNF- ⁇ and IFN- ⁇ , miRNA- 155, previously implicated in immune modulation, was increased in their EVs compared to non-primed control MSC-EVs (21, 53). Similarly, IL-1 ⁇ priming of MSCs upregulated miR- 146a, which has previously been shown to regulate the T cell response through the NF-NB pathway (54, 55). MSCs cultured in hypoxia have increased miR-223, miR-146b, miR126, and miR199a (20). Of these, miR-223 is involved in driving anti-inflammatory macrophage polarization, and miR-146b has been shown to monocyte inflammation (56).
  • MSC Mesenchymal Stem Cells
  • G-MDSC Granulocytic-Myeloid-Derived Suppressor Cells
  • Tan MCB Goedegebuure PS, Belt BA, Flaherty B, Sankpal N, Gillanders WE, et al. Disruption of CCR5-dependent homing of regulatory T cells inhibits tumor growth in a murine model of pancreatic cancer. Journal of immunology (Baltimore, Md : 1950). 2009;182(3):1746-55. 41. Wang X, Lang M, Zhao T, Feng X, Zheng C, Huang C, et al. Cancer-FOXP3 directly activated CCL5 to recruit FOXP3+Treg cells in pancreatic ductal adenocarcinoma. Oncogene.2016;36:3048. 42.
  • Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane. Journal of extracellular vesicles.2017;6(1):1378056. 45. Tan SS, Yin Y, Lee T, Lai RC, Yeo RW, Zhang B, et al. Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane. Journal of extracellular vesicles.2013;2. 46. Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, Aradi B, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.

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Abstract

The present invention is directed to extracellular vesicles prepared from adherent cells (AC-EVs) and methods for manufacturing these AC-EV s. Pursuant to the present invention, AC-EVs, often MSC-EVs are subjected to various conditions to enhance quality and therapeutic function of these extracellular vesicles. Pursuant to the present invention, the inventors examined hypoxia (L02-EV), acidosis (LPH-EV), and inflammatory cytokines (INF-EV) on AC-EV (MSC-EV) biogenesis and secretion as compared to the unprimed, serum-free culture conditions, in embodiments, the present invention is directed to a method to create immune-suppressive Adherent cell (preferably MSC) exosomes as well as pH stimulated adherent cell (preferably, MSC) exosomes.

Description

Mesenchymal Cell (MSC) Exosomes Increase T Cell Differentiation Towards T Regulatory Cells Related Applications and Government Rights This application claims the benefit of priority of United States provisional application serial number 63/005,695, filed April 6, 2020, the entire contents of which application is incorporated by reference herein. This invention was made with government support under Grant No.1648035 awarded by the NSF/CMAT. The Government has certain rights in the invention.” (37 CFR 401.14 f (4)). Field of the Invention The present invention is directed to extracellular vesicles prepared from adherent cells (AC-EV) and methods for manufacturing these AC-EVs. Pursuant to the present invention, AC-EVs, often MSC-EVs are subjected to various conditions to enhance quality and therapeutic function of these extracellular vesicles. Pursuant to the present invention, the inventors examined hypoxia (LO2-EV), acidosis (LPH-EV), and inflammatory cytokines (INF-EV) on AC-EV (MSC-EV) biogenesis and secretion as compared to the unprimed, serum-free culture conditions. In embodiments, the present invention is directed to a method to create immune-suppressive Adherent cell (preferably MSC) exosomes as well as pH stimulated adherent cell (preferably, MSC) exosomes. Background of the Invention Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types including osteoblasts, chondrocytes, myocytes, and adipocytes. These cells are being used by researchers to artificially reconstruct human tissue which has been previously damaged. Although promising, the use of these cells also raises safety concerns such as undesired tissue or promotion of tumor growth during therapy. When used in preclinical models, MSCs have been shown to greatly reduce inflammation and while the mechanism for this reduction may not have been elucidated, it is known that MSCs reduce the levels of many pro-inflammatory cytokines such as TNF-α, IL- 1β, and IL-6 while increasing the levels of cytokines which reduce inflammation like IL-4, IL-5, and IL-10. In addition, MSCs promote bacterial clearance both directly by secreting antimicrobial peptides and proteins such as LL-37 and lipocalin as well as indirectly by activating host monocytes, macrophages and neutrophils which then phagocytose the bacteria. MSC secretion of substances such as keratinocyte growth factor (KGF) has been shown to be essential in alveolar fluid clearance and restoration of epithelial permeability. MSCs have been proven to have an immunomodulatory effect through multiple mechanisms. Although initially, it was thought that these cells would promote regeneration of the injured tissue through engraftment and trans-differentiation, now it has become apparent that engraftment plays little to no role in their therapeutic action. However, it is known that these cells modulate host cells through direct cell-to-cell interactions and through the release of factors (paracrine) including active agents such as KGF, indoleamine 2,3-dihydrogenase, and prostaglandin E2. Accumulating evidence suggests that one of the most important effectors in paracrine mechanisms of MSC effect are extracellular vesicles (EVs) which seem to be able to recapitulate the therapeutic effect of their parent MSCs. Cell-free extracellular vesicles (EV) secreted by MSC (MSC-EVs) possess the similar therapeutic function of MSC therapy, but the MSC-EVs do not divide or engraft like MSCs, thus addressing concerns with tumorigenicity and ectopic tissue growth. Thus, in contrast to the use of whole cell MSCs, extracellular/extrasomal vesicles are being actively explored as an alternative to whole-cell therapy. Novel therapies for disease states and/or conditions where there is high mortality and no pharmacological therapeutic approach to providing therapy such as osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders are critically needed. Additional disease states and/or conditions which can be treated using EVs according to the present invention include, eosinophil-associated gastrointestinal diseases, noninfectious posterior uveitis and cancers, especially including myeloma, multiple myeloma, lymphoma and opsoclonus-myoclonus syndrome (OMS). Evidence suggests that MSC-EVs demonstrate potent protective effects mediated through a variety of mechanisms related to the transfer of the EVs cargo to the recipient cells. As MSC-EVs may suppress pro-inflammatory responses, the biomanufacturing of MSC-EVs remains an emerging field with great potential. The lack of standardized methods may result in final products with functional heterogeneity. Acidic priming is an easy approach to increase the production of MSC-EVs with anti- inflammatory function. Further research into the mechanism of action, biodistribution, standardization, and biomanufacturing is needed to facilitate clinical translation of this new cell therapy to more general therapeutic outcomes. Brief Description of the Invention In order to determine the effects of manufacturing conditions on extracellular vesicles (EVs) produced from adherent cells (AC-EVs) preferably from mesenchymal stem cells (MSC-EVs) and the quality and therapeutic function obtained therefrom, the inventors examined the effects of hypoxia (LO2-EV), acidosis (LPH-EV), and inflammatory cytokines (INF-EV) on extracellular vesicle biogenesis and secretion as compared to AC-EVs produced using unprimed, serum-free culture conditions. While acidosis (LPH-EVs) and hypoxia (LO2-EVs) did not produce AC-EVs (in particular, MSC-EVs) which differed in size from the control, the exposure of the MSCs to inflammatory cytokines produced INF-EVs which were significantly larger than the control vesicles. While both acidic and hypoxic priming increase the EV production per cell, no significant result was observed in the inflammatory priming group. This is evidenced in the experimental section of the present application. The expression of surface markers was also significantly affected by inflammatory priming and hypoxic priming, but not by acidic priming. AC-EVs, and in particular, MSC-EVs pursuant to the present invention, are useful for treating auto-immune diseases, traumatic injuries and vascular injuries, including osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as enhancing skeletal muscle repair, bone repair, treating cancer, cardiovascular diseases, and other neurological disorders. These methods comprise administering to a patient or subject in need a composition comprising an effective amount of AC-EVs, preferably MSC-EVs, by intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal routes of administration in order to effect therapy in said patient or subject. In embodiments, the present invention is directed to extracellular vesicles prepared from adherent cells (ACEVs) which are exposed to hypoxic conditions to produce low oxygen extracellular vesicles (LO2-EVs), acidic conditions to produce low pH extracellular vesicles (LPH-EVs) and inflammatory conditions (exposure to concentrations of cytokines, including interferons, interleukins and tumor necrosis factors) to produce inflammatory extracellular vesicles (INF-EVs). Once produced, these EVs find use in the treatment of disease states and/or conditions as otherwise described herein. In embodiments, the EVs pursuant to the present invention may be formulated in pharmaceutical compositions comprising an effective amount of EVs in combination with a pharmaceutically acceptable carrier, additive and/or excipient. In embodiments, compositions according to the present invention may also include an effective amount of one or additional bioactive agent which is useful in the treatment of disease. In embodiments, the bioactive agent is an anti-cancer agent. In embodiments, adherent cells (including MSCs as otherwise described herein) are first grown to 70-95% confluence in media (confluent cells), harvested and replated at 2,000- 10,000 cells/cm2 (preferably, 5,000 cells/cm2) on plating material. The replated cells are exposed to hypoxic, low pH or inflammatory conditions in a priming step for approximately one minute to 72 hours, often 24-28 hours, rinsed, and subsequently grown in growth medium for another 12 hours to 6 days or more, preferably 24-48 hours, after which the cells are collected and filtered to remove cells and large debris from the extracellular vesicles in the medium. Generally, the period of priming for all conditions is that which is sufficient to affect the cell secretome, to maximize EV production with characteristics of the priming conditions. The period of time sufficient for the adherent cells to release EVs after priming is about 12-36+ hours, more often 24-48 hours. The filtered media containing extracellular vesicles is then subjected to ultrafiltration (e.g., at 4000g for 10-20 minutes). The EVs are then subsequently washed, collected, aliquoted and frozen to be used subsequently. In an embodiment which is directed to producing LO2-EVs, confluent adherent cells, (preferably, MSCs) are exposed to a hypoxic priming step wherein the confluent cells are exposed to gas/atmosphere mixtures in culture containing from 0.5-21% oxygen, more often about 0.75% to 15% oxygen, often about 1.0% to 10% oxygen, about 1.5% to 5.0% oxygen, 2.0-3.5% oxygen, about 0.25% to about 12.5% oxygen, and about 5% oxygen, the remainder of the gas/atmosphere consisting essentially of nitrogen (from 75% to 98%) and a small percentage of carbon dioxide (about 1% to 10%, often about 5%). Often, the adherent cells (preferably, MSCs) are primed in the hypoxic atmospheric conditions for a period sufficient to affect the cell secretome. This period may range from one minute up to 72 hours, often 2- 15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 12-48 hours before washing, collecting and freezing the EVs which are released from the cells. In an embodiment of the invention which is directed to producing LPH-EVs, confluent adherent cells (preferably, MSCs) are primed under acidic conditions to provide a pH of the priming medium ranging from 6.0-7.35, often 6.9-7.3 for a period ranging from one minute up to 72 hours, often 2-15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells. In an embodiment of the invention which is directed to producing INF-EVs, confluent adherent cells (preferably, MSCs) are primed under conditions with effective concentrations of cytokines (e.g. interferon type I, including IFN-α, IFN-β, IFN-ε IFN-κ, IFN-δ, IFN-τ, IFN- ω and IFN-v, interleukins 1, 1α, 1β, and 2-36 and/or tumor necrosis factors, TNF 1-19 and 13B) at effective concentrations ranging from 1 ng/ml to 100 ng/ml or more, often about 5 ng/ml to about 50 ng/ml, about 10 ng/ml to about 30 ng/ml, often about 15 ng/ml to 25ng/ml for a period ranging from one minute up to 72 hours, often 2-15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells. Brief Description of the Figures Abbreviations on Figures which follow: • NC: from normal culture MSCs • INF: from inflammatory culture MSCs • LO2: from hypoxic culture MSCs • LPH: from hypoxic culture MSCs • EV: extracellular vesicle • MSC: mesenchymal stem/stromal cell FIGURE 1 shows EV characterization. The size and concentration of MSC-EVs is affected by priming. Hypoxic and acidic preconditioning increased EV release. (A) Size distributions of EV groups as measured by NTA (B): EV diameter across MSC culture conditions as determined by NTA. (C): EV concentration across MSC culture conditions as determined by NTA. One-way ANOVA. Data is presented as means ± SEM. N = 3 independent experiments. (*,**, ***,****) indicate significant difference from Normal Culture at p < 0.05, 0.01, 0.001, 0.0001 by Dunnett’s post-hoc test. FIGURE 2 shows (A) Relative expression of selected EV surface markers across MSC culture conditions as determined by MACSPLEX analysis. MSC priming affects the surface marker expression of released EVs. (B) Plot of PC1 vs PC2 following PCA of all MACSPLEX markers. (C) Plot of PC1 and PC2 values. Two-way ANOVA. Data is presented as means ± SEM. N = 3 independent experiments. (*,**, ***,****) indicate significant difference from Normal Culture at p < 0.05, 0.01, 0.001, 0.0001 by Dunnett’s post-hoc test. FIGURE 3 shows the differential uptake of MSC-EVs by T cell subsets. LPH-EV treated PBMCs had significantly higher CFSE+ frequency than untreated cells, indicating uptake of EVs. (A): Frequency of CD4+ cells positive for CFSE. (B): Frequency of CD8+ cells positive for CFSE. (C): Frequency of CD4+/CD25+ cells positive for CFSE. (D): Frequency of CD8+/CD25+ cells positive for CFSE. (E): Frequency of CD4+/CD25+/FOXP3+ cells positive for CFSE. (F): Frequency of CD8+/CD25+/FOXP3+ cells positive for CFSE. (G): Frequency of