NEURAL CELL EXTRACELLULAR VESICLES (2025)

This application is a continuation of U.S. application Ser. No. 15/770,881, filed Apr. 25, 2018, issued as U.S. Pat. No. 11,993,787, which is a National Stage Entry of International Patent Application No. PCT/US2016/062245, filed Nov. 16, 2016, which claims benefit of U.S. Provisional Application No. 62/256,823, filed Nov. 18, 2015, each of which is hereby incorporated herein by reference in its entirety.

This invention was made with government support under grant number CBET0939511 awarded by the NSF and grant number EP-D-13-018 awarded by the EPA. The government has certain rights in the invention. 37 CFR 401.14 f (4).

Diseases and injuries to the nervous system, including congenital disorders, cancers, degenerative diseases, and spinal cord injury, affect millions of people of all ages. Congenital disorders occur when the brain or spinal cord does not form correctly during development. Cancers of the nervous system result from the uncontrolled spread of aberrant cells. Degenerative diseases occur when the nervous system loses functioning of nerve cells. There is evidence that damage can be reversed by replacing lost cells with new ones derived from cells that can mature into nerve cells, called neural stem cells. However, transplanted stem cells may and often do migrate from the site of implantation and undergo some cell transformation that lead to a teratoma formation. In addition, due to their size, stem cells are often directly injected into the CNS which can induce complications. Invasive CNS surgeries place the patient at risk due to complication that occur during and after delivery, including but not limited to hemorrhages and edema. Stem cells administered systemically often end up being lodged in small capillaries which can induce undesired effects in the lung and may not even reach the disease or injury. Stem cell therapy can also trigger an elicit and adverse immune response.

Disclosed herein are neural extracellular vesicles (EVs) and methods of using these EVs in the treatment of spinal cord injury, stroke, and traumatic brain injury and neurodegenerative diseases.

The disclosed EVs can be obtained in some embodiments, by culturing neural progenitor (NP) cells that were produced from pluripotent stem cells (e.g. human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)) in cell culture medium under conditions and for a time sufficient for the NP cells to produce EVs, and isolating said EVs from the culture medium. In some embodiments, the NP cells are SOX 1+, SOX 2+, OCT4−, and NESTIN+.

The disclosed EVs can be obtained in some embodiments by culturing neural cells derived directly or indirectly from pluripotent stem cells (PSCs) (e.g. ESCs or iPSCs) in cell culture medium under conditions and for a time sufficient to produce EVs, and isolating said EVs from the culture medium. In some embodiments, the PSC-derived neural cell comprises a glial cell, such as an astrocyte or oligodendrocyte. In some embodiments, the PSC-derived neural cell comprises a neuron. In some embodiments, the PSC-derived neural cell are differentiated from NP cells derived from the hES cells. In some embodiments, the PSC-derived neural cells are differentiated directly from PSCs.

Also disclosed are EVs obtained by culturing astrocytes of any origin in cell culture medium under conditions and for a time sufficient for the astrocytes to produce EVs, and isolating said EVs from the culture medium.

The disclosed EVs can alternatively be obtained in some embodiments, by culturing mesenchymal stem cells (MSCs) that were produced from PSCs in cell culture medium under conditions and for a time sufficient for the MSC cells to produce EVs, and isolating said EVs from the culture medium.

As disclosed herein, EVs produced from hNP cells (also referred to herein as NPEX) had 1653 proteins (see Table 9) that were not identified in EVs from stem-cell-derived astrocytes (also referred to herein as APEX) or mesenchymal stem cells (also referred to herein as MSCEX). Therefore, in some embodiments, the EVs comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600 or more protein biomarkers listed in Table 9.

As disclosed herein, EVs produced from APEX had 596 proteins (see Table 8) that were not identified in NPEX or MSCEX. Therefore, in some embodiments, the EVs comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more protein biomarkers listed in Table 8.

As disclosed herein, EVs produced from MSCEX had 536 proteins (see Table 7) that were not identified in APEX or MSCEX. Therefore, in some embodiments, the EVs comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or more protein biomarkers listed in Table 7.

In some embodiments, the disclosed EVs are produced from a substantially homogeneous population of cells. In some embodiments, the disclosed EVs are produced from non-transformed cells.

Also disclosed are compositions containing the disclosed EVs. In some embodiments, the composition comprises the disclosed EVs in 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™. The hydrogel can, for example, include any of the following: polysaccharides, proteins, polyphosphazenes, poly(oxyethylene)-poly(oxypropylene) block polymers, poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine, poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate), and sulfonated polymers.

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), ciliary neurotrophic factor (CTNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CTNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-Ira, 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 composition comprising the disclosed EVs further comprises a protease inhibitor, RNAse inhibitor, or combination thereof.

Also disclosed is a method of treating a subject with a with a spinal cord injury, stroke, traumatic brain injury or a neurodegenerative disease comprising administering to the subject an effective amount of a composition containing a neural EVs disclosed herein. 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).

There are several protein families represented in the contents of the disclosed EV that could be beneficial in the context of several age related diseases. These include catalytically active enzymes like metalloproteases, several calcium-mediated signaling proteins, and other ion channels. Several DNA and RNA polymerase subunits are present as well as ubiquitin ligases, and proteasome subunits. Therefore, also disclosed is a method of treating a subject with a protein homeostasis disorder or a proteinopathy comprising administering to the subject an effective amount of a composition containing a neural EVs disclosed herein.

Also disclosed is the use of a composition containing a neural EVs in the manufacture of a medicament for treating a patient or subject with a spinal cord injury, who has suffered a stroke or traumatic brain injury or has a neurodegenerative disease.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

FIG. 1 illustrates potential sources of cells from which extracellular vesicles (EVs) (e.g. exosomes) may be obtained.

FIGS. 2A-2H an ultrastructural analysis indicating that multivesicular bodies (MVBs) which contain EVs are common in neural progenitors (NPs). Traditionally described as “vesicles containing smaller spherical and ellipsoidal vesicles and other inclusions described as filaments, granules, irregular dense masses, and membrane components”, are apparent near the limiting membrane of NPs. Also as reported, the vesicles were frequently found in clusters of 3 or more vesicles (FIGS. 2A, 2B). The inset from FIG. 2B is shown in FIG. 2C and this MVB actual has a vesicle budding inward into the larger multivesicular body, supporting a role for these vesicles in protein recycling from the plasma membrane. These vesicles do appear to bind with the plasma membrane, releasing EVs into the extracellular space (FIGS. 2D, 2E). Previous reports indicate that the vesicles can coalesce, and this seems common in NPs, and can be seen in most cells with clusters of MVBs (FIGS. 2A, 2F, 2G). FIG. 2H is a transmission electron microscopic (TEM) image of purified EVs from NPs.

FIGS. 3A to 3D show that EVs can be purified and detected by nanoparticle tracking analysis. FIG. 3A shows proteins from exosomes separated by SDS-PAGE and gels stained with coomassie stain. Protein profiles from EVs overlapped but was distinctive from the cell pellet, supporting that cargoes are specifically trafficked to EVs. FIG. 3B shows that protein content when compared by BCA Assay reveals that amount of protein in the profile changes when cells are exposed to stressful conditions, like nutrient deprivation. FIGS. 3C and 3D show NanoSight analysis of purified EVs from neural progenitor cells (FIG. 3C) and astrocytes (FIG. 3D). These EVs show overlapping but different size profiles, with both types of cells producing a peak indicating 55 nm vesicles, and ranging from 25-250 nm.

FIG. 4 shows differentiated neural cells internalize DiI-labeled EVs from neural progenitor cells. By adding 10 uM DiI to the cellular supernatant for 30 minutes prior to the final EVs spin and PBS wash, it was possible to label EVs. When added into the culture medium of differentiated cells, uptake of EVs was evident within 5 minutes.

FIGS. 5A and 5B show EVs from neural progenitors protect more differentiated neural cells from starvation stress. FIG. 5A shows EVs harvested by ultrafiltration from neural progenitor cells (NPs), astrocytes, or human umbilical MSCs. Protein content was measured by BCA, and EVs were serially diluted and transferred in equal volumes phosphate buffered serum (PBS) into wells containing NeuroNet cells (6-8 week differentiated). Cells were subjected to starvation stress for 10 days, when cells were fixed, and stained for 3-III tubulin (Tuj). The center 20 fields of view per well were imaged using the Cellomics Arrayscan (representative images from 4 technical reps shown). FIG. 5B shows NP and Astrocyte EVs protected the cells from starvations stress, and largely maintained integrity of the monolayer and extensions. While more cells and debris are present in the MSC treated wells, the cells had largely lost their neural morphology. Few cells were still detectable in the wells that received only PBS. NP and Astrocyte EVs samples had higher protein concentrations, so were treated with 50 g protein/well. Protein was limiting in the MSC samples, so 6.25 was the highest concentration possible, but still appeared to kill the cells in a concentration dependent way, so it is unlikely that the higher doses (50, 25, or 12.5 g/well) would produce a different result.

FIGS. 6A to 6C show indium-111 labeled EVs can be found in proximity of stroke tissue within 1 hour of injection. FIGS. 6A to 6C show biodistribution of indium-111 labeled EVs (FIGS. 6B, 6C) compared to free indium (FIG. 6A), which indicates that EVs are present in the brain in proximity of the stroke within 1 hour of injection either when injected immediately following stroke (FIG. 6B circles, left panels), or 24 hours after the stroke occurred (FIG. 6C, circles, left panels). Regardless of the timing of the initial injection, EVs were largely cleared from the area 24 hours after injection (FIGS. 6B, 6C; circles, right panels) although a smaller amount of radioactivity was still detectable, likely indicating EVs were metabolized by surrounding tissues.

FIG. 7 shows biodistribution of DiR labeled EVs in piglet brain. DiR labeled EVs (approximately 2.7×1010 vesicles/kg) were detectable from both the dorsal and ventral via direct delivery into the brain parenchyma by stereotaxic injection (top panels), and into the CSF in the subarachnoid space (lower panels). Fluorescence was detectable in the dorsal and ventral aspects of the brain in both instances, but more pronounced in the dorsal region when injected IP (top panels), and ventral areas of the brain following CSF injection (lower panels).

FIGS. 8A to 8E show treatment using EVs derived from neural cells improves infarct size and functional outcomes in the mouse embolic stroke model. APEX, MSCEX, PBS, and NPEX aliquots were provided to blinded investigators for injection into mice following induction of embolic stroke (3 repeated doses of approximately 2.7×1011 vesicles/kg at 2, 14, and 28 hours after stroke). TTC staining of brains harvested 96 hours post-stroke revealed substantially decreased infarct size following NPEX or APEX treatment while MSCEX had no effect (FIGS. 8A, 8C). NPEX and APEX also decreased mortality over the course of the study with over 40% of control and MSCEX treated mice succumbing to complications over the course of the study while approximately 25-30% of APEX and NPEX treated animals were lost (FIG. 8B). Phenotypic benefits including improved sensory capacity detected by adhesive tape test (FIG. 8E), and fewer neurological deficits as summarized by neurological deficit score (FIG. 8D) were also noted in APEX and NPEX treated animals.

FIGS. 9A to 9C show EVs derived from neural cells modulate the immune response following stroke. Blood sample flow cytometry 96 hours post-stroke indicates that APEX and NPEX increase regulatory T-cells relative to MSCEX and control (vehicle) treated groups (FIG. 9A), decrease inflammatory T-helper cells (FIG. 9B), and increase anti-inflammatory M2 macrophages in the circulation (FIG. 9C).

FIGS. 10A to 10D show NPEX treatment results in decreased infarct volume within 28±4 hours post-stroke as measured by MRI. Animals subjected to stroke received either NPEX (approximately 2.7×1010 vesicles/kg) or PBS (vehicle) at 2, 14, and 24 hours post-stroke. While anesthetized, animals were also subjected to MRI analysis. NPEX treated animals had substantially reduced infarct volume compared to those that received PBS (FIGS. 10A-C). When the size difference between the ipsilateral and contralateral sides was evaluated, there was less difference between hemispheres in the NPEX group, indicating less edema and swelling as compared to controls. FIG. 10D shows NPEX treatment results in less change in volume in the ipsilateral and contralateral hemispheres after stroke.

FIG. 11 shows NPEX treatment improves molecular deficits 12 weeks after stroke. T2 and T2 flair images both indicate less detectable damage in NPEX treated pigs 12 weeks post-stroke. The absence (death) of tissue was significantly reduced at the 12 week timepoint and areas of density incongruent with host tissue were far less evident in treated animals.

FIG. 12 is a Venn diagram showing the number of proteins unique to, and shared by NPEX, APEX, and MSCEX EVs.

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 “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.

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 neuronal stem and progenitors, neural crest cells, mesenchymal stem cells (MSCs) and related proliferative and non-proliferative neural 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 “neural progenitor cell” refers to cells capable of dividing a limited number of times that have the capacity to differentiate into neuronal and glial cell types.

The terms “extracellular vesicle” and “EV” are used herein to refer to a vesicle of about 10 nm to 10 μm in size consisting of fluid, macro-molecules, solutes, and metabolites from a cell contained by a lipid bilayer or micelle. In some cases, the EV is a cell-derived EV. The term “EV” also includes lipid vesicle engineered to contain bioactive molecules found in a cell-derived EVs, such as a neural EVs. These terms encompass both exosomes and ectosomes. Exosomes are 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 20 nm to 10 μm, 20 nm to 1 μm, 20 nm-500 nm, 30 nm-100 nm, 30 nm-160 nm, or 80-160 nm in size. In some embodiments, the EVs are exosomes that are about 20 to 150 nm in size.

The term “autologous EV” is used to describe a population of EVs which are obtained from cells from a subject or patient to whom the EVs are to be administered.

The term “neural EV” is used to refer to a cell-derived EV produced from neural progenitor cells derived in vitro from pluripotent stem cells or neural cells derived in vitro from said neural progenitor cells or from pluripotent stem cells. The term also refers to vesicles engineered to contain a sufficient number of the bioactive molecules found in the cell-derived neural EV to have substantially the same bioactivity.

Disclosed herein are neural EVs (e.g. exosomes) and methods of using these EVs in the treatment of spinal cord injury, stroke, and traumatic brain injury and neurodegenerative diseases.