CD4-/CD8-cells positive for CFSE. Data is presented as means ± SEM. N = 3 independent experiments. (*,**, ***,****) indicate significant difference from 0-EV at p < 0.05, 0.01, 0.001, 0.0001 by Dunnett’s post-hoc test of one-way ANOVA. FIGURE 4 shows the inhibition of T cell proliferation 5 days after treatment with EVs or MSCs as measured by CFSE dilution. EVs did not affect the proliferation of T cells, while MSCs greatly decreased it. (A): CD4+ cells. (B): CD8+ cells. Data is presented as means ± SEM. (*,**, ***,****) indicate significant difference from (+) CTRL at p < 0.05, 0.01, 0.001, 0.0001 by Dunnett’s post-hoc test of one-way ANOVA. FIGURE 5 shows the comparative activation of T-cell subsets 5 days after treatment with EVs or MSCs. Overall, MSCs had significant effects on T effector cells frequency, while LPH-EVs had significant effects on Treg frequency. (A): Frequency of CD4+/CD25+/FOXP3- cells. (B): Frequency of CD8+/CD25+/FOXP3- cells. (C): Frequency of CD4+/CD25+/FOXP3+ cells. (D): Frequency of CD8+/CD25+/FOXP3+ cells. (E): Frequency of CD4+/TNF-α+ cells. (F): Frequency of CD8+/ TNF-α+ cells. (G): Frequency of CD4+/IFN- y+ cells. (H): Frequency of CD8+/ IFN-y+ cells. Data is presented as means ± SEM. (*,**, ***,****) indicate significant difference from (+) CTRL at p < 0.05, 0.01, 0.001, 0.0001 by Dunnett’s post-hoc test of one-way ANOVA. FIGURE 1S shows experimental workflow. MSCs were split into groups that each underwent different priming steps. MSCs were then either co-cultured with PBMCs for 5 days or used for EV isolation. Isolated EVs were used in NTA or MACSPLEX analysis, stained with CFSE and incubated for 24 hours with PBMCs, or incubated for 5 days with PBMCs. FIGURE 2S shows an antibody table. All antibodies used in the experimental study, were titrated to optimal concentration prior to experiments. FIGURE 3S shows a fFlow analysis diagram. After successively gating out debris (A), activating beads (B) and doublets (C), live PBMCs (D) were gated based on CD4 and CD8 expression (E). Single-positive cells were then analyzed based on the panel they were stained with. Panel 1 was first gated by CD25 expression (F), then CD25+ cells were gated by FOXP3 expression (G). Panel 2 was gated separately by TNF-α and IFN-γ. The median fluorescence intensity of CFSE (J) was calculated for all samples to be used in proliferation calculations. For EV uptake, the populations from Panel 1 were determined at 24 hours post- treatment and T-cell subpopulations were gated by CFSE intensity (K). All gates were determined by FMO controls after compensation. FIGURE 4S shows the dynamic Light Scattering of EVs. Histograms of EV size distribution confirm the existence of a large-diameter population present in INF-MSC-EV at a high concentration. FIGURE 5S shows the comparative activation of T cell subsets 24 hours after treatment with CFSE-EVs. There were no significant differences between treatment groups. (A): Frequency of CD4+ cells. (B): Frequency of CD8+ cells. (C): Frequency of CD4+/CD25+ cells. (D): Frequency of CD8+/CD25+ cells. (E): Frequency of CD4+/CD25+/FOXP3+ cells. (F): Frequency of CD8+/CD25+/FOXP3+ cells. Data is presented as means ± SEM. No significant differences from 0-EV group by Dunnett’s post-hoc test of one-way ANOVA. FIGURE 6S shows the top 10 contributing surface markers to PC1 and PC2 of MACSPLEX PCA. Detailed Description of the Invention Definitions In accordance with the present invention there may be employed conventional cell culture methods, chemical synthetic methods and other biological and pharmaceutical techniques within the skill of the art. Such techniques are well-known and are otherwise explained fully in the literature. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It is to be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the following terms shall have the definitions set out below. It is understood that in the event a specific term is not defined herein below, that term shall have a meaning within its typical use within context by those of ordinary skill in the art. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The term “effective” is used to describe an amount of a composition or component which is included to effect an intended result, including the production of MSC-EVs as otherwise described herein . The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. The terms “treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a patient at risk for or afflicted by a disease state, condition or deficiency which may be improved using cellular compositions according to the present invention. Treating a condition includes improving the condition through lessening or suppression of at least one symptom, delay in progression of the effects of the disease state or condition, including the prevention or delay in the onset of effects of the disease state or condition, etc. In the present application, treatment can involve reducing the impact of a spinal cord injury or stroke, including reversing and/or inhibiting the effects of such injury, reversing, improving, inhibiting and/or stabilizing a neurodegenerative disease such that the disease improves and/or does not progress or worsen. The term “prophylactic” is used to describe a method which “reduces the likelihood” that a particular result will occur, often the progression and/or worsening of a disease state and/or condition. Standard techniques for growing cells, separating cells, and where relevant, cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (Ed.) 1993 Meth. Enzymol.218, Part I; Wu (Ed.) 1979 Meth. Enzymol.68; Wu et al., (Eds.) 1983 Meth. Enzymol.100 and 101; Grossman and Moldave (Eds.) 1980 Meth. Enzymol.65; Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols.1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein. As used herein, the term “culture media” is used to describe a growth medium in which MSC’s are grown, such media especially including media in which MSCs are grown to produce EVs as otherwise described herein. Culture media describeds media which comprises sufficient components to promote the growth and health of cells. Culture media are well known in the art and comprise at least a minimum essential medium plus one or more optional components such as growth factors, including fibroblast growth factor (FGF), ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine (often at 2mM L-glutamine), penicillin (often, for example at 50U/ml), streptomycin (often 50μg/ml), insulin (where indicated and not excluded), transferrin, beta mercaptoethanol, and other agents well known in the art and as otherwise described herein. Culture media may include basal cell media which may contain between 1% and 20% (often 10%) fetal calf serum (FCS), or for defined medium an absence of fetal calf serum (serum free). In embodiments, the culture medium defined and is serum free. Other components which optionally may be added to culture medium are as otherwise described herein, especially including the experimental section which follows. Each of these components, when included, are included in effective amounts, i.e., in amount sufficient to promote the growth and health of cells. These additional components which optionally may be added to the medium include, depending on the cell type grown in the media, for example, any one or more agents selected from the group consisting of leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factors (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), transforming growth factors beta (TGFβ-1) and TGFβ-3, nicotinamide, members of TGF-β family, including TGF-β 1, 2, and 3, Bone Morphogenic Proteins (BMP 2 to 7), members of the fibroblast growth factor (FGF) family, platelet- derived growth factor-AA, and -BB, insulin growth factor (IGF-I, II, LR-IGF), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, betacellulin, ITS, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP) or combinations thereof, among a number of other components. Each of these components, when included, are included in effective amounts. By way of further example, suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco # 10829- 018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non- essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029. Preferred embodiments of culture media used in the present invention are as otherwise described herein. Culture media useful in the present invention are commercially available and can be supplemented with commercially available components, available from Invitrogen Corp. (GIBCO), Cell Applications, Inc., among numerous other commercial sources, including Calbiochem. One of ordinary skill in the art will be able to readily modify the cell media to grow MSCs or to produce any one or more of the target MSC-EVs pursuant to the present invention. The term “human Pluripotent Stem Cells”, of which ”human Embryonic Stem Cells” (hESCs) and human induced pluripotent stem cells (hiPSCs) are a subset, are derived from pre-embryonic, embryonic, fetal tissue or adult stem cells (in the case of human induced pluripotent stem cells) at any time after fertilization, and have the characteristic of being capable under appropriate conditions of producing progeny of several different cell types, especially including mesenchymal stem cells (MSCs) and related proliferative and non- proliferative cells. The term includes both established lines of stem cells of various kinds, and cells obtained from primary tissue that are pluripotent in the manner described. The term “embryonic stem cell” refers to pluripotent cells, preferably of primates, including humans, which are isolated from the blastocyst stage embryo. The term “extracellular vesicle producing adherent cells” or “EVPAC” is used to describe adherent cells which can be used to produce LO2-EVs, LPH-EVs and INF-EVs “adherent cell extracellular vesicles” or “EC-EVs”, preferably MSC-EVs pursuant to the present invention. These adherent cells include primary cells (e.g. mesenchymal stem cells/MSCs, pericytes, fibroblasts, and immortalized cells lines, such as HEK-293T), among others. In preferred embodiments, MSCs are preferred adherent cells for providing LO2- EVs, LPH-EVs and INF-EVs pursuant to the present invention. The term “mesenchymal stem cell” or “MSC” refers to multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes. These cells are multipotent non-hematopoietic, self-renewable cells that are capable of trilineage differentiation into mesoderm, ectoderm, and endoderm cells. These cells also characterized by their pluripotency and immunomodulatory features, and their ability to be cultured over lengthy periods of time. In preferred embodiments according to the present invention, MSCs are used to produce the various extracellular vesicles/extrasomes pursuant to the present invention. MSCs may be purchased from commercial sources (e.g. Lifeline Cell Technologies, Oceanside, California USA, among others). In addition, MSCs can be readily produced following literature preparations well-known in the art. US patent no.5,486,359 and Pittenger et al., Science, Vol.284, pg.143 (1999) provide methods for the production of MSCs. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate readily into lineages including osteoblasts, myocytes, chondrocytes, and adipocytes (Pittenger, et al., Science, Vol.284, pg.143 (1999); Haynesworth, et al., Bone, Vol.13, pg.69 (1992); Prockop, Science, Vol.276, pg.71 (1997)). Studies with a variety of animal models have shown that MSCs may be useful in the repair or regeneration of damaged bone, cartilage, meniscus or myocardial tissues (DeKok, et al., Clin. Oral Implants Res., Vol. 14, pg.481 (2003)); Wu, et al., Transplantation, Vol.75, pg.679 (2003); Noel, et al., Curr. Opin. Investig. Drugs, Vol.3, pg.1000 (2002); Ballas, et al., J. Cell. Biochem. Suppl., Vol. 38, pg.20 (2002); Mackenzie, et al., Blood Cells Mol. Dis., Vol.27 (2002)). Several investigators have used MSCs with encouraging results for transplantation in animal disease models including osteogenesis imperfecta (Pereira, et al., Proc. Nat. Acad. Sci., Vol.95, pg. 1142 (1998)), parkinsonism (Schwartz, et al., Hum. Gene Ther., Vol.10, pg.2539 (1999)), spinal cord injury (Chopp, et al., Neuroreport, Vol.11, pg.3001 (2000); Wu, et al., J. Neurosci. Res., Vol.72, pg.393 (2003)) and cardiac disorders (Tomita, et al., Circulation, Vol.100, pg.247 (1999). Shake, et al., Ann. Thorac. Surg., Vol.73, pg.1919 (2002)). The terms “extracellular vesicle” and“EV” are used herein to refer to a vesicle of about 10nm to 10μm in size consisting of fluid, macro-molecules, solutes, and metabolites from a cell contained by a lipid bilayer or micelle. In preferred embodiments, the EV is a cell-derived EV (from adherent cells, preferably MSCs). The term “EV” may also include lipid vesicles engineered to contain bioactive molecules found in a cell-derived EVs, such as MSC-EVs. These terms encompass both exosomes and ectosomes. Exosomes are extracellular vesicles released on the exocytosis of multivesicular bodies (MVBs). Ectosomes are vesicles assembled at and released from the plasma membrane. In some cases, the EV is about 20nm to 10μm, 20nm to 1μm, 20 nm-500 nm, 30 nm-100nm, 30 nm-160nm, or 80-160 nm in size. In some embodiments, the EVs are exosomes that are about 20 to 150 nm in size. Often the exosomal vesicles range in size from 10 nm to 250 nm or more. The term “autologous EV” is used to describe a population of EVs which are obtained from MSC cells from a subject or patient to whom the EVs are to be administered. The term “MSC-EV” is used to refer to a cell-derived EV produced from mesenchymal stem cells derived in vitro from pluripotent stem cells or progenitor cells. The term also refers to vesicles engineered to contain a sufficient number of the bioactive molecules found in the cell-derived MSC-EVs to have substantially the same bioactivity. The term “LO2-EV” is used to describe extracellular vesicles which are prepared using a hypoxic priming step of the adherent cells prior to incubating. Preferred Methods of Producing AC-EVs (Preferably MSC-EVs) The following preferred methods are provided for producing AC-EVs: LO2-EV Extracellular vesicles produced from adherent cells (preferably MSCs) exposed to hypoxic conditions; LPH-EV Extracellular vesicles produced from adherent cells (preferably MSCs) exposed to acidosis/acidic conditions; and INF-EV Extracellular vesicles produced from adherent cells (preferably MSCs) exposed to inflammation cytokines. MSCs are plated at approximately 1,000-10,000, or 2,500-10,000, often 5,000 cells cells/cm2 on tissue culture flasks in complete medium (Alpha-Minimum Essential Medium (Gibco), 10% defined fetal bovine serum (Hyclone), 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco)) and allowed to grow to 80% confluence (approximately 20,000–25,000 cells/cm2). The cells were harvested using 0.05% trypsin (Gibco) and replated at 5000 cells/cm2. All proliferation cultures