The disclosed EVs can be obtained in some embodiments, by culturing neural progenitor (NP) cells or 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 NP cells or MSCs to produce EVs.

Methods for the production of human neural progenitor (hNP) cells from human embryonic stem cells (ESCs) are described, for example, in U.S. Pat. No. 7,531,354, which is hereby incorporated by reference in its entirety for the teaching of these cells. Human neuroprogenitor cells (hNPs) are known to express markers associated with the earliest multipotent neural stem cells, including Nestin, Musashi-1, Sox1, Sox2 and Sox3. It is noted that although feeder cell free neural progenitor cells may be used to produce EVs, any neuroprogenitor cell as otherwise described herein may also be used. Preferred neuroprogenitor cells are produced according to the methods presented in U.S. Pat. No. 7,531,354, are adherent feeder cell free as well as free from embryoid bodies.

The disclosed EVs can be obtained in some embodiments by culturing differentiated neural cells, such as astrocytes, 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. In some embodiments, the differentiated neural cells are hN2™ neuronal cells (ArunA Biomedical), NeuroNet™ neurons, or AstroPro™ astrocytes (ArunA Biomedical Inc).

Pluripotent stem cells used to produce the EV-producing NP cells, neural cells, or 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 (<2,0000 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.” 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 NP cells and/or neural cells 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 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. 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.

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 NP or neural cells to be grown in the growth media, the inclusion of LIF is preferred but may not be required. 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 neural cell to be grown, all of which are well known in the art. Particularly preferred media is media and supplement from ArunA. The AB2™ Neural Cell Culture Media Kit contains AB2™ Basal Neural Medium and ANS™ Neural Medium Supplement. The medium and supplement are specifically engineered for versatility to meet all neural cell culture needs. The AB2™ Basal Neural Medium and ANS™ Neural Medium Supplement can be used as the base for specialized mediums to direct differentiation of the hNP1™ line toward various neural phenotypes. Each lot of medium and supplement is pre-qualified for use by testing for cell growth, sterility, pH, osmolarity, and endotoxins.

Formulations from ArunA allow neural 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, Beth HaEmek, Israel, and Calbiochem. 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 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 cells 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 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 proliferative and non-proliferative neural cells as otherwise described herein.

In certain embodiments, EVs are harvested and collected by 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.

Disclosed is a 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 1 g to about 1 g or more of total EVs, about 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), 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. Example 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 Ig (1,000 mg) 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 spinal cord injury, stroke, traumatic brain injury and/or neurodegenerative disease. 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-Ira, 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 imidizoles 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.

Also disclosed is a method of treating a subject with a with a spinal cord injury, stroke, traumatic brain injury or a neurodegenerative disease comprising administering to the subject an effective amount of a composition containing a population of neural EVs disclosed herein. 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 “spinal cord injury” is used to describe a spinal cord injury which results in a temporary or permanent change in the normal motor, autonomic or sensory function of the cord. The damage often results from physical trauma, such as sports injuries, slip and fall accidents or motor vehicular accidents but can also result from diseases such as spina bifida, Friedrich's ataxis and/or transverse myelitis. Injury to the spinal cord resulting in a loss of function does not have to be the result of complete severing of the spinal cord. Depending on where the spinal cord and its nerve roots are damaged, the symptoms and degree of injury can vary widely, from pain to incontinence to paralysis. Spinal cord injuries are described at various levels of incomplete to complete injury, resulting in a total loss of function. The spinal cord injury can result in paraplegia or tetraplegia.

Traditional treatment of spinal cord injuries starts with stabilizing the spine and controlling inflammation associated with the spin cord damage to prevent further damage. Other interventions can vary widely depending on the location and extent of the injury. In many cases, using conventional therapy, spinal cord injuries require substantial, long-term physical therapy and rehabilitation, especially if the injury interferes with activities of daily life.

Spinal cord injury can be classified into three types based on its cause: mechanical forces, toxic, and ischemic, from lack of blood flow. Spinal cord damage can also be divided into primary and second injury. Primary injury is caused by the cell death that occurs immediately in the original injury (physical trauma, exposure to toxins, or ischemia), and secondary injury is caused by the resultant cascades that are caused by the original insult and cause further tissue damage. These secondary injury pathways include inflammation, swelling, neurotransmitter deficiencies/imbalances, the results of ischemia and cell suicide. The present invention may be used to treat all forms of spinal cord injury, including complete and incomplete injuries, ischemia, spinal cord injury without radiographic abnormality, central cord syndrome, anterior cord syndrome, Brown-Séquard syndrome, posterior cord syndrome, tabes dorsalis and conus medullaris, among others.

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 “traumatic brain injury” (TBI) is used to describe an injury to the brain caused by movement of the brain within the skill or an injury to the brain caused by a foreign object. Causes of TBI may include falls, a motor vehicle crash or being struck by or with an object. TBI may also be caused by a penetrating object—an injury to the brain caused by a foreign object entering the skull. Causes may include firearm injuries or being struck with a sharp object. TBI may cause a concussion, a period of unconsciousness (coma) or amnesia. TBI may impair one or more of cognitive function (e.g., attention and memory), motor function (e.g., extremity weakness, impaired coordination and balance), sensation (e.g., hearing, vision, impaired perceptin and touch and emotion (e.g., depression, anxiety, aggression, impulse control, personality changes).

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 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 a neurodegenerative disease, stroke and/or spinal cord injury) agent. 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 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 routes of administration.

In some embodiments, the disclosed EVs are administered within 24 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. 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 neurodegenerative disease, the EVs are administered continuously (e.g., once every 1, 2, 3, or 4 weeks) 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 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 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.

More generally, non-steroidal anti-inflammatory drugs (NSAIDS) and other anti-inflammatory agents may be used in the treatment of neurodegenerative diseases as described herein.

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 have been 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 following claims.

Studies were conducted to determine whether EVs confer paracrine benefits on neural stem cells and play a role in both optimal in vitro neural cultures conditions and in therapeutic outcomes of neural stem cell treatments. The objective of the study was to determine if it was possible to isolate EVs from human stem cells of a neural lineage, specifically neural progenitor cells and/or differentiated post-mitotic neuronal cells. EVs were purified from neural progenitor cells (SOX 1+ and 2+, OCT4−; hNP1™ ArunA Biomedical), derived from human pluripotent stem cell lines or differentiated neuronal cells (β-III tubulin (Tuj1)+, MAP2+, Oct4−); hN2™ ArunA Biomedical).