were maintained at 37°C and at 5% CO2. All cells used in experiments had undergone fewer than 10 passages and had over 90% viability at harvest as assessed by Trypan blue staining. Confluent MSCs (as grown above to about 70-95% confluence, preferably 80% confluence and 80-100%, often 90-95% or more viability) are then exposed (primed) to hypoxic, acidosis or inflammatory conditions for between one minute up to 72 hours, often 2- 15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for a time sufficient to release EVs into culture media, often between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells. Alternatively, the primed cells can be placed in co-culture with human Peripheral Blood Mononuclear Cells (PBMCs). Extracellular Vesicle/Exosome Isolation After priming, MSCs are rinsed twice with PBS before adding fresh serum free medium (Alpha-Minimum Essential Medium (Gibco), 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin (all from Gibco/Invitrogen) and incubating cultures for 24- 48 hours. The resulting conditioned media containing cells were collected and filtererd (e.g. 0.22 μm filters) to remove cells and large debris. The filtered media were subjected to ultrafiltration (with, for example a 100kDa MWCO (Amicon, Millipore-Sigma) at 4000g for 10 minutes as previously published (26). The EVs remained on top of the filter and were then washed twice with PBS +/+ (Thermo Fisher Scientific, Waltham, MA) at 2000g for 10 minutes. The EVs in PBS+/+ were then collected, aliquoted, and frozen at -20°C. Characterization of Extracellular Vesicles Nanoparticle tracking analysis; Size and distribution of EVs; Flow cytometry- marker expression For each EV isolation, nanoparticle tracking analysis (NTA) was performed using a Nanosight NS3200 (Nanosight, Salisbury UK) according to the manufacturer’s recommendations. Briefly, aliquots of EV suspensions were thawed at room temperature and diluted to 107-109 particles/mL with the same lot of PBS +/+ the EVs were isolated in. A minimum of three samples and five one-minute videos were recorded for each exosome isolation. All videos were captured at the same camera level and analyzed with the same detection threshold. The size and size distribution of vesicles was further verified via Dynamic Light Scattering using a Malvern Zetasizer Nano ZS Analyzer (Malvern Instruments, Malvern, UK). Samples were diluted to a total vesicle concentration of approximately 2x108 vesicles/ml in 0.22μm filtered Phosphate Buffered Saline containing Calcium and Magnesium, pH 7.4 prior to measurements. Disposable polystyrene cuvettes were rinsed with 1mL of filtered PBS +/+ prior to adding sample. Measurements were taken using cuvette with 800uL of prepared sample. EV surface marker characterization was performed using the MACSPLEX Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s directions. Briefly, an equal number of exosomes as determined by NTA were analyzed from each isolation in triplicate. Flow cytometry analysis was performed using a CytoFLEX S (Beckman Coulter, Hialeah, Florida) alongside bead only controls, with FlowJo being used for data analysis. Data was processed with background subtraction and normalized to the median of the average value of CD9, CD63, and CD81 for each sample (27). The data was transformed to be a percentage of the difference between the maximum and minimum relative expression of a marker. Principal component analysis (PCA) was performed on the transformed data using JMP (SAS Institute, Cary NC). Hypoxic Conditions- prime confluent adherent cells (preferably MSCs) in gas mixtures containing 0.5-21% oxygen, more often about 0.75% to 15% oxygen, often 1.0% to 10% oxygen, 1.5-5.0% oxygen, 2.0-3.5% oxygen, 0.25% to 12.5% oxygen, 2.5% to 7.0% oxygen, about 5% oxygen, the remainder of the atmosphere consisting essentially of nitrogen (75-98%) and a smaller percentage of carbon dioxide (2% to 10%, often 5%). Often the MSCs are primed using a hypoxic gas/atmospheric mixture as described above for a period ranging from about 1 minute up to 1 hour or much longer in certain instances, more often 2- 15 minutes, more often 5-10 minutes before growing the MSCs in serum free medium for a period between 12 hours and 6 days or more, more often 24 hours to 72 hours, 36 to 60 hours, often about 48 hours or as otherwise described herein. Acidosis/Acidic Conditions- confluent adherent cells (MSCs) in media under acidic conditions (use of protic acid such as HCl, phosphoric acid, organic acid or other acid to reduce pH of priming media to about 6.0 to 7.35, often 6.9 to about 7.3, often 7.1 + 0.05). After priming (preferably for a period ranging from about 1 minute up to 1 hour or much longer in certain instances, more often 2-15 minutes, more often 5-10 minutes), these cells are grown in serum free medium for from 12 hours to 6 days or more, often 24 hours to 72 hours, more often 24 to 48 hours before isolating the EVs by ultrafiltration followed by filtration. Inflammation Conditions- prime confluent adherent cells (preferably, MSCs) are primed under conditions with effective concentrations of cytokines (e.g. interferon type I, including IFN-α, IFN-β, IFN-ε IFN-κ, IFN-δ, IFN-τ, IFN-ω and IFN-ν, interleukins 1, 1α, 1β, and 2-36 and/or tumor necrosis factors, TNF 1-19 and 13B) at effective concentrations ranging from 1 ng/ml to 100 ng/ml or more, often about 5 ng/ml to about 50 ng/ml, about 10 ng/ml to about 30 ng/ml, often about 15 ng/ml to 25ng/ml for a period ranging from one minute up to 72 hours, often 2-15 minutes, often 15 minutes up to 12 hours, more often 5-10 minutes before growing the primed adherent cells in growth medium (preferably, serum free medium) for between 12 hours and 6 days or more, more often 24 hours to 72 hours or 36 to 60 hours, more often about 48 hours, before washing, collecting and freezing the EVs which are released from the cells. Compositions Disclosed herein are AC-EVs, preferably MSC-EVs (e.g. extracellular vesicles or exosomes) and methods of using these EVs in the treatment of disease states and conditions, including osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders. The disclosed EVs can be obtained in some embodiments, by culturing mesenchymal stem cells (MSCs) that were produced in vitro from pluripotent stem cells (e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs)) in cell culture medium under conditions and for a time sufficient for the MSCs to produce EVs. These conditions may be modified as described herein to provide EV’s produced under conditions of hypoxia (LO2-EV), acidosis (LPH-EV), and inflammatory cytokines (INF-EV) for purposes of providing EV’s which are standardized, reproducible, therapeutically effective and useful in the treatment of diseases states and conditions where immunomodulatory and other therapy may provide therapeutic benefit. The disclosed EVs can be obtained in some embodiments by culturing MSCs, derived directly or indirectly from pluripotent stem cells in cell culture medium under conditions and for a time sufficient to produce EVs, and isolating said EVs from the culture medium. These MSCs may be purchased from commercial sources or produced by known methods, long known in the art. In a preferred method, MSCs are cultured in hypoxic, acidic (acidosis) or in the presence of inflammatory cytokines conditions for a sufficient period (generally about 12 hours to 72 hours or more (about 1 week), often 24-60 hours, more often about 48 hours (about 2 days) to produce MSC-EVs in the media and the resulting media is subjected to filtration to remove cellular debris to isolate the MSC-EVs which are further subjected to ultrafiltration to isolate the MSC-EVs. Pluripotent stem cells used to produce the EV-producing MSCs include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR. The types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non- limiting examples are established ethical lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines WA01, WA07, and WA099 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BG01v (BresaGen, Athens, Ga.), as well as normal human embryonic stem cell lines such as WA01, WA07, WA09 (WiCell) and BG01, BG02 (BresaGen, Athens, Ga.). Human embryonic stem cells (hESCs) may be prepared by methods which are described in the in the art as described for example, by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995). Alternatively, they may be obtained commercially. Epiblast stem cells (EpiScs) and induced pluripotent stem cells (iPSCs) isolated from early post-implantation stage embryos. They express Oct4 and are pluripotent. iPSCs are made by dedifferentiating adult somatic cells back to a pluripotent state by retroviral transduction of four genes (c-myc, Klf4, Sox2, Oct4). As described in U.S. Patent Application Document No.20140356382, “[e]xosomes produced from cells can be collected from the culture medium and/or cell tissue by any suitable method. Typically a preparation of EVs can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods. For example, EVs can be prepared by differential centrifugation, that is low speed (<2,000g) centrifugation to pellet larger particles followed by high speed (>100,000 g) centrifugation to pellet EVs, size filtration with appropriate filters (for example, 0.22 μm filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods.” In preferred aspects, the MSCs are cultured over a sufficient period (12 hours to a week or more as otherwise described herein) to produce EVs and the EVs are isolated by filtration (to remove cellular debris), followed by ultrafiltration to separate EVs from remaining fluid. After washing the EVs may be collected and aliquoted and used directly or frozen (at preferably -10ºC to -30ºC) for subsequent use after thawing. It is noted that the contents of EVs, i.e., EVs in which the lipid bilayer has been removed or eliminated and the contents obtained may also be used to engineer artificial EVs. Further, as described in U.S. Patent Application Document No.20140356382, exogenous protein and/or peptide and other cargo can be introduced into the EVs by a number of different techniques including electroporation or the use of a transfection reagent. Electroporation conditions may vary depending on the charge and size of the biotherapeutic cargo. Typical voltages are in the range of 20V/cm to 1,000V/cm, such as 20V/cm to 100V/cm with capacitance typically between 25 μF and 250 μF, such as between 25 μF and 125 μF. A voltage in the range of 150 mV to 250 mV, particularly a voltage of 200 mV is preferred for loading EVs with an antibody. Alternatively, the EVs may be loaded with exogenous protein and/or peptide using a transfection reagent. Despite the small size of the EVs, conventional transfection agents may be used for transfection of EVs with protein and/or peptide. EVs may also be loaded by transforming or transfecting a host cell with a nucleic acid construct which expresses therapeutic protein or peptide of interest, such that the therapeutic protein or peptide is taken up into the EVs as the EVs are produced from the cell. In illustrative embodiments, the EV-producing MSCs disclosed herein are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or for as long as about 1, 2, 3, 4, 5, 6, 7, 8 weeks or about 1, 2, 3, 4, 5, or 6 months, depending on the cell and its ability to produce EVs. In preferred embodiments, the MSCs are cultured for between about 12 hours and one week, often 24-72 hours, more often about 24-60 hours, even more often about 48 hours to produce the MSC-EVs according to the present invention. The EV-producing cells may be cultured in suitable media and grown under conditions that are readily determined by one of ordinary skill in the art. Cell culture conditions may vary with cell type and the examples presented hereinafter illustrate suitable media and conditions as otherwise described herein. For example, CMRL 1066 medium (from Invitrogen) with fetal bovine serum (e.g., at 10%) and optionally supplemented with glutamine or glutamine-containing mixtures and antibiotics could be used. Cells can be grown on a surface (feeder cells) in some embodiments, e.g. they can be grown as a monolayer on the surface (feeder cell free) and may be grown until 30, 40, 50, 60, 70, 80, 90, 95 or 100% confluent. As presented, EV producing adherent cells may include primary cells (mesenchymal stem cells, pericytes, fibroblasts, immortalized cell lines (HEK-293T). The density of the cells (which are primed) sufficient to produce EVs include a range of 1000-10000 cells/cm2 on plating material (often 2D culture, tissue culture plastic, although other plating material may be used). The media which is used to promote the growth and health of adherent cells often includes a minimum essential medium, which may include alpha-Minimum Essential Medium (Gibco), 10% defined fetal bovine serum (Hyclone), 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco) among other antibiotics. The adherent cells are generally grown at body temperate. As discussed, cell growth media are well known in the art and comprise at least a minimum essential medium plus one or more optional components such as growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (where indicated and not excluded), Activin A, transferrin, beta mercaptoethanol, and other agents well known in the art and as otherwise described herein. A preferred media is a low protein, serum-free based growth medium that supports neural cells. The growth factor used can be fibroblast growth factor 2 (FGF2), alone or preferably in combination with leukemia inhibitor factor (LIF). Depending on the MSCs to be grown in the growth media, the inclusion of may be preferred. Additional media includes basal cell media which may contain serum, for example, between about 0.1% and 20% (preferably, about 2-10%) fetal calf serum, or for defined medium, an absence of fetal calf serum and KSR, and optionally including bovine serum albumin (about 1-5%, preferably about 2%). Preferred medium is defined and is serum-free and low protein. The components of the growth media depends on the type of MSC to be grown, all of which are well known in the art. Particularly preferred media is media and supplement is identified in the experimental section hereinbelow. The medium and supplement are engineered for versatility to meet all mesenchymal stem cell culture needs. Each lot of medium and supplement is pre-qualified for use by testing for cell growth, sterility, pH, osmolarity, and endotoxins. Cell culture formulations allow mesenchymal cell cultures to maintain a stable karyotype over multiple passages without the need for feeder cells, making them an excellent choice for a wide variety of research applications including early stage drug discovery. Other agents which optionally may be added to the medium include, depending on the cell type grown in the media, for example, any one or more of nicotinamide, members of TGF-β family, including TGF-β 1, 2, and 3, Activin A, nodal, Bone Morphogen Proteins (BMP 2 to 7) serum albumin, members of the fibroblast growth factor (FGF) family, platelet- derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II, LR-IGF), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na- Butyrate, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, heregulin, or combinations thereof, among a number of other components. Each of these components, when included, are included in effective amounts. By way of further example, suitable media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco # 10829- 018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non- essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029. Cell media are commercially available and can be supplemented with commercially available components, including defined xeno-free components, such as those available from Invitrogen Corp. (GIBCO), Cell Applications, Inc., Biological Industries and Calbiochem, among numerous others. One of ordinary skill in the art will be able to readily modify the cell media to produce any one or more of the target cells pursuant to the present invention. The disclosed MSC-EV-producing cells may be cultured on a layer of feeder cells that support the cells in various ways. Approaches for culturing cells on a layer of feeder cells are well known in the art. The cells may be grown on a cellular support or matrix, as adherent monolayers, rather than as embryoid bodies or in suspension. In certain embodiments, the use of a cellular support may be preferred, depending upon the cells used to produce the EVs. When used, cellular supports preferably comprise at least one substrate protein. Substrate proteins include, for example, an extracellular matrix protein, which is a protein found in the extracellular matrix, such as laminin, tenascin, thrombospondin, and mixtures thereof, which exhibit growth promoting and contain domains with homology to epidermal growth factor (EGF) and exhibit growth promoting activity. Other substrate proteins which may be used include for example, collagen, fibronectin, vibronectin, polylysine, polyornithine and mixtures thereof. In addition, gels and other materials such as methylcellulose of other gels which contain effective concentrations of one or more of these embryonic stem cell differentiation proteins may also be used. Exemplary differentiation proteins or materials which include these differentiation proteins include, for example, laminin, BD Cell-Tak™ Cell and Tissue Adhesive, BD™ FIBROGEN Human Recombinant Collagen I, BD™ FIBROGEN Human Recombinant Collagen III, BD Matrigel™ Basement Membrane Matrix, BD Matrigel™ Basement Membrane Matrix High Concentration (HC), BD™ PuraMatrix™ Peptide Hydrogel, Collagen I, Collagen I High Concentration (HC), Collagen II (Bovine), Collagen III, Collagen IV, Collagen V, and Collagen VI, among others. Alternatively, these MSCs may be cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of the cells to produce EVs. The growth of cells in feeder-free culture can be supported using a medium conditioned by culturing previously with another cell type. Alternatively, the growth of EV-producing cells in feeder-free culture without differentiation can be supported using a chemically defined medium. These approaches are well known in the art. In certain embodiments of the present invention, the cells are grown in feeder cell free medium. EVs can be harvested at various time intervals (e.g. at about 12 hours, 1, 2, 4, 6, 8 or 3, 6, 9, 12 day or longer intervals, depending upon the rate of production of EVs). Exemplary yields of EVs can range from at least about 1 ng EVs/1 million cells, at least about 10 ng EVs/1 million cells, at least about 50 ng EVs/1 million cells, at least about 100 ng EVs/1 million cells, at least about 500 ng EVs/1 million cells, at least about 750 ng EVs/1 million cells, at least about 800 ng EVs/1 million cells, at least about 900 ng EVs/1 million cells, at least about 1.0 μg EVs/1 million cells, at least about 1.5 μg EVs/1 million cells, at least about 2.0 μg EVs/1 million cells, at least about 2.5 μg EVs/1 million cells, at least e.g. about 3.0 μg EVs/1 million cells, at least about 5.0 μg EVs/1 million cells, and at least about 10.0 μg EVs/1 million cells, during a time period of about 24 hours to seven days of culture of MSCs as otherwise described herein. In certain embodiments, EVs are harvested and collected by filtration, ultrafiltration, ultracentrifugation or differential centrifugation or any combination thereof, pelleted EVs are collected, and, optionally, collected pelleted EVs are washed with a suitable medium. For example, a preparation of EVs can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods. In some embodiments, the EVs can be prepared by differential centrifugation, that is low speed (<2,0000 g) centrifugation to pellet larger particles followed by high speed (>100,000 g) centrifugation to pellet EVs, size filtration with appropriate filters (for example, 0.22 μm filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods. EVs may be purified by differential centrifugation, micro and ultra-filtration, polymeric precipitation, microfluidic separation, immunocapture and size-exclusion chromatography. These and/or related methods for isolating and purifying EVs are described by Théry, et al., Current Protocols in Cell Biology, (2006) 3.221-3.22.29, copyright 2006 by John Wiley & Sons, Inc.; Sokolova, et al., Colloids and Surfaces B: Biointerfaces, 2011, 87, 146-150; Wiklander, et al., Journal of Extracellular Vesicles, 2015, 4, 26316, pp.1-13; and Böing, et al., Journal of Extracellular Vesicles, 2014, 3, 23430, pp.1-11. Other methods for isolation may be developed such as electrical field radiofrequency and acoustics. In certain preferred embodiments, the EVs pursuant to the present invention are isolated by filtration (e.g.0.22 μM), followed by ultrafiltration. The EVs which remain (often on top of the filter) are washed for several minutes collected, aliquoted and used directly or frozen for later use. Pharmaceutical compositions Disclosed are pharmaceutical compositions containing therapeutically effective amounts of one or more of the disclosed EVs and a pharmaceutically acceptable carrier. Formulations containing the disclosed EVs may take the form of liquid, solid, semi-solid or lyophilized powder forms, such as, for example, solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. Pharmaceutical compositions typically include a conventional pharmaceutical carrier and/or excipient and may additionally include other medicinal agents, carriers, adjuvants, additives and the like. The weight percentage ratio of the EVs to the one or more excipients can be between about 20:1 to about 1:60, or between about 15:1 to about 1:45, or between about 10:1 to about 1:40, or between about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1 to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, and preferably is about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1 or 5:1. In some embodiments, the disclosed composition comprises between about 0.1 μg to about 1 g or more of total EVs, about 1 μg to aobut 750 mg, 500 μg about 500 mg, about 1 mg to about 500 mg of total EVs, about 5 to about 500 mg, about 10 to about 500 mg, about 25 to about 500 mg, about 50 mg to about 350 mg, about 75 mg to about 450 mg, about 50 mg to about 450 mg, or about 75 mg to about 325 mg or about 100 mg to about 650 mg of total EVs and may optionally contain one or more suitable pharmaceutical carriers, additives and/or excipients. An injectable composition for parenteral administration (e.g. intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal, among other routes), will typically contain the EVs and optionally additional components in a suitable i.v. solution, such as sterile physiological salt solution. The composition may also be formulated as a suspension in an aqueous emulsion. Liquid compositions can be prepared by dissolving or dispersing the pharmaceutical composition comprising the EVs, and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension. For use in an oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline. In the case of intranasal, intratracheal or intrapulmonary administration, the compositions may be provided as liquid composition which can be sprayed into the nose, trachea and/or lungs. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers. When the composition is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like. In a tablet formulation, the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations. Methods for preparing such dosage forms are known or are apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co. 1985). The composition to be administered will contain a quantity of the selected compound in a pharmaceutically effective amount for therapeutic use in a biological system, including a patient or subject according to the present invention. Intravenous formulations can comprise the EVs described herein, an isotonic medium and one or more substances preventing aggregation of the EVs. Exemplary intravenous/ intrathecal/ intracerebrospinal fluid formulations may contain saline solutions (e.g. normal saline (NS); about 0.91% w/v of NaCl, about 300 mOsm/L) and/or dextrose 4% in 0.18% saline, and optionally 1%, 2% or 3% human serum albumin. In addition, the EVs may be disrupted to obtain the contents and the contents used in compositions according to the present invention. In exemplary embodiments, formulations of the invention may comprise about 50 ng EVs/ml intravenous/intrathecal/intracerebrospinal fluid medium, including about 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1.0 μg, 1.5 μg, 2.0 μg, 2.5 μg, 3.0 μg, 5.0 μg, 10.0, 15.0 μg, 20.0 μg, 100 μg, or more EVs/ml intravenous/intrathecal/intracerebrospinal fluid medium for use in treating spinal cord injury, stroke, traumatic brain injury and/or neurodegenerative diseases. In some embodiments, intravenous formulations may comprise about 0.1 μg EVs/ml medium, about 0.2 μg EVs/ml intravenous medium, about 0.3 μg EVs/ml intravenous medium, about 0.4 μg EVs/ml intravenous medium, about 0.5 μg EVs/ml intravenous medium, about 0.6 μg EVs/ml intravenous medium, about 0.7 μg EVs/ml intravenous medium, about 0.8 μg EVs/ml intravenous medium, about 0.9 μg EVs/ml intravenous medium, about 1.0 μg EVs/ml intravenous medium, about 1.5 μg EVs/ml intravenous medium, about 2.0 μg EVs/ml intravenous medium, about 2.5 μg EVs/ml intravenous medium, such as at least e.g. about 3.0 μg EVs/ml intravenous medium, such as e.g. at least about 5.0 μg EVs/ml intravenous medium, about 10.0 μg EVs/ml intravenous medium, 15.0 μg EVs/ml intravenous medium or about 20.0 μg or more EVs/ml intravenous medium. In some embodiments, the pharmaceutical composition is in a dosage form comprising at least 25 mg of EVs, at least 50 mg of EVs, at least 60 mg of EVs, at least 75 mg of EVs, at least 100 mg of EVs, at least 150 mg of EVs, at least 200 mg of EVs, at least 250 mg of EVs, at least 300 mg of EVs, about 350 mg of EVs, about 400 mg of EVs, about 500 mg of EVs, about 750 mg of EVs, about 1g (1,000mg) or more of EVs, alone or in combination with a therapeutically effective amount of at least one additional bioactive agent, which agent may be useful in the treatment of osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis and Parkinson’s disease, as well as skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders. In some embodiments, the pharmaceutical composition comprises between about 10 mg to about 750 mg, about 25 mg to about 650 mg, or between about 30 mg to about 500 mg, or about 35 mg to about 450 mg, most often about 50 to about 500 mg of EVs. In some embodiments, an intravenous formulation comprises the EVs described herein, an isotonic medium, and one or more substances preventing aggregation of the EVs. Intravenous formulations may therefore contain saline solutions (e.g. normal saline (NS); about 0.91% w/v of NaCl, about 300 mOsm/L) and/or dextrose 4% in 0.18% saline, and optionally 1%, 2% or 3% human serum albumin. In some embodiments, the composition comprising the disclosed EVs further comprises one more neurotrophic agents. The composition can further comprises one or more agents selected from the group consisting of leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte colony- stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factors (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF- β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB), transforming growth factors beta (TGFβ-1) and TGFβ-3. In some embodiments, the disclosed EVs are contained in or on a biocompatible scaffold, such as a hydrogel. Suitable hydrogels include temperature dependent hydrogels that solidify or set at body temperature, e.g., PLURONICS™; hydrogels crosslinked by ions, e.g., sodium alginate; hydrogels set by exposure to either visible or ultraviolet light, e.g., polyethylene glycol polylactic acid copolymers with acrylate end groups; and hydrogels that are set or solidified upon a change in pH, e.g., TETRONICS™. Examples of materials that can be used to form these different hydrogels include polysaccharides such as alginate, polyphosphazenes, and polyacrylates, which are cross-linked ionically, or block copolymers such as PLURONICS™ (also known as POLOXAMERS™), which are poly(oxyethylene)- poly(oxypropylene) block polymers solidified by changes in temperature, or TETRONICS™ (also known as POLOXAMINES™), which are poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine solidified by changes in pH. Suitable hydrogels also include undefined extracellular matrix derived hydrogels that originated from tissues including but not limited to bladder intestine, blood and brain. In some embodiments, the disclosed EVs are contained in or on a biocompatible scaffold comprising collagen, fibrin, silk, agarose, alginate, hyaluronan, chitosan, a biodegradable polyester such as polylactic-co-glycolic acid, polylacic acid, or polyglycolic acid, polyethylene glycol, polyvinylpyrrolidone, polyethersulfone, a peptide-based biomaterial, glycose amino glycan, fibronectin, laminin, or any combination thereof. In some cases, the hydrogel is produced by cross-linking the anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with ions, such as calcium cations. The strength of the hydrogel increases with either increasing concentrations of calcium ions or alginate. For example, U.S. Pat. No.4,352,883 describes the ionic cross-linking of alginate with divalent cations, in water, at room temperature, to form a hydrogel matrix. EVs are mixed with an alginate solution, the solution is delivered to an already implanted support structure and then solidifies in a short time due to the presence in vivo of physiological concentrations of calcium ions. Alternatively, the solution is delivered to the support structure prior to implantation and solidified in an external solution