Human pluripotent stem cells [see, Chambers, et al., Methods Mol Biol, 2016. 1307: p. 329-43] lines were cultured in media absent of serum such as MTeSR or E8 which is commercially available from vendors such Stem Cell Technology using feeder free conditions or media composed of knock-out serum replacement (KSR) media (DMEM=F12, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids, 50 U/mL penicillin, 50 mg/mL streptomycin, and 20% KSR) (all from Gibco, Carlsbad, CA) and 4 ng/mL basic fibroblast growth factor bFGF; R&D Systems, Minneapolis, MN). Cells were cultured on Mitomycin-C(Sigma, St. Louis, MO) mitotically inactivated murine embryonic fibroblasts (MEF) or without feeders, manually or enzymatically dissociated, and passaged to new feeder layers every 2-5 days. For feeder-free culture of hPSC in Conditioned media, cells grown on MEF were washed once with PBS (without Ca2+ and Mg2+), and then incubated with 0.25% trypsin (Gibco) until the MEF layer began to lift off the dish. The floating MEF layer was discarded after agitating it to release adherent stem cells that were collected, centrifuged, and resuspended in MEF-conditioned media (CM). CM was prepared by placing 20% KSR media on MEF for 24 h and then supplementing the collected media with an additional 4 ng/mL of bFGF. Cells were plated on tissue culture dishes coated with laminin substrate (1 mg/cm2; Sigma) and grown to ˜90% confluence. The cells were passaged at least three times to minimize MEF contamination [see, Boyd, et al., Tissue Eng Part A, 2009. 15(8): p. 1897-907, Mumaw, et al., Microsc Microanal, 2010. 16(1): p. 80-90 and Young, et al., Neuroscience, 2011. 192: p. 793-805,]. Regardless of the culture method all media is collected and EVs are collected from the media and used to treat patients

After manual passage onto fresh feeder cells, hESCs were allowed to proliferate in ES medium for 7 days (stage 1). Cell differentiation was then induced with either DN2, MEDII, or ES medium for another 7 days (stage 2). DN2 medium is DMEM/F12-based medium supplemented with N2 (Gibco), L-glutamine, penicillin/streptomycin (P/S), and 4 ng/ml bFGF. MEDII medium for this study is DN2 medium supplemented at 50% (unless otherwise noted) with conditioned medium. To understand and follow the differentiation steps applied here, phenotype marker expression was examined at various time intervals. At stages 1, 2, and 3, populations were harvested and the markers Musashi-1, Nestin, and Oct-4 were observed. Immunocytochemical analysis was also performed on the adherent cell population. The cells at both stages were double-stained with Nestin and Oct-4 and observed under the fluorescence microscope for immunocytochemical examination associated with morphology. Groups that displayed phenotypic difference were then subjected to quantitative analysis for these same markers using flow cytometry. All experiments were replicated three times unless otherwise noted. After manual passage onto fresh feeder cells, hESCs were allowed to proliferate in ES medium for 7 days (stage 1). Cell differentiation was then induced with either DN2, MEDII, or ES medium for another 7 days (stage 2). DN2 medium is DMEM/F12-based medium supplemented with N2 (Gibco), L-glutamine, penicillin/streptomycin (P/S), and 4 ng/ml bFGF. MEDII medium for this study is DN2 medium supplemented at 50% (unless otherwise noted) with conditioned medium (described above). Stages 1, 2, and 3, populations were harvested and the markers Musashi-1, Nestin, and Oct-4 were observed. Immunocyto-chemical analysis was also performed on the adherent cell population. The cells at both stages were double-stained with Nestin and Oct-4 and observed under the fluorescence microscope for immunocytochemical examination associated with morphology. Groups that displayed phenotypic difference were then subjected to quantitative analysis for these same markers using flow cytometry. All experiments were replicated three times unless otherwise noted.

After manual passage onto fresh feeder cells, hESCs were allowed to proliferate in ES medium for 7 days (stage 1). Cell differentiation was then induced with either DN2, MEDII, or ES medium for another 7 days (stage 2). DN2 medium is DMEM/F12-based medium supplemented with N2 (Gibco), L-glutamine, penicillin/streptomycin (P/S), and 4 ng/ml bFGF. MEDII medium for this study is DN2 medium supplemented at 50% (unless otherwise noted) with conditioned medium (described above). At stages 1, 2, and 3, populations were harvested and the markers Musashi-1, Nestin, and Oct-4 were observed. Immunocytochemical analysis was also performed on the adherent cell population. The cells at both stages were double-stained with Nestin and Oct-4 and observed under the fluorescence microscope for immunocytochemical examination associated with morphology. Groups that displayed phenotypic difference were then subjected to quantitative analysis for these same markers using flow cytometry. All experiments were replicated three times unless otherwise noted.

hPSC were allowed to proliferate in hPSC medium (any of those described above) for approximately 5 to 10 days (stage 1) as a monolayer or as embryoid bodies. Cell differentiation was then induced with medium typically DMEM/F12-based medium supplemented with N2 (Gibco), L-glutamine, penicillin/streptomycin (P/S), and usually 4 ng/ml bFGF. Nestin, and Oct-4 were observed and neural rosettes form. Neural rosettes are isolated (manually or enzymatically and are immunocytochemical analysis was also performed on the adherent rosette population. The cells at both stages were double-stained and are Nestin+ and Oct-4− and observed under the fluorescence microscope for immunocytochemical examination associated with morphology [Shin, et al., Stem Cells, 2006. 24(1): p. 125-38]. The rosettes when isolated from most of the contaminating cells are considered human neural progenitor cells. Briefly, hNP cells were grown on poly-ornithine (20 mg/mL)/laminin (Sigma-Aldrich, Inc.) (5 mg/mL) coated plates or other ECM such as Matrigel, and maintained and expanded in media with 2 mM L-glutamine and 20 ng/mL b-FGF. hNP Cells were passaged approximately every 48 h and split 1:2 following manual dissociation [see, Mumaw, et al., Id., Young, et al. Id. and Dhara, et al., Methods Mol Biol, 2011.767: p. 343-54].

Alternatively, neural induction may be stimulated by inhibition of SMAD signaling using inhibitors of Activin/Nodal pathway, and/or BMP signaling (examples of inhibitors might be Noggin, SB431542 [Chambers, et al. Nat Biotechnol, 2009. 27(3): p. 275-80], Compound C [Zhou, et al., Stem Cells, 2010. 28(10): p. 1741-508], or other strategies alone or in combination). Cells may be cultured on matrigel or other extracellular matrices, in AB2, Neurobasal, or other mediums listed above, in the presence of absence of Sonic Hedgehog [Chambers, et al., Id., and Zhou, et al., Id.].

hNP cells differentiated into neurons on poly-ornithine and laminin coated plates or other ECM such as Matrigel under maintenance media described above without bFGF and LIF. Alternatively LIF or EGF are added hNP cells were allowed to differentiate under these two conditions for 1 to 7 weeks [Mumaw, et al., Id., and Dodia, et al., PLoS One, 2011. 6(8): p. e232669].

After 1 to 4 weeks of neuronal differentiation such as hNP cells differentiated into neurons on poly-ornithine and laminin coated plates (under two conditions in maintenance media without bFGF or bFGF and LIF or EGF), astrocytic differentiation was induced by switching the neural to cells from hNSC Maintenance Medium to the basic medium (DMEM HAM's F12 medium, glutamine, Penicillin/Streptomycin) supplemented with 1% FCS (Gibco). See Mumaw, et al., Id. and Young, et al., Id. Finally, multilinear differentiation was achieved by replacing the maintenance medium by the basic medium containing 10% of FCS. After 45-50 days cultures were nearly 100% positive for glial markers S100β and vimentin [Palm, et al., Sci Rep, 2015. 5: p. 1632110].