containing calcium ions. In general, these polymers are at least partially soluble in aqueous solutions, e.g., water, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof. There are many examples of polymers with acidic side groups that can be reacted with cations, e.g., poly(phosphazenes), poly(acrylic acids), and poly(methacrylic acids). Examples of acidic groups include carboxylic acid groups, sulfonic acid groups, and halogenated (preferably fluorinated) alcohol groups. Examples of polymers with basic side groups that can react with anions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinyl imidazole). Polyphosphazenes are polymers with backbones consisting of nitrogen and phosphorous atoms separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two side chains. Polyphosphazenes that can be used have a majority of side chains that are acidic and capable of forming salt bridges with di- or trivalent cations. Examples of acidic side chains are carboxylic acid groups and sulfonic acid groups. Bioerodible polyphosphazenes have at least two differing types of side chains, acidic side groups capable of forming salt bridges with multivalent cations, and side groups that hydrolyze under in vivo conditions, e.g., imidazole groups, amino acid esters, glycerol, and glucosyl. Bioerodible or biodegradable polymers, i.e., polymers that dissolve or degrade within a period that is acceptable in the desired application (usually in vivo therapy), will degrade in less than about five years and most preferably in less than about one year, once exposed to a physiological solution of pH 6-8 having a temperature of between about 25° C. and 38° C. Hydrolysis of the side chain results in erosion of the polymer. Examples of hydrolyzing side chains are unsubstituted and substituted imidazoles and amino acid esters in which the side chain is bonded to the phosphorous atom through an amino linkage. Methods for synthesis and the analysis of various types of polyphosphazenes are described in U.S. Pat. Nos.4,440,921, 4,495,174, and 4,880,622. Methods for the synthesis of the other polymers described above are known to those skilled in the art. See, for example Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz, editor (John Wiley and Sons, New York, N.Y., 1990). Many polymers, such as poly(acrylic acid), alginates, and PLURONICS™, are commercially available. Water soluble polymers with charged side groups are cross-linked by reacting the polymer with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups, or multivalent anions if the polymer has basic side groups. Cations for cross-linking the polymers with acidic side groups to form a hydrogel include divalent and trivalent cations such as copper, calcium, aluminum, magnesium, and strontium. Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels. Anions for cross-linking the polymers to form a hydrogel include divalent and trivalent anions such as low molecular weight dicarboxylate ions, terepthalate ions, sulfate ions, and carbonate ions. Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels, as described with respect to cations. For purposes of preventing the passage of antibodies into the hydrogel, but allowing the entry of nutrients, a useful polymer size in the hydrogel is in the range of between 10,000 D and 18,500 D. Temperature-dependent, or thermosensitive, hydrogels have so-called “reverse gelation” properties, i.e., they are liquids at or below room temperature, and gel when warmed to higher temperatures, e.g., body temperature. Thus, these hydrogels can be easily applied at or below room temperature as a liquid and automatically form a semi-solid gel when warmed to body temperature. As a result, these gels are especially useful when the support structure is first implanted into a patient, and then filled with the hydrogel-EV composition. Examples of such temperature-dependent hydrogels are PLURONICS™ (BASF-Wyandotte), such as polyoxyethylene-polyoxypropylene F-108, F-68, and F-127, poly(N-isopropylacrylamide), and N-isopropylacrylamide copolymers. These copolymers can be manipulated by standard techniques to affect their physical properties such as porosity, rate of degradation, transition temperature and degree of rigidity. For example, the addition of low molecular weight saccharides in the presence and absence of salts affects the lower critical solution temperature (LCST) of typical thermosensitive polymers. In addition, when these gels are prepared at concentrations ranging between 5 and 25% (W/V) by dispersion at 4°C., the viscosity and the gel-sol transition temperature are affected, the gel-sol transition temperature being inversely related to the concentration. U.S. Pat. No.4,188,373 describes using PLURONIC™ polyols in aqueous compositions to provide thermal gelling aqueous systems. U.S. Pat. Nos.4,474,751, '752, '753, and 4,478,822 describe drug delivery systems which utilize thermosetting polyoxyalkylene gels; with these systems, both the gel transition temperature and/or the rigidity of the gel can be modified by adjustment of the pH and/or the ionic strength, as well as by the concentration of the polymer. pH-dependent hydrogels are liquids at, below, or above specific pH values, and gel when exposed to specific pHs, e.g., 7.35 to 7.45, the normal pH range of extracellular fluids within the human body. Thus, these hydrogels can be easily delivered to an implanted support structure as a liquid and automatically form a semi-solid gel when exposed to body pH. Examples of such pH-dependent hydrogels are TETRONICS™ (BASF-Wyandotte) polyoxyethylene-polyoxypropylene polymers of ethylene diamine, poly(diethyl aminoethyl methacrylate-g-ethylene glycol), and poly(2-hydroxymethyl methacrylate). These copolymers can be manipulated by standard techniques to affect their physical properties. Hydrogels that are solidified by either visible or ultraviolet light can be made of macromers including a water soluble region, a biodegradable region, and at least two polymerizable regions as described in U.S. Pat. No.5,410,016. For example, the hydrogel can begin with a biodegradable, polymerizable macromer including a core, an extension on each end of the core, and an end cap on each extension. The core is a hydrophilic polymer, the extensions are biodegradable polymers, and the end caps are oligomers capable of cross- linking the macromers upon exposure to visible or ultraviolet light, e.g., long wavelength ultraviolet light. Examples of such light solidified hydrogels include polyethylene oxide block copolymers, polyethylene glycol polylactic acid copolymers with acrylate end groups and 10K polyethylene glycol-glycolide copolymer capped by an acrylate at both ends. As with the PLURONIC™ hydrogels, the copolymers comprising these hydrogels can be manipulated by standard techniques to modify their physical properties such as rate of degradation, differences in crystallinity, and degree of rigidity. Methods of Treatment Also disclosed is a method of treating a subject with any one of a number of disease states and/or conditions as described herein including osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis, Parkinson’s disease, skeletal muscle or bone in need of repair, cancer, cardiovascular disease, including stroke and other neurological disorders comprising administering to the subject in need an effective amount of a composition containing a population of neural EVs disclosed herein, optionally in combination with at least one additional bioactive agent. In some embodiments, the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, a Parkinson’s-related disorder, Huntington’s disease, prion disease, motor neuron disease (MND), spinocerebellar ataxia (SCA) or spinal muscular atrophy (SMA). The term “stroke” is used to describe a cerebrovascular accident (CVA), cerebrovascular insult (CVI), or brain attack, occurs when poor blood flow to the brain results in cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Both of these types of stroke result in part of the brain not functioning properly. Signs and symptoms of a stroke may include an inability to move or feel on one side of the body, problems understanding or speaking, a sense of spinning, or loss of vision to one side, among others. Signs and symptoms often appear soon after the stroke has occurred. If symptoms last less than one or two hours it is known as a transient ischemic attack. Hemorrhagic strokes may also be associated with a severe headache. The symptoms of a stroke can be permanent. Long term complications of stroke may include pneumonia or loss of bladder control. The main risk factor for stroke is high blood pressure. Other risk factors include tobacco smoking, obesity, high blood cholesterol, diabetes mellitus, previous transient ischemic attack (TIA), and atrial fibrillation, among others. An ischemic stroke is typically caused by blockage of a blood vessel. A hemorrhagic stroke is caused by bleeding either directly into the brain or into the space surrounding the brain. Bleeding may occur due to a brain aneurysm. Both ischemic and hemorrhagic stroke are treated pursuant to the present invention. The term “neurodegenerative disease” is used throughout the specification to describe a disease which is caused by damage to the central nervous system and which damage can be reduced and/or alleviated through transplantation of neural cells according to the present invention to damaged areas of the brain and/or spinal cord of the patient. Exemplary neurodegenerative diseases which may be treated using the neural cells and methods according to the present invention include for example, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, lysosomal storage disease (“white matter disease” or glial/demyelination disease, as described, for example by Folkerth, J. Neuropath. Exp. Neuro., 58, 9, Sep., 1999), Tay Sachs disease (beta hexosamimidase deficiency), other genetic diseases, multiple sclerosis, brain injury or trauma caused by ischemia, accidents, environmental insult, etc., spinal cord damage, ataxia and alcoholism. In addition, the present invention may be used to reduce and/or eliminate the effects on the central nervous system of a stroke or a heart attack in a patient, which is otherwise caused by lack of blood flow or ischemia to a site in the brain of said patient or which has occurred from physical injury to the brain and/or spinal cord. The term neurodegenerative diseases also includes neurodevelopmental disorders including for example, autism and related neurological diseases such as schizophrenia, among numerous others. The term “osteoarthritis” is used to describe a type of joint disease that results from the breakdown of joint cartilage and underlying bone. Osteoarthritis (OA) is caused by aging joints, injury, and obesity. OA symptoms include joint pain and stiffness. Traditional treatment depends on the affected joint, including the hand, wrist, neck, back, knee, and hip, and has involved medication and exercise. If the subject is overweight, weight loss may be recommended to improve OA symptoms. The term “acute respiratory distress syndrome (ARDS)” is used to describe a respiratory condition that causes fluid to build up in the lungs so that oxygen is unable to get to the organs. In ARDS, fluid leaks from small blood vessels and collects in tiny air sacs in the lungs so the air sacs can’t fill with enough air. Because of this, the blood can’t pick up the oxygen it needs to carry to the rest of the body. Organs such as the kidneys and brain often become dysfunctional or may shut down. ARDS is sometimes life-threatening and can get worse quickly. It’s generally treatable, and most people recover, but the therapies are limited and somestimes not effective. Fast diagnosis and treatment are important. ARDS is usually triggered by another health problem, so most people who have it are already in the hospital for something else. Causes of ARDS include sepsis, accidents and breathing in toxins such as dense smoke or chemical fumes. The term “multiple sclerosis” or “MS” is used to describe a potentially disabling disease of the brain and spinal cord (central nervous system). In MS, the immune system attacks the protective sheath (myelin) that covers nerve fibers and causes communication problems between the brain and the rest of the body. Eventually, the disease can cause permanent damage or deterioration of the nerves. Signs and symptoms of MS vary widely and depend on the amount of nerve damage and which nerves are affected. Some people with severe MS may lose the ability to walk independently or at all, while others may experience long periods of remission without any new symptoms. Traditiional treatments can help speed recovery from attacks, modify the course of the disease and manage symptoms, but there is a need for more current therapy regarding same. The term “auto-immune” disease is used to describe disease in which a subject’s own immune system produces an abnormal immune response to a normal organ or tissue of a subject. Exemplary auto-immune diseases which can be treated using the extracellular vesicles (EVs) pursuant to the present invention include rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticarial, Sjogren’s disease, autoimmune-related Type 1 diabetes, rheumatoid arthritis (RA), psoriasis/psoriatic arthritis, multiple sclerosis, inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, Addison’s disease, Grave’s disease, Hashimoto’s thyroiditis, Myasthenia gravis, autoimmune vasculitis, pernicious anemia and celiac disease. The herein disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Methods of treating subjects involve administration of a pharmaceutical composition comprising an effective amount of EVs described herein and optionally at least one additional bioactive (e.g. an agent which is useful in the treatment of osteoarthritis, acute respiratory distress syndrome (ARDS), multiple sclerosis, Parkinson’s disease, skeletal muscle repair, bone repair, cancer, cardiovascular diseases, and other neurological disorders, among others) agent and otherwise as described herein. For example, the compositions could be formulated so that a therapeutically effective dosage of between about 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg of patient/day or in some embodiments, greater than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/kg of the disclosed EVs can be administered to a patient receiving these compositions. The dose of EVs administered to a subject can be less than 10 μg, less than 25 μg, less than 50 μg, less than 75 μg, less than 0.10 mg, less than 0.25 mg, less than 0.5 mg, less than 1 mg, less than 2.5 mg, less than 5 mg, less than 10 mg, less than 15 mg, less than 20 mg, less than 50 mg, less than 75 mg, less than 100 mg, less than 500 mg, less than 750 mg, less than 1 g or more than 1 g. Administration may be by numerous routes of administration, but intravenous, intrathecal, intranasal and/or intracerebrospinal