Alternatively, hNP cells were propagated as adherent monolayer cultures in a proliferation media (such as AB2™, ANS™ Neurobasal™, 1× B27, 1× Glutamax™ P/S, FGF2 (10 ng/mL) as described (Shin et al., Id.), and differentiated by removal of FGF. For astrocytic differentiation of hNP cells, neuronal differentiation media were supplemented with recombinant proteins, such as BMP2 and combinations of chemicals such as Azacytidine, Trichostatin A, or similar molecules for 1-5 days, with complete media changes in between, followed by differentiation media supplemented with the molecules separately or in combination. Cells were harvested prior to analysis at 5, 15 or 30 days of treatment or for cryopreservation at d6 or d10 of differentiation. For cryopreservation, cells were dissociated with Accutase™ and frozen in differentiation media containing 10% DMSO [Majumder, et al., Stem Cell Res, 2013. 11(1): p. 574-86].

When hPSC cultured without feeders as described above reached approximately 90% confluence, the 100 mm dishes were washed with PBS++(with Ca2+ and Mg2+) and replaced with 10 mL of fresh endothelial growth media 2 microvascular (EGM2− MV) (Lonza; 5% FBS, proprietary endothelial basal media 2 (EBM2) basal media and concentrations of bFGF, VEGF, EGF, and R3-IGF-125). The media was changed every 2-3 days over a period of 20-30 days. After transition from hESC to epithelial sheet was completed, the cells were trypsin passaged to a T75 flask and grown to confluence. To expand the initial cell culture, cells were passaged and seeded at a target density of approximately 4×104 cells/cm2 per flasks. For subsequent culture for experimentation, cells were subcultured at 106 cells=T75 flask (approximately 1.3×105 cells/cm2) and grown to confluence over 5-7 days [Boyd, et al., Id].

Cell medium was collected from confluent cultures 24 hours post media change and frozen at −20° C. Medium was thawed at 4° C. overnight and filtered through a 0.22 μm Steriflip unit prior to EV purification.

Isolation of extracellular vesicles from cell culture media was performed according to the protocols published by Théry, C., et al., Isolation and Characterization of EVs from Cell Culture Supernatants and Biological Fluids, in Current Protocols in Cell Biology. 2001, John Wiley & Sons, Inc. Briefly, filtered media was serially centrifuged at 300× g for 10 minutes, and supernatant was transferred to a fresh tube for centrifugation at 2,000× g for 10 minutes. Collected supernatants were then centrifuged at 10,000× g for 30 minutes, and resultant supernatant collected into a fresh tube. To label EVs, DiI was added to the purified supernatants at a final concentration of 10 μM and incubated at room temperature for 30 minutes. Supernatants were distributed into 11.5 ml Sorvall Ultracrimp tubes and sealed before transfer into a Sorvall T880 fixed angle rotor for centrifugation at 100,000× g for 70 minutes at 4° C. Supernatant was carefully removed and pelleted material resuspended in PBS and transferred into another ultracrimp tube, and again centrifuged at 100,000× g for 70 minutes at 4° C. The PBS was removed and pelleted material from each tube was resupended in 100 μl PBS. All purified EVs from the same cell type were pooled, triturated, and aliquoted into DNase/RNase free tubes (20-50 μl aliquots) for storage at −20° C.

Ultrafiltration of extracellular vesicles was performed according to the procedure developed for purification of cardiomyocyte derived extracellular vesicles. Amicon Ultra-15 100 kDa molecular weight cutoff filters were wetted with 10 ml PBS and centrifuged in a swinging bucket rotor at 4,000× g for 10 minutes. The PBS was discarded and the cell culture medium was added to the filter approximately 15 ml/tube and centrifuged at 4,000× g for 10 minutes. Another 15 ml culture medium was added to the filter when filtering stem cell derived extracellular vesicles, because less media was retained in the filter from the first run through, so approximately 30 ml of medium total was filtered for the H9 derived NP, Astrocyte, and MSC lines, while only 15 ml of medium was filtered for SH SY5Y cells; the media in the filter was centrifuged at 4,000× g in 5 minute increments to obtain approximately 1 ml of retentate. This was then either moved to a 1.5 ml tube for DiI labeling (at 10 μM for 30 minutes) or for unlabeled purification diluted to 15 ml with PBS and washed twice before repeating 5 minute centrifugation increments until a final volume of 1-1.5 ml was obtained. The purified extracellular vesicle preparation was then pooled from the same cell type and dispersed into approximately 100 μl aliquots (DNase/RNase free tubes) and stored at −20° C.

Labeled vesicles were generated by ultrafiltration. After the ultrafiltration was complete, the filter retentate was moved to a centrifuge tube, and 10 M DiI was added to the supernatant for 30 min. PBS was added to the filtration unit during this time to prevent the filter from drying out. After incubation with the labeling agent, supernatant was transferred back into the filtration device and washed three times with PBS (approximately 45 ml) to remove free label. After the final wash the retentate was concentrated to approximately 1 ml, which was aliquoted and stored at −20° C.

Vesicle preparations were mixed 1:1 with 4% paraformaldehyde (PFA) (to yield 2% PFA final) and incubated for 15 minutes. 5 μl droplets of fixed vesicle suspensions were transferred to Formvar-coated grids for 20 min., and then washed by transferring to drops of PBS. Grids were transferred onto drops of 1% glutaraldehyde for 5 min, and then moved over several drops of water to remove residual glutaraldehyde before transferring to uranyl-oxylate. Grids were imaged by electron microscopy at 80 kV.

While initial reports indicate that neural progenitor cells secrete fewer EVs than other cell types, ultrastructural analysis of the cells revealed prominent vesicles of endocytic origin (FIGS. 2A-2C), and many were in close proximity or associated with the outer limiting membrane of the cell (FIGS. 2D, 2E). Cargoes within the multivesicular bodies (MVBs) vary in size (FIGS. 2B, 2C) and are occasionally visualized budding off into the vesicle (arrow). Smaller vesicles seem to coalesce into larger MVBs as they move to the periphery of the cell (FIGS. 2F, 2G). It was possible to purify and visualize vesicles from the media of neural progenitor cells (FIG. 2H). These data suggest that neural progenitor cells do release extracellular vesicles into the cellular medium, and these vesicles can be purified using published EV purification protocols. All scale bars are 500 nm.

After realizing that neural progenitor cells have the capability of producing EVs that could be purified and visualized by electron microscopy, the process of characterizing the purified vesicles from multiple cell types began with protein profiling by Coomassie stain (FIG. 3A), and BCA Analysis of total protein content (FIG. 3B). The protein profiles were compared from neural progenitor cells, differentiated neural cells, and astrocytes, all derived from the same ES cell line, and SH SY5Y cells, a human neuroblastoma cell line used as a positive control. Early experiments indicate that the protein profile overlaps, but there are distinct proteins in the neural progenitor and differentiated neural cells, even though the cells are of the same genetic origin. Similarly, comparing the size profiles of the vesicles from neural progenitors, and astrocyte cells from the same genetic origin indicates that the size of vesicles from the 2 cell types overlap (including a large percentage of 55 nm vesicles), but there are distinct subpopulations that vary between the two cell types, with astrocytes showing unique vesicle sizes including a 25 nm population, and slightly larger 135 nm population (FIGS. 3C, 3D). Taken together, these data indicate that cargoes are specifically targeted into MVBs, and these cargoes change in the differentiation process, supporting a role for EVs in cell to cell communication throughout the process of development.