fluid are often used as preferred routes of administration. In some embodiments, the disclosed EVs are administered within 24 hours after a stroke or trauma. However, in some embodiments, the EVs are administered at least 1, 2, 3, or 4 weeks after a stroke or trauma, including a heart attack (myocardial infarction). In some embodiments, the disclosed EVs are administered in multiple doses 1, 2, 3, or more days apart. In some cases, such as cases of osteoarthritis, neurodegenerative disease, ARDS, multiple sclerosis, cancer, muscle repair, bone repair and the like, the EVs are administered continuously (e.g., once every 1, 2, 3, or 4 weeks or more often) over the course of the disease. EVs may be loaded with small molecules, antisense oligonucleotides, siRNAs, peptides, proteins or antibodies that target, peptides or peptide translation products which are involved in therapeutic, including neurodegenerative processes. In certain embodiments, the disclosed EVs are loaded with additional bioactive agents or are co-administered with additional bioactive agents, especially agents which are useful in the treatment of cancer and/or neurodegenerative diseases. The term “coadministered”, “coadministration” or “combination therapy” is used to describe a therapy in which at least two active compounds/compositions in effective amounts are used to treat neural injury and/or a neurodegenerative disease. Although the term co- administration preferably includes the administration of EVs and at least one additional active compound to the subject at the same time, it is not necessary that the compounds/compositions be administered to the patient simultaneously, only that effective amounts of the individual compounds/compositions be present in the patient at the same time. Thus, the term co-administration includes an administration in which the EVs and the bioactive agent(s) are administered at approximately the same time (contemporaneously), or from about one to several minutes to about eight hours, about 30 minutes to about 6 hours, about an hour to about 4 hours, or even much earlier than the other compound/composition as otherwise described herein including up to a day or substantially more. Agents which may be loaded or coadministered along with EVs may include, for example aricept, namenda, donepezil, excelon, razadyne, glantamine, rivastigmine, memantine, ergoloid, namzaric and mixtures thereof for Alzheimer’s disease, biperiden, apomorphine, trihexyphenidyl, carbidopa/levodopa, rasagline, belladona, levodopa, benztropine, entacapone, selegiline, rivastigmine, pramipexole, rotigotine, bromocriptine, pergolide, ropinirole, carbidopa/entacapone/levodopa, amantadine, tolcopone, trihexiphenidyl and mixtures thereof, for Parkinson’s disease, tetrabenazine, haloperidol, chlorpromazine, olanzapine, fluoxetine, sertraline, nortriptyline, benzodiazpines, paroxetine, venlafaxin, beta- blockers, lithium, valproate, carbamazepine, botulinum toxin and mixtures thereof for the treatment of Huntington’s disease, anticholinergic drugs, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, pain medications, stimulants and mixtures thereof for the treatment of motor neuron disease, selective serotonin reuptake inhibitors (SSRI’s), selective norepinephrine-serotoning reuptake inhibitors (SNRI’s), acetazolamide, baclofen, clonazepam, flunarizine, gabapentin, meclizine, memantine, ondansetron, scopolamine, modafinil, armodafinil, amantadine, atomoxetine, buproprion, carnitine, creatine, modafinil, armodafinil, pyrudistigmine, selegiline, venlafaxine, desvenlafaxine, buspirone, riluzole, verenicline, memantine, baclofen, tizanidine, cymbalta, lyrica, acetazolamide, carbamazepine, clonazepam, isoniazid, droxidopa, ephedrine, fludrocortisones, midodrine, levodopa, pramipexole, fluoxetine, n-acetylcysteine, baclofen, dantrolene sodium, diazepam, ropinirole, tizanidine, trihexylphenidyl, clonazepine, flunarazine, levetiracetam, primidone, topiramate, valproic acid, phenytoin, 4-aminopyridine and mixtures thereof for the treatment of spinocerebellar ataxia and riluzole for the treatment of spinal muscular atrophy. Agents for the treatment of stroke include salicylates, such as aspirin, a thrombolytic agent (alteplase) and a platelet aggregation inhibitor (clopidogrel), among others. Anticancer agents may also be used in conjunction with EVs according to the present invention. The term “anti-cancer compound”, “anti-cancer drug” or “anti-cancer agent” is used to describe any compound (including its derivatives) which may be used to treat cancer. The “anti-cancer compound”, “anti-cancer drug” or “anti-cancer agent” can be an anticancer agent. In many instances, the co-administration of another anti-cancer compound according to the present invention results in a synergistic anti-cancer effect. Exemplary anti-cancer compounds for co-administration with formulations according to the present invention include anti-metabolites agents which are broadly characterized as antimetabolites, inhibitors of topoisomerase I and II, alkylating agents and microtubule inhibitors (e.g., taxol), as well as tyrosine kinase inhibitors (e.g., surafenib), EGF kinase inhibitors (e.g., tarceva or erlotinib) and tyrosine kinase inhibitors or ABL kinase inhibitors (e.g. imatinib). Anti-cancer compounds for co-administration include, for example, agent(s) which may be co-administered with EVs according to the present invention in the treatment of cancer. These agents include chemotherapeutic agents and include one or more members selected from the group consisting of everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101 , pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111 , 131-I-TM-601 , ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001 , IPdR1 KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311 , romidepsin, ADS- 100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, liposomal doxorubicin, 5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901 , AZD-6244, capecitabine, L-Glutamic acid, N -[4-[2-(2- amino-4,7-dihydro-4-oxo-1 H - pyrrolo[2,3- d ]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11 , CHIR-258,); 3-[5-(methylsulfonylpiperadinemethyl)- indolylj-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D- Ser(Bu t ) 6 ,Azgly 10 ] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t )-Leu-Arg-Pro- Azgly-NH 2 acetate [C59H84N18Oi4 -(C2H4O2)X where x = 1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK- 165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI- 166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951 , aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette- Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291 , squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, cremophor-free paclitaxel, docetaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA- 923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR- 3339, ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP- 23573, RAD001 , ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa- 2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11 , dexrazoxane, alemtuzumab, all- transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa, darbepoetin alfa, ipilimumab, nivolomuab, pembrolizumab, dabrafenib, trametinib and vemurafenib among others. The activities of EVs described herein can be evaluated by methods known in the art. The amount of EVs required for use in treatment can vary not only with the particular cell from which the EVs are prepared, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and can be ultimately at the discretion of the attendant physician or clinician. In general, however, a dose can be in the range of from about 0.01 mg/kg to about 10 mg/kg of body weight per day. Identifying EVs useful in the present methods for treating a spinal cord injury, stroke, traumatic brain injury and/or a neurodegenerative disease which occurs by modulating the activity and expression of a disease-related protein and biologically active fragments thereof can be made by screening EV activity in any of a variety of screening techniques. The screening can be made for whole EVs or their contents. Fragments employed in such screening tests may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The blocking or reduction of biological activity or the formation of binding complexes between the disease-related protein, the EVs and/or one or more components of the EVs may be measured by methods available in the art. A number of embodiments of the invention are described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the claims which follow. MSCs-based therapy is considered as a promising approach for a number of disease states and conditions because of their ability to target major aspects of the pathophysiology associated with these disease states. MSC-EVs are expected to act similarly by way of effectiveness but with fewer of the side effects of MSC therapy, such as tumorogenicity. Experimental Overview Mesenchymal stromal cells (MSCs) are being explored as an immunomodulatory therapy to treat immune diseases such as osteoarthritis, multiple sclerosis, and Parkinson’s disease (1, 2); however, cell therapies face challenges associated with manufacturing consistent, high quality products. Although promising as an allogeneic ‘off-the-shelf’ therapy, cryopreserved and thawed MSCs have diminished efficacy and require a recovery period at point of care when compared to fresh, non-thawed MSCs (3). Transplantation of any cell therapy, even MSCs, can also raise safety concerns in the event of uncontrolled differentiation into undesired tissue or promotion of tumor growth following engraftment (4- 6). Cell-free extracellular vesicle (EV) preparations from MSC cultures that possess similar MSC therapeutic function are a potential alternative to MSC therapy. Secreted factors from MSCs are responsible for much of their regenerative and immunomodulatory functions (7). MSC immunomodulation via secreted factors has been directly linked to T cell suppression, as shown in a transwell system that prevents cell-cell contact between MSCs and T cells (8)(9, 10). MSC-derived extracellular vesicles (MSC-EVs) are secreted nanoscale vesicles that participate in intercellular signaling through transference of bioactive molecules including RNA, proteins, and lipids (11). MSC-EVs do not divide, engraft, or dynamically respond to their environment like MSCs, thus addressing concerns with tumorigenicity and ectopic tissue growth (5, 6). MSC-EVs have demonstrated preliminary evidence of regenerative effects ranging from recovery from myocardial ischemia and reperfusion injury (12), stroke (13), gentamicin induced acute kidney injury (14), and allogeneic skin grafts (15) and MSC-EV clinical trials are being conducted for a debilitating skin disease The injury microenvironment is often characterized by inflammation, involving local hypoxia, acidosis, as well as the presence of cytokines such as Tumor Necrosis Factor alpha (TNF-α) and Interferon gamma (IFN-γ) (16-18). MSCs are known to respond to priming by inflammatory environments by switching to an “activated” immunosuppressive phenotype. This often involves upregulating expression of regenerative and anti-inflammatory factors such as Vascular Endothelial Growth Factor, Indolamine 2,3-Dioxygenase, Transforming Growth Factor Beta, and Prostaglandin E2 (19). More recently, inflammatory and hypoxic priming have been shown to increase the potency of MSC-EV immunomodulation (20-22). EVs derived from MSCs primed with hypoxia were more effective than EVs from non- primed MSCs in inducing macrophage proliferation and type 2 macrophage polarization (20). TGF-β and IFN-γ primed MSCs produced EVs that were more effective in inducing Treg formation than those from resting MSCs (22). This suggests that MSC-EVs are involved in the MSC response to inflammatory priming. However, acidic priming has not been investigated in this manner, and different treatments have not been compared within the same study. To determine the effects of manufacturing conditions (i.e. priming) on MSC-EV quality and their potential role in a therapeutically-relevant function (T cell suppression) we performed the following studies. First, we examined the effects of hypoxia, acidosis, and inflammatory cytokines on MSC-EV biogenesis and secretion as compared to unprimed, serum-free MSC culture conditions. We then comprehensively profiled the activation of different T cell subsets treated with both MSCs and MSC-EVs. We found that inflammatory cytokine priming increased EV size, decreased relative expression of a panel of surface markers. Meanwhile, acidosis and hypoxia increased EV yield while having little effect on their surface markers. Additionally, while MSCs in direct contact demonstrated greater suppression of effector T cells than MSC-EVs, MSC-EVs derived from MSCs primed with acidosis induced the formation of Tregs while other MSC-EV groups had no significant effect on T cell activation. Therefore, precise control and monitoring of the pH during MSC-EV manufacturing should be further explored as a means to enhance MSC-EV immunomodulatory function. EXAMPLES Methods Cell culture and priming Human female wharton’s jelly MSCs (Lifeline Cell Technologies) referred to as MSC here, were plated at 5000 cells/cm2 on tissue culture flasks in complete medium (Alpha- Minimum Essential Medium (Gibco), 10% defined fetal bovine serum (Hyclone), 2 mM L- glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco) and allowed to grow to 80% confluence (20,000–25,000 cells/cm2). They were harvested using 0.05% trypsin (Gibco) and replated at 5000 cells/cm2. All proliferation cultures were maintained at 37°C and at 5% CO2. All cells used in experiments had undergone fewer than 10 passages and had over 90% viability at harvest as assessed by Trypan blue staining. Several environments were used to prime MSCs when they reached 80% confluence (FIGURE S1). Metabolic acidosis was induced through the addition of HCl to complete media to lower the pH to 7.1 ± 0.05 (23). These MSCs were termed LPH-MSCs, with their EVs being LPH-EV. A hypoxic environment was created by placing the cell culture vessels in a hypoxia incubator chamber (STEMCELL Technologies, Cambridge MA), which was then filled with a gas mixture containing 2% O2, 5% CO2, and 93% N2 (Airgas) for 5 minutes at 2 psi according to the manufacturer’s recommendation (24, 25). The MSCs undergoing this priming were termed LO2-MSCs, and their EVs were LO2-EV. An inflammatory environment was created by adding the cytokines TNF-α and IFN-γ (Sigma) to the medium at 15 and 20 ng/mL, respectively. These MSCs were termed INF-MSCs, with their EVs being INF-EV. These environments were used separately to precondition MSCs for 48 hours at 37°C prior to receiving exosome isolation media or being placed in co-culture with human Peripheral Blood Mononuclear Cells (PBMCs). A final group of MSCs remained in complete growth medium at pH 7.4 and 20% oxygen as described above for 48 hours. The normal culture MSCs and their EVs were NC-MSC and NC-EV, respectively. PBMCs (STEMCELL Technologies, Cambridge MA) were thawed into RPMI media (RPMI 1640, 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin) and cultured for 16hrs at 37°C and at 5% CO2 prior to use in experiments. Exosome isolation and characterization After priming, MSCs were rinsed twice with PBS before adding fresh serum free medium (Alpha-Minimum Essential Medium (Gibco), 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin (all from Gibco/Invitrogen)) and incubating cultures for 24 hours. The resulting conditioned media were collected and passed through 0.22 μm filters to remove cells and large debris. The media were subjected to ultrafiltration with a 100kDa MWCO (Amicon, Millipore-Sigma) at 4000g for 10 minutes as we previously published (26). The EVs remained on top of the filter and were then washed twice with PBS +/+ (Thermo Fisher Scientific, Waltham, MA) at 2000g for 10 minutes. The EVs in PBS+/+ were then collected, aliquoted, and frozen at -20°C. For each EV isolation, nanoparticle tracking analysis (NTA) was performed using a Nanosight NS3200 (Nanosight, Salisbury UK) according to the manufacturer’s recommendations. Briefly, aliquots of EV suspensions were thawed at room temperature and diluted to 107-109 particles/mL with the same lot of PBS +/+ the EVs were isolated in. A minimum of three samples and five one-minute videos were recorded for each exosome isolation. All videos were captured at the same camera level and analyzed with the same detection threshold. The size and size distribution of vesicles was further verified via Dynamic Light Scattering using a Malvern Zetasizer Nano ZS Analyzer (Malvern Instruments, Malvern, UK). Samples were diluted to a total vesicle concentration of approximately 2x108 vesicles/ml in 0.22μm filtered Phosphate Buffered Saline containing Calcium and Magnesium, pH 7.4 prior to measurements. Disposable polystyrene cuvettes were rinsed with 1mL of filtered PBS +/+ prior to adding sample. Measurements were taken using cuvette with 800uL of prepared sample. EV surface marker characterization was performed using the MACSPLEX Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s directions. Briefly, an equal number of exosomes as determined by NTA were analyzed from each isolation in triplicate. Flow cytometry analysis was performed using a CytoFLEX S (Beckman Coulter, Hialeah, Florida) alongside bead only controls, with FlowJo being used for data analysis. Data was processed with background subtraction and normalized to the median of the average value of CD9, CD63, and CD81 for each sample (27). The data was transformed to be a percentage of the difference between the maximum and minimum relative expression of a marker. Principal component analysis (PCA) was performed on the transformed data using JMP (SAS Institute, Cary NC). EV uptake assay PBMCs were added to each well at 500,000 cells per well in 48 well plates. Stimulating anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA) were then added at 500,000 per well. EVs were stained with CFSE (Thermo Fisher Scientific, Waltham, MA) following a protocol modified from Morales-Kastresana (28).40 μM CFSE in PBS +/+ was added to an equivalent volume of EVs and incubated for 2 hours in the dark at 37°C. Excess dye was quenched with an equivalent volume of 0.1% BSA, and the whole mixture was rinsed with PBS +/+ and concentrated via ultrafiltration as in the initial EV isolation.109 CFSE-EVs were then added to the appropriate wells. The assays took place in complete RPMI medium formulated as above, but with EV- depleted FBS. EVs were depleted by centrifuging FBS at 100,000g for 1 hour at 4°C (Sorvall WX Ultra 80, Thermo Fisher Scientific, Waltham, MA) and using the supernatant (29). The cultures incubated for 24 hours at 37°C, 5% CO2 for the duration of the experiments. Following incubation, the PBMCs were harvested and stained for flow cytometry using Pacific Blue anti-CD4, APC anti-CD8, Brilliant Violet 711 anti-CD25, and PE anti- FOXP3. Antibodies and clones are listed in Table S2. PBMCs were first washed, then stained with Zombie Yellow viability dye, blocked with 2% FBS and Fc receptors blocked with Trustain FcX. The PBMCs were then stained for CD4, CD8, and CD25 as appropriate at room temperature in the dark for 30 minutes and fixed in FOXP3 TrueNuclear fix before storing overnight in the dark at 4C. PBMCs were permeabilized with TrueNuclear permeabilization buffer and stained with PE anti-FOXP3 according to the manufacturer’s directions. Samples were resuspended in 2% FBS at 4C in the dark for up to 2 days before flow analysis. All antibodies and reagents were from Biolegend (San Diego, CA) unless otherwise specified and were used at previously titrated optimal concentrations. Immunomodulation assay MSCs were plated at 20,000 cells/cm2 in 48 well plates in complete medium and allowed to adhere for 24 hours. The cells were then subjected to priming as previously described, followed by two PBS -/- washes. PBMCs were labeled with CFSE (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions and 500,000 PBMCs were added to each well. After the addition of PBMCs, stimulating anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA) were added at 500,000 each per well, and 109 EVs were added to the appropriate wells. The assays took place in complete RPMI medium formulated as above, but with EV- depleted FBS. EVs were depleted by centrifuging FBS at 100,000g for 1 hour at 4°C (Sorvall WX Ultra 80, Thermo Fisher Scientific, Waltham, MA) and using the supernatant (Li 2017).The cultures incubated for 5 days at 37°C, 5% CO2 for the duration of the experiments. Following incubation, the PBMCs were harvested and analyzed by flow cytometry using one of two panels of conjugated antibodies. Panel 1 was composed of Pacific Blue anti- CD4, APC anti-CD8, Brilliant Violet 711 anti-CD25, and PE anti-FOXP3. Panel 2 included Pacific Blue anti-CD4, APC anti-CD8, PE anti-IFN-γ, and Brilliant Violet 711 anti-TNF-α. Antibodies and clones are listed in Table S2. PBMCs were first washed, then stained with Zombie Yellow viability dye, blocked with 2% FBS and FC receptors blocked with Trustain FcX. The PBMCs were then stained for CD4, CD8, and CD25 as appropriate at room temperature in the dark for 30 minutes and fixed in 4% PFA for Panel 2 or FOXP3 TrueNuclear fix for Panel 1 before storing overnight in the dark at 4°C. Panel 1 was then permeabilized with TrueNuclear permeabilization buffer and stained for FOXP3 according to the manufacturer’s directions. Panel 2 was permeabilized with Permwash (BD Biosciences) and stained for IFN-γ and TNF-α. Samples were resuspended in 2% FBS at 4°C in the dark for up to 2 days before flow analysis. All antibodies and reagents were from Biolegend (San Diego, CA) unless otherwise specified and were used at previously titrated optimal concentrations. Flow Cytometry All flow analysis was performed using a CytoFLEX S (Beckman Coulter, Hialeah, Florida), with 20,000 events collected per sample for the uptake and immunosuppression assays (S3). All data was analyzed using FlowJo software (Treestar, Inc., Ashland, Oregon). Cellular debris, activating beads, and doublets were gated out via scatter properties. Single-stain controls were used to generate compensation matrices, and Fluorescence-minus one controls were used to determine positive populations of CD4, CD8, CD25, TNF-α, IFN-γ, and FOXP3. Example scatter plots for the gating strategy are shown in FIGURE S3. All activation parameters of T cells including CD25, FOXP3, TNF-α, and IFN-γ expression were normalized according to the formula below, so that fully activated samples have an average value of 100 and fully suppressed samples have an average value of 0 (30).
Figure imgf000045_0001
T cell proliferation was calculated according to the formula below, where MI is the median fluorescence intensity of CFSE stained samples, and PS is the proliferation score (31):
Figure imgf000045_0002
Statistics All data is expressed as mean +/- SEM, with all experiments performed in triplicate. All statistical tests were one-way ANOVA against controls unless stated otherwise with Dunnett’s post-hoc test using Prism (Graphpad, San Diego CA). Results Effects of priming on MSC-EV yield and size distributions NC-EV, LO2-EV, and LPH-EV had unimodal size distributions centered at approximately 100nm (FIGURE 1A-C). The size distribution of INF-EV was bimodal, with one population centered at 100 nm as well as a larger diameter population between 150 and 400 nm (FIGURE 1D). INF-EVs had a significantly higher mean diameter than NC-EVs (p<0.0001), while LPH-EV and LO2-EV did not differ in size from the control (FIGURE 1E). Dynamic Light Scattering (DLS) analysis was used to confirm the presence of a larger population in the INF-EV group and generated similar results to NTA, despite interference from the bimodal distribution (S4). NTA analysis also revealed significantly higher EV production per cell from LO2-MSC (p<0.0001) and LPH-EV (p = 0.0028) compared to NC- MSC (FIGURE 1F). Overall, hypoxic and acidosis priming increased EV release, while only inflammatory priming affected EV size. Inflammatory priming affects MSC-EV surface marker expression Surface marker characterization via MACSPLEX analysis revealed significant changes in the relative expression of EV surface markers in the preconditioned groups compared to the NC-EV group (FIGURE 2A). It is important to note that the MACSPLEX assay does not differentiate between higher expression of markers on each EV and a greater percentage of total EVs expressing those markers. CD9, CD63, and CD81 exosome markers were present in all EV groups, although to varying degrees. INF-EV had elevated expression of CD63 (p < 0.0001), while expression of CD9 (p < 0.0001) and CD81 (p < 0.0001) were decreased. Additionally, INF-EV had greatly decreased expression of CD29 (Integrin beta-1, p < 0.0001), CD44 (p < 0.0001), CD49e (Integrin alpha-5, p < 0.0001), CD105 (Endoglin, p < 0.0001), and melanoma-associated chondroitin sulfate proteoglycan (MCSP, p < 0.0001). In contrast, LO2-EV had significantly elevated expression of MCSP (p = 0.0478), CD44 (p = 0.0201), and CD29 (p = 0.0031) compared to NC-EV. LPH-EV did not have significantly different expression of any surface marker compared to NC-EV. PCA enabled visualization of the high dimensional MACSPLEX data to better discriminate differences in the overall expression of surface markers with priming (FIGURE 2B). The first and second principal components (PC1, PC2) were responsible for 28% and 10% of the variance in the data set, respectively. INF-EV separated from the other groups along PC1, and INF-EV’s mean value of PC1 was significantly lower than that of NC-EV (p<0.0001, FIGURE 2C). Additionally, LO2-EV had a significantly higher mean value of PC2 than NC-EV (p = 0.0018, FIGURE 2C). The largest contributors to PC1 were cell adhesion markers, while PC2 was largely made up of cell membrane and immune signaling proteins (Table S5). Exosome Uptake CFSE-labeled exosome uptake was determined by %CFSE+ cells from T cell sub- groups at 24 hours post-treatment. LPH-EV had the highest uptake of all groups (FIGURE 3). Treatment with LPH-EV led to significantly greater percent CFSE+ cells than the untreated controls for helper T cells (p < 0.05, FIGURE 3A), cytotoxic T cells (p < 0.05, FIGURE 3B), activated helper T cells, (p < 0.05, FIGURE 3C), activated cytotoxic T cells (p <0.01, FIGURE 3D), CD4+ Tregs (FIGURE 3E, p < 0.05), and CD8+ Tregs (FIGURE 3F, p < 0.01). No EV group had significant uptake by CD4-/CD8- cells (FIGURE 3G). LPH-EV was the only EV group to have significant rates of uptake by T cells. Suppression of T cell activation by MSCs and MSC-EVs PBMCs were subjected to flow analysis to determine T cell activation after 5 days incubation with either MSCs or MSC-EVs. Proliferation of CD8+ cells after 5 days was significantly decreased when co-cultured with INF-MSC (p = 0.0012), LO2-MSC (p < 0.0001), LPH-MSC (p < 0.0001), and NC-MSC (p < 0.001) (FIGURE 4B). While the MSC groups trended towards decreased CD4+ proliferation as well, this was not significant (FIGURE 4A). There was no effect on T cell proliferation by any MSC-EV group. CD25 expression in helper T cells was significantly reduced by INF-MSC (p<0.0001), LO2-MSC (p < 0.001), LPH-MSC (p < 0.01), and NC-MSC (p < 0.001) (FIGURE 5A). CD25 expression was similarly reduced in cytotoxic T cells exposed to INF-MSC (p < 0.05), LO2- MSC (p < 0.05), LPH-MSC (p < 0.05), and NC-MSC (p < 0.05) (FIGURE 5B). The frequency of CD8+ Tregs was increased by LPH-EV (p < 0.05) cells and trended towards a significant increase in CD4+ Tregs (p = 0.061), while there was no significant change in Treg composition for the MSC only groups (FIGURE 5C-D). To determine whether MSC-EVs exhibited time dependent changes in T cell activation, we profiled T cell subset activation following 24 hours of EV treatment and found no significant differences (S5). All MSC groups trended towards a decrease in TNF-α and IFN-γ expression in both helper T cells and cytotoxic T cells, but the only significant reductions were by LPH-MSC (p < 0.05) and NC- MSC (p < 0.05) on IFN-γ in cytotoxic T cells (FIGURE 5E-H). Overall, MSC co-culture resulted in significant reduction of T cell activation as measured by proliferation, CD25 expression, and cytokine expression, regardless of priming condition. On the other hand, MSC-EVs had a less pronounced effect with the exception of increased Treg frequency due to LPH-EV treatment. Discussion Although significant evidence exists for the immunomodulatory roles of MSCs and MSC-EVs, the mechanisms of action governing these therapeutic functions are still largely unknown. There is increasing evidence that the MSC microenvironment has substantial effects on the function of their released EVs in vitro (20, 21). We investigated the effects of different aspects of the injury microenvironment on MSCs and their released EVs. We found that while the yield, size, and surface marker composition of released EVs varied substantially with priming treatments, only those from acidosis primed MSCs had any immunomodulatory effects. These results further emphasize the effect of disease-relevant microenvironment cues on MSCs and could inform the development of future MSC-EV therapeutics. To our knowledge, this is the first study showing changes in MSC-EV immunomodulation through acidic priming. However, the effect of environmental pH on MSCs has previously been investigated regarding their interactions with various cancers. Tumors are known to create an immunosuppressive microenvironment, and it is hypothesized that MSCs might be involved in this phenomenon (32-34). MSCs cultured in an acidic environment enhanced in vivo melanoma growth, partly through their increased expression of TGF-β (35). TGF-β is a