In order to determine if it was possible to label EVs, and visualize their uptake by another cell, differentiated neural cells (6-8 weeks in differentiation medium without fibroblast growth factor (FGF) were treated with astrocyte derived vesicles at timepoints ranging from 30 seconds through 30 minutes, after which, cells were fixed, and stained for β-III tubulin. At the earliest time points, few vesicles were found in the cells, (FIGS. 4A-4C). By 5 minutes in the culture it was possible to find neural cells with prominent red fluorescence, indicating vesicle uptake in these cells (FIGS. 4D-4F).

To determine if these vesicles could elicit an effect in recipient cells, differentiated neural cells were treated with serially diluted concentrations of vesicles derived from neural progenitors, astrocytes, or MSCs all derived from the same ES cell line, or SH SY5Y cells. These cells were then subjected to nutrient deprivation over 10 days and analyzed for neurite outgrowth. As expected, in wells that received only PBS the monolayer was disrupted and few cells were left (FIG. 5D). However, cells that received the highest concentrations of EVs from either neural progenitors or astrocytes were able to survive this nutrient deprivation, and the monolayer of cells with intact neurites were largely still intact (FIGS. 5A, 5B). Wells treated with MSC derived EVs contained more cells than untreated wells, but the monolayer was no longer intact and neurites were largely lost (FIG. 5C). These data indicate that vesicle treatment protected the recipient cells from nutrient deprivation, and importantly, indicate that cells respond differently to vesicles that originate from different cell types, even if the vesicles are from an isogenic or autologous source.

Taken together, these data support the idea that the parental source of origin has an impact on the vesicles that result. This has huge implications for considering extracellular vesicles as a therapeutic source that can potentially be exploited for regenerative medicine, and highlights the need for vesicles derived from neural sources for the treatment of CNS injuries and/or disease. Importantly, these data also indicate that not only neuron derived vesicles, but also glial vesicles provide benefit in vitro.

Biodistribution Methods: Rodent biodistribution by single-photon emission spectroscopy. 1.5-2 mCi of Indium-111-oxine in PBS was added to 200 μl doses containing EVs (−2.7×1011 vesicles/kg) and incubated at 37° C. for 20 minutes. Free indium was removed by three repeated PBS washes through an Amicon 100 kDa ultrafiltration device. Collected EVs were diluted to 200 μCi of radioactivity per dose, and injected intravenously into the mouse tail vein, either 1 hour or 24 hours post-stroke. Control animals received injection of free indium-111-oxine. Whole body and head single photon emission spectroscopy (SPECT) images were acquired by Mediso's nanoScan microSPECT/CT system 1 and 24 hours after injection, and projection images were reconstructed according to maximum intensity to determine radioactivity in the brain and throughout the body.

Piglet biodistribution: EVs were concentrated by ultrafiltration using Centricon units as previously described and then moved to 50 ml falcon tubes for DiR labeling (5 μM) in the dark for 30 minutes. Labeled EVs were collected by ultracentrifugation at 100,000× g for 4 hours. Pelleted EVs were washed with PBS and again collected by ultracentrifugation. EVs were resuspended in PBS and diluted into 2.7×1010 vesicle/kg body weight (in 200 ul PBS) doses for individual piglets based on NanoSightNS 300 nanoparticle tracking analysis. Piglets were anesthetized with isoflourane for intravenous injection (tail vein), intranasal delivery, cerebrospinal fluid EV injection into the subarachnoid cistern, or injection directly into the brain parenchyma. For intraparenchymal injection EVs were delivered at a flow rate of 5 μl per minute. Animals were euthanized by isoflourane followed by CO2 asphyxiation 30 minutes after completion of EV delivery. Brain, heart, liver, kidneys, lungs, and spleen were removed and imaged using Lumina IVIS (model) to detect DiR fluorescence.

Mouse: EV biodistribution was evaluated after either free indium-111 or indium-111 labeled EVs were injected into the mouse tail vein either immediately (FIG. 6B), or 24 hours post-stroke (FIG. 6C). SPECT scans were performed 1 hour after injection (FIGS. 6A, 6B, 6C, left panels), and again 24 hours after injection (FIGS. 6A, 6B, 6C, right panels). Images reconstructed by maximum intensity indicate that indium-111 labeled EVs are distributed throughout the bodies filtration organs in lungs, liver, spleen, and kidneys at both the initial timepoint, and 24 hours later (FIGS. 6B, 6C; whole body scans) while free indium-111 is initially localized mostly to the lungs before disbursing into the liver, spleen, and kidneys 24 hours later (FIG. 6A; whole body scans) likely indicating clearance through the renal system. Uniquely, labeled EVs were present in the brain in proximity of the stroke within 1 hour of injection when injected either immediately or 24 hours after stroke (FIGS. 6B, 6C; circles), indicating either access from the circulation due to disruption of the blood brain barrier, homing to the damaged tissue, or some combination of the two. Free indium did not localize to the stroked tissue at either time point assessed (FIG. 6A, circles).

Uniqueness: This is the first time that anyone has shown that IV injection of EVs are actually distributed in and around the infarcted brain following stroke. The majority of the EVs were distributed in other organs such as heart, liver, lung, and kidney, and we have data to suggest that the EVs for the first time have a systemic effect on the immune response post stroke (data in other sections). Thus it is likely that systemic effects of peripheral EVs have a positive effect on molecular and phenotypic benefits following stroke. Thus the EVs may have a direct effect at the site of injury, could be via local immune cells or via direct effect on the neurons, as well as a system effect on the immune system. This is the first data showing biodistribution in a large animal brain.

Piglet: In order to optimize EV delivery to the brain following stroke, biodistribution of fluorescently labeled (DiR, 5 μM) was evaluated in uninjured piglets. Labeled EVs were detected in the brain after injection directly into the parenchyma (FIG. 7, top panels), or into the CSF of the subarachnoid cistern at the base of the skull (FIG. 7, lower panels). This is the first demonstration of EV biodistribution in a large animal study.

Middle-aged C57/B6 male mice (retired breeders of 9-11 months) were pre-trained for adhesive tape test (ATT) for 3 days prior to the stroke surgery (3 trials/day). Mice were marked for identity, numbered, and randomized for therapy after stroke in a block size of 4 (4 animals from the same cage) to different treatment groups following induction of embolic stroke. The surgeon performing the stroke surgery and cerebral blood flow (CBF), and the investigator performing neurobehavior and neurologic deficit scoring remained blinded to the identity of the groups. All four therapies received were thawed and injected intravenously at 2, 14 and 38 hrs post stroke with doses 1, 2 and 3, respectively. Relative cerebral blood flow (CBF) was measured at 6 and 48 hrs post stroke. Neurologic deficit score (NDS) was assessed at 48 hrs post stroke; the ATT that reflects the somatosensory function was performed at 96 hrs post stroke just prior to euthanasia. Mortality was monitored daily and recorded until day 4 and prior to euthanasia.