potent growth factor and has been shown to induce the maturation of Tregs (36). MSC-EV have been shown to associate with TGF-β, and EVs may bind TGF-β on their surface (37, 38). Acidosis primed MSCs upregulated osteosarcoma expression of CXCL5 and CCL5 (39). These chemokines have also been implicated in the formation and recruitment of Tregs, respectively (40-42). MSC-EV may play a role in the MSC driven immunosuppressive role since we observed increased Tregs in PBMC cultures treated with acidic preconditioned MSC-EVs. EV biogenesis and release is known to take place through several mechanisms, including sphingomyelinases (43). Sphingomyelinase activity has been shown to be increased in an acidic environment (44, 45). While increased EV release from MSCs under hypoxic and inflammatory conditions have previously been demonstrated, to our knowledge acidosis has not been previously shown to increase EV release from MSCs (20, 21). Our study describes the first instance of increased EV release by MSCs in an acidic environment, as well as further demonstrating the effect of other priming strategies such as hypoxia and cytokine stimulation. It was evident that inflammatory priming of MSCs resulted in production of distinct EV populations from other priming conditions. NC-EV, LPH-EV, and LO2-EV had average sizes consistent with exosomes, which range from 50-150 nm. By comparison, INF-EVs exhibited a biomodal size distribution that included a population with size range similar to microvesicles, which bud from the plasma membrane and are between 100nm and 1μm in diameter (43, 46). EVs from both IFN-γ and TNF-α/IFN-γ preconditioned MSCs have previously been observed with larger size distributions tending towards the microvesicle size range (21, 47). The biogenesis of microvesicles differs from that of exosomes, and their immunomodulatory potency has been shown to be less than that of exosomes (48). Additionally, we observed decreases in adhesion-related markers of INF-EV compared to NC-EV. This included integrins beta-1 and alpha-5, as well as endoglin, which is an auxiliary receptor for TGF-β (49). The broad reduction in cell adhesion marker expression of INF-EV was especially evident when viewing the first principal component of the PCA, which was largely composed of these markers (Supplemental Figure 5). Although the MACSPLEX assay lacks a reference standard for absolute quantification, results are comparable within an experiment using equal numbers of EVs (27). the dimensionality reduction of PCA aids in drawing out overall trends in surface marker expression. Cellular recognition and uptake of EVs is regulated in part by their surface markers, which can vary based on the environment of their source cells (21). If INF-EV contain multiple heterogeneous vesicle populations as indicated here, this could dilute the overall potency, possibly explaining the lack of immunomodulation by INF-EVs compared to other studies. We observed the highest uptake of MSC-EVs in T cells compared to non-T cells across all EV groups (Figure 3). Our results agree with other studies in which MSC-EVs delivered to PBMCs consistently associated with T cells compared to macrophages or NK cells (50). Our study found very little MSC-EV uptake by CD4-/CD8- cells. However, MSC- EVs were reported to be primarily taken up by monocytes (21) (47). Previous studies investigated MSC-EV uptake by subsets of immune cells but not the differential uptake by effector and regulatory T cells. The greater uptake of LPH-EV by Tregs may be related to their subsequent increased frequency. The decreased expression of cell signaling and adhesion markers in INF-EV may contribute to the observed low uptake for that priming condition. The inventors observed significantly greater suppression of T cells when they were cultured in direct contact with MSCs versus indirect culture using MSC-EVs. This is in line with previous studies, which have indicated that MSCs interact with T cells differently than their isolated EVs. EV dosing may play a role in this, although the dose used in our study, 2000 EV/PBMC, is higher than studies reporting T cell suppression by MSC-EVs (50, 51). As we observed, MSCs are more effective than their EVs alone at inhibiting T cell proliferation (21, 48, 50, 51). However, inhibition of EV release impairs the suppression of T cell proliferation by MSC co-culture, so EVs likely play some role in this process (21). Isolated MSC-EVs also induce Treg formation, while their source cells do not (48, 50), which occurred for the LPH-EV group in this study. Interestingly, there were no significant differences in T cell suppression between any of the direct contact MSC/PBMC co-culture priming conditions. As activated PBMCs create an inflammatory environment of their own, shown here by their production of TNF-α and IFN-γ (Figure 5), it could be that any effects from the initial priming conditions prior to co-culture were overridden by cytokines and other signals produced by the PBMCs. This study was focused on differences in the release, uptake, and surface composition of EVs; however, we did not look at other aspects of EV potency such as their intravesicular nucleic acid content. MSC-EVs can contain a wide range of micro RNAs (miRNAs), many of which are associated with angiogenesis and tissue remodeling (52). Certain miRNAs have been shown to have increased frequency in EVs derived from MSCs primed with inflammation-relevant signals. When MSCs were primed with TNF-α and IFN-γ, miRNA- 155, previously implicated in immune modulation, was increased in their EVs compared to non-primed control MSC-EVs (21, 53). Similarly, IL-1β priming of MSCs upregulated miR- 146a, which has previously been shown to regulate the T cell response through the NF-NB pathway (54, 55). MSCs cultured in hypoxia have increased miR-223, miR-146b, miR126, and miR199a (20). Of these, miR-223 is involved in driving anti-inflammatory macrophage polarization, and miR-146b has been shown to monocyte inflammation (56). Further studies will determine whether upregulation of Tregs by LPH-EVs is due to change in their miRNA content. EV biomanufacturing remains an emerging field with enormous potential; however, the lack of standardized methods for characterization and processing further compounds functional heterogeneity observed for MSCs. Differences in manufacturing conditions such as harvesting, isolation, and purification can result in loss of EV subpopulations, exposure of EVs to different stresses, and result in final products with significant heterogeneity (57). For example, EVs frozen at -80°C after isolation and thawed before use have been found to decrease in immunomodulatory potency (48). It will be important to optimize these processing methods to enable proper assessment of EV properties and comparison of EV studies. Contributors to EV release and function are still being explored. This study adds to a growing body of evidence demonstrating that EV immunosuppressive function can be enhanced by priming MSCs with inflammation-relevant microenvironment signals. However, there is not yet a consensus on which signals significantly impact EV function, and the mechanisms of action through which MSC-EVs exert their potential therapeutic effects. Comparison of the miRNA, protein, and lipid cargo of EVs from different priming methods could be a future avenue to examine any possible mechanisms for their variable immunosuppressive potential, as well as comparison of MSC-EVs produced by MSCs derived from different donors and tissue sources. Here, MSCs exposed to an acidic environment produce EVs with anti-inflammatory function (i.e. promotion of Tregs), which could hold great potential to both our understanding of EVs and their eventual clinical translation. References 1. 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Claims

Claims: 1. A population of extracellular vesicles (EVs) obtained by priming adherent cells under hypoxic, low pH or inflammatory conditions for a period ranging from one minute to 72 hours followed by growing the primed cells in growth media for a period ranging from 12 hours to 6 days or more, collecting and filtering the media to remove cells and large debris from the extracellular vesicles in the medium, filtering the media containing extracellular vesicles using ultrafiltration and subsequently washing and collecting the vesicles.
2. The population of extracellular vesicles according to claim 1 wherein the collected vesicles are aliquoted and frozen to be used subsequently.
3. The population of extracellular vesicles according to claim 1 wherein the adherent cells are mesenchymal stem cells (MSCs), pericytes, fibroblasts or immortalized cells lines.
4. The population of extracellular vesicles according to any of claims 1-3 wherein said inherent cells are mesenchymal stem cells.
5. The population of extracellular vesicles according to any of claim 1-3 wherein said adherent cells are immortalized cells lines.
6. The population of extracellular vesicles according to any of claims 1-3 wherein immortalized cells lines are HEK-293T cells.
7. The population of extracellular vesicles according to any of claims 1-6 wherein said adherent cells are primed under hypoxic conditions wherein the level of O2 in the atmosphere in which the cells are primed ranges from 0.5-21% oxygen, more often about 0.75% to 15% oxygen, often about 1.0% to 10% oxygen of the total volume of gasses in the atmosphere.
8. The population of extracellular vesicles according to any of claims 1-6 wherein said adherent cells are primed in media under acidic conditions at a pH ranging from 6.0 to 7.35 or 6.9-7.3. 9. The population of extracellular vesicles according to claim 8 wherein said adherent cells are primed in media under acidic conditions at a pH ranging from 6.
9-7.3.
10. The population of extracellular vesicles according to any of claims 1-6 wherein said cells are primed in inflammatory cytokines at effective concentrations.
11. The population of extracellular vesicles according to claim 10 wherein said cytokines are selected from the group consisting of interferon type I, including IFN-α, IFN-β, IFN-ε IFN-κ, IFN-δ, IFN-τ, IFN-ω and IFN-ν, interleukins 1, 1α, 1β, and 2-36 and/or tumor necrosis factors, TNF 1-19 and 13B at effective concentrations ranging from 1 ng/ml to 100 ng/ml.
12. The population of extracellular vesicles according to any of claims 7-11 wherein said primed adherent cells are grown in culture media for a time sufficient to release extracellular vesicles into said culture medium to provide EV-containing culture medium.
13. The population of extracellular vesicles according to claim 12 wherein said EV- containing medium is collected and filtered to remove cells and large debris from the extracellular vesicles in the medium to produce an EV-rich filtered sample.
14. The population of extracellular vesicles according to claim 13 wherein said EV-rich sample is further subjected to an ultrafiltration step.
15. The population of extracellular vesicles according to claim 14 wherein said sample obtained by ultrafiltration is thereafter washed and collected.
16. The population of extracellular vesicles according to claim 15 wherein said sample is frozen.
17. The population of extracellular vesicles according to claim 15 which is combined with a pharmaceutically acceptable carrier or additive.
18. The population of extracellular vesicles according to claim 16 which is thawed and then combined with a pharmaceutically acceptable carrier or additive.
19. A pharmaceutical composition comprising a therapeutically effective amount of a population of extracellular vesicles according to claim 15 in combination with a pharmaceutically acceptable carrier or additive.
20. A pharmaceutical composition comprising a therapeutically effective amount of a population of extracellular vesicles according to claim 16 which are thawed and combined with a pharmaceutically acceptable carrier or additive.
21. The pharmaceutical composition population of extracellular vesicles according to any one of claims 17-19 wherein said pharmaceutical composition is adapted for intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal routes of administration.
22. The composition according to claim 20 wherein said pharmaceutical composition is adapted for intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal routes of administration.
23. The composition according to any one of claims 17-22 wherein said composition is adapted for intravenous route of administration.
24. A method of treating a disease state or condition selected from the group consisting of osteoarthritis, acute respiratory distress syndrome (ARDS), skeletal muscle tissue damage, bone damage, cancer, cardiovascular disease, a neurological disorder or an autoimmune disease in a patient in need, comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition according to any one of claims 17-23.
25. The method according to claim 24 wherein said cardiovascular disease is stroke.
26. The method according to claim 24 wherein said neurological disorder is multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, a Parkinson’s-related disorder, Huntington’s disease, prion disease, motor neuron disease (MND), spinocerebellar ataxia (SCA) or spinal muscular atrophy (SMA).
27. The method according to claim 24 wherein said autoimmune disease is rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticarial, Sjogren’s disease, autoimmune-related Type 1 diabetes, rheumatoid arthritis (RA), psoriasis/psoriatic arthritis, multiple sclerosis, inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, Addison’s disease, Grave’s disease, Hashimoto’s thyroiditis, Myasthenia gravis, autoimmune vasculitis, pernicious anemia and celiac disease^ 28. The method according to claim 24 wherein said cancer is myeloma, multiple myeloma, lymphoma or opsoclonus-myoclonus syndrome (OMS). 29. A method of treating a disease state or condition selected from the group consisting of eosinophil-associated gastrointestinal diseases, noninfectious posterior uveitis and cancer in a patient in need, comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition according to any one of claims 17-23. 30. A population of extracellular vesicles (EVs) obtained by priming mesenchymal stem cells under hypoxic, low pH or inflammatory conditions for a period ranging from one minute to 72 hours, growing the primed cells in growth media for a sufficient period and isolating extracellular vesicles from said growth media. 31. The population according to claim 30 wherein the cells are grown in growth media for a period ranging from 12 hours to 6 days. 32. The population according to claim 31 wherein said growth media is collected, filtered to remove cells and large debris from the extracellular vesicles in the medium, filtered to obtain said extracellular vesicles using ultrafiltration and subsequently washing and collecting the vesicles.
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