Mice were sedated with Buprenorphine (0.05 mg/kg SC) 20 min prior to stroke surgery, and anesthetized with 3.5% isofluorane. Surgical plain of anesthesia was maintained with 1.5-2.0% during the surgery. Body temperature was maintained at 37° C. by a thermo-regulated surgery pad. By a midline incision on the ventral side of the neck, the right common carotid artery (CCA), external carotid artery (ECA), and the internal carotid artery (ICA) were assessed. A temporary atraumatic clip was placed on the CCA to prevent loss of blood during catheter insertion. A modified PE-10 catheter containing a single fibrin rich clot (9±0.5 mm length) was introduced into the ECA and advanced into the ICA. The clot was gently injected with 100 μL of PBS, the catheter was removed immediately after embolization, and the arterial wound was secured to prevent blood loss. Induction of stroke was confirmed using on-site portable single point cortical laser Doppler flowmetry (PeriMed Inc.). Finally, the temporary clip was removed and the blood flow in the CCA was reinstated. The site of surgery was closed using #6 sterile monofilament nylon suture, and Buprenorphine (0.05 mg/kg SC) was again injected. Mice were transferred to clean recovery cages and animal temperature was maintained. Conscious mice were transferred to clean regular cages with free access to food and water. NAPA gel and lactated ringers solution were provided as needed, in case of any sign of dehydration; otherwise 1 ml of regular sterile saline pre-warmed at 37° C. was injected SC every 12 hours.

Briefly, 6 hrs after stroke, mice were anesthetized using isofluorane, while body temperature was maintained at 37±0.5° C. The skull was shaved and a midline skin incision was made to expose the middle cerebral region. Perfusion images were acquired using PeriCam high resolution Laser Speckle Contrast Imager (LSCI; PSI system, Perimed) with a 70 mW built-in laser diode for illumination and 1388×1038 pixels CCD camera installed 10 cm above the skull (speed 19 Hz, and exposure time 6 mSec, 1.3×1.3 cm). Acquired video and images were analyzed for dynamic changes in CBF. Overall perfusion of the ischemic region will be compared to an equally sized region of interest from the uninjured contralateral hemisphere to estimate relative CBF. The skin wound was closed using tissue glue. At 48 hrs post-stroke, the skin wound was again opened, cleaned, and the middle cerebral artery region was exposed to repeat the LSCI procedure as previously described.

Neurologic deficits in mice were assessed by investigators blinded to the therapeutic group at 48 hr post stroke on a 5-point scale with the highest number indicating the worst outcomes and lower number indicating better neurological outcomes according to the following criteria: 0, no deficit (normal mice); 1, forelimb flexion deficit on contralateral side; 2, flexion deficit along with decreased resistance to lateral push and torso turning to the ipsilateral side when held by tail; 3, All deficits as in Score 2, including very significant circling to the affected side during the move inside the cage, and reduced capability to bear weight on the affected side; 4, All deficits as above but rarely willing to move spontaneously, and prefer to stay in rest; 5, considered terminal and euthanized in accordance with animal care requirements.

Adhesive tape test (ATT) was used as a test of somatosensory motor function, and was performed at 96 hrs post-stroke immediately prior to euthanasia. Briefly, naïve mice were acclimatized to the procedure of the test for 3 days prior to surgery by placing them in a transparent acrylic box (15 cm×25 cm). Two pieces of adhesive tape (0.3 cm×0.4 cm) were used as bilateral tactile stimuli after they were attached at the distal-radial region on each forelimb such that it covered the hairless part (3 pads, thenar and hypothenar). Within 180 seconds, the tape removal time was recorded as the sensorimotor function. If a mouse failed to remove the tape within 180 seconds, it was given a score of 180 seconds. Therefore, a shorter time score indicates a better outcome while longer time indicates an outcome with higher deficit.

TTC-stain differentiates between metabolically active (live or penumbra) and inactive (dead or core) tissues after stroke. TTC is a colorless solution, which is reduced to red 1,3,5-triphenylformazan (TPF) by the enzymatic action of various dehydrogenases primarily mitochondrial dehydrogenase from the living tissues, while the core (dead tissue) remains white. Therefore, larger white area indicates higher injury and infarction volume. At 96 hrs post stroke and after performing ATT, mice were deeply anesthetized with isofluorane (5%). Blood was collected via direct cardiac puncture to isolate and obtain serum later. Brains were very quickly perfused with 25 mls of cold 0.01 M phosphate-buffered saline (PBS), harvested fresh, and immediately transferred to a metallic mouse brain matrix. Looking at the infarcted area, 5-blades were placed in alternate gaps to obtain 2-mm coronal slices (4 sections per brain). Sections were individually placed in a 35-mm dish containing pre-warmed (37° C.) 3 ml of 5% TTC in PBS (Sigma) for 20-30 minutes at 37° C., followed by 2× washing with cold PBS and fixation with 10% formalin. In order to image, fixed sections were taken out of the dish and placed in order on a high-resolution Cannon Scanner. Images were cropped and saved for analysis. Corrected infarct volume was estimated using gray scale image and Scion Image software, and presented as the % volume of the uninjured side.

Prior to euthanasia blood was collected and purified cells were subjected to fluorescence activated cell sorting to identify populations of immune cells present systemically including T-helper (CD4+/FOX3P+) populations, regulatory T-cells (CD4+/IL17+), and M2 macrophage (IL10+/CD206+) populations.

EVs were purified from isogenically derived astrocyte progenitors (APEX), MSCs (MSCEX), and neural progenitor (NPEX) cells using standard methods (see manufacturing section), evaluated by nanoparticle tracking analysis (NanoSight-NS300) and stored in individual dose aliquots at −20° C. until they were thawed at room temperature immediately prior to tail vein injection following embolic stroke. The 3 EV types and PBS were administered by blinded investigators following embolic stroke, in 3 doses of approximately 2.7×1011 vesicles/kg (or vehicle) at 2, 14, and 28 hours post-stroke. In all parameters evaluated both APEX and NPEX outperformed the vehicle treated controls as well as the MSCEX treatment group. Immediately after euthanasia, 2,3,5-Triphenyltetrazolium Chloride (TTC) was used to differentiate metabolically active (live, red) and inactive (dead, colorless) tissues. Importantly, TTC indicates decreased injury and infarction volume following APEX or NPEX™ treatment (FIG. 8A), while MSCEX treatment was comparable to control. Within the 96 hrs following stroke APEX and NPEX treatments decreased mortality by 20 and 17% respectively (FIG. 8B). Neurologic deficits evaluated 48 hrs post stroke (on a 5-point scale from no deficit [0] with increasing severity through terminal deficits [5]) indicated significantly better behavioral outcomes for mice that received APEX or NPEX (FIG. 8D). Ability to remove adhesive tape used as bilateral tactile stimuli attached at the distal-radial region of each forelimb indicated improved sensorimotor function as well (FIG. 8E). Taken together, these data indicate improved survival, molecular, and functional outcomes in NPEX treated thromboembolic stroke rodent models compared to contemporary vehicle controls.

Flow cytometric analysis of blood cells at the 96 hour time point just prior to euthanasia indicates that neural cell type derived EVs, APEX and NPEX treatment resulted in an increase in the presence of protective regulatory T cells in circulation (FIG. 9A), while MSCEX were indistinguishable from controls. Pro-inflammatory T-helper cells were reduced in the APEX and NPEX groups (FIG. 9B), while anti-inflammatory M2 macrophages were increased in these groups (FIG. 9C). Taken together, these data indicate that neural cell type derived EVs exert part or all of their effects by modulating the immune response following stroke.

The production and quality control methods used to produce isogenically (genetically identical) derived neural progenitor, astrocyte progenitor, and MSC cells from the same ES cell line produce a unique opportunity to compare the EVs from these 3 cell types without the confounding variable of genetic variation due to source of the donor material. The vast majority of stem cell derived EV literature centers on use of MSC derived EVs. Here, for the first time neural cell derived EVs (APEX and NPEX) were evaluated and compared directly with MSC-derived EVs. There is a substantial improvement in molecular benefit (infarct volume), also increased survival and improved functional outcomes of neural derived EVs (APEX and NPEX) over the MSCEX. These improvements were immediate in older animals which is more prone to death. Thus no other group has shown that any EV (MSC or neural) therapy has such stark and immediate improvements in a mouse model that replicates the human stroke condition (embolic stroke) and factors in co-morbidly factors such as age (middle age mice). It appears that these early strong effects can only be obtained with a neural EV as disclosed herein.

These studies suggest that the mechanism of action in part may be through immune modulation suppressing the inflammatory M1 response including but not limited to suppressing IL17 cytokine while enhancing the M2 response perhaps through IL10 or other cytokines.

Landrace barrows (5-6 months, 150-170 lbs) were subjected to injury as previously described using the only fully developed porcine stroke model [1]. Briefly, a portion of the zygomatic arch was resected and the underlying muscle was elevated dorsally revealing the parietal bone. A window was generated in the bone surface exposing the dura mater. The proximal MCA was permanently occluded resulting in infarction spanning the most caudal aspect of the frontal lobe as well as significant portions of the temporal, parietal, and occipital lobes.

Magnetic resonance imaging (MRI) was performed 24 hours and 90 days post-MCAO surgery on a GE 16-channel fixed-site Twin gradient Signa HDx 3.0 Tesla MRI system. Under anesthesia, MRI of the neurocranium was performed using a multichannel phase array spine coil, with the patient in dorsal recumbency. Standard multiplanar MR brain imaging series were acquired. These included T2-weighted (T2w), T2-weighted fluid attenuated inversion recovery (FLAIR), and T1-weighted (T1w) FLAIR, as well as diffusion-weighted imaging (DWI) series. DWI was acquired with b=0 and b=1000. DWI, apparent diffusion coefficient (ADC) maps and T1w-FLAIR images were evaluated using Osirix® software for presence of cerebral infarction and changes in cerebral hemisphere volume. Specifically, the volume of the ischemic area was manually derived from the ADC maps generated from DWI sequences. The ischemic area was defined by two levels of ADC number reduction, with the ADC number from the contralateral cerebral hemisphere providing normal ADC values. Ischemic areas, defined by those with 80% and 40% ADC values of normal, were manually traced on the sequential ADC images. Each area was multiplied by slice thickness to produce a volume of ischemic tissue. This method was chosen because it has been demonstrated to strongly correlate with histologically defined areas. The cerebral hemisphere volume was determined through a similar process, whereby the cerebral hemisphere volume was quantified (excluding sulci and the lateral ventricular spaces) on Tiw FLAIR images. The T2w FLAIR images were used for reference to differentiate areas filled with CSF and parenchymal areas of hyperintensity (Platt, S. R., et al., Experimental & Translational Stroke Medicine, 2014. 6:5).

NPEX EVs containing ˜2.7×1010 vesicles/kg in 50-60 ml PBS were thawed at 4° C. and transferred into a 60 cc syringe using aseptic technique while in a biological safety cabinet. Samples were inverted a minimum of 25 times immediately prior to intravenous injection via an ear vein catheter. Pigs received either 3 doses (50-60 ml) of NPEX or PBS (vehicle) at 2, 14, and 24 hours post-MCAO.

Following surgery, animals were moved to a clean recovery pen and monitored continuously until extubation. Rectal temperature, heart rate, and respiration rate (TPR) were recorded every 15 minutes until the pig was awake and vital signs were stable within normal limits. Thereafter, TPR measurements were initially reduced to 1-2 hour intervals unless vital signs deviated from normal (for example, a fever), then to longer intervals over the next 48 hours as the pig recovered. During the first 36 hours, pigs were never left for more than 4 hours without observation, and generally for no more than 2 hours. In addition to TPR, other observations recorded included time from arrival in recovery until the animal first stood up on its own, time to drink and eat with assistance, and time to eat and drink unassisted. Events such as fevers (rectal temperature 103° F. or greater), circling behavior, and seizures were also monitored and documented.

As seen in Table 1, routine evaluation indicates improved survival and functional outcomes in immediate recovery post-stroke with NPEX treatment. Survival was substantially better in the NPEX treatment group, with 7/7 pigs surviving more than 72 hours post-stroke, while only 5/8 survived in the control group. Time until the animals could stand unassisted was reduced by about 2 hours with NPEX treatment. Fevers, which are very common within 72 hours post-stroke, were reduced in the NPEX treatment group where 4/7 animals had one or more fever episodes as compared to control group where 6/8 animals had at least one fever. Time to eat and drink unassisted, number of animals exhibiting circling behavior, and number of animals with documented seizure activity were similar between groups.

TABLE 1
PercentTime toTime toAssistedUnassisted
SurvivalstandFeverseatdrinkingDrinking
(72 hrs(hrs)(%)(days)(days)(days)
Treated1004.2157.141.241.563.13
Control62.56.2875.001.360.963.12

As seen in Table 2, gait analysis indicates improved motor function in NPEX treated pigs compared to controls. At 7 days post-stroke, NPEX treated pigs move faster and with more cadence (rhythmicity) as they move throughout their stride. Due to stroke in the right hemisphere, the left side specific deficits were more pronounced. Left side specific measurements indicate greater step length, shorter cycle time, greater stride length, and swing percent of cycle time. Treated animals placed more pressure on each foot as they moved through their stride and displayed more pronounced movement of the hind limbs past the front limb evidenced by the reach, indicating a more natural movement compared to controls.

TABLE 2
StepCycleStrideSwing
LengthTimeLength% ofFootHind
VelocityCadence(cm)(sec)(cm)CyclePressureReach
NPEX131.4591.9941.430.7980.8941.0662.87−12.34
Control106.6379.9938.600.8377.1438.2857.18−9.55

Initial MRI analysis at 28±4 hours indicated a smaller lesion volume in NPEX treated pigs compared to controls (FIGS. 10A-10C). Volume measurements of the ipsilateral and contralateral hemispheres indicate less change in volume after stroke with NPEX treatment (FIG. 10D), indicating less swelling in the ipsilateral hemisphere in the first 24 hours after stroke, consistent with the rodent data indicating an early modulation of the immune response and indicative of a neuroprotective effect in the porcine model as well. Physiological parameters, most notably survival, was also increased in the 72 hours following induction of stroke. Treated animals were also able to stand unassisted −2 hours faster than controls. Gait analysis at 7 days post-stroke indicated improved motor function as detected by increased velocity and cadence (rhythmicity), more pressure being applied to each foot throughout the motion, as well as increased step and stride length, an increased percentage of time in swing stance (foot off of ground) per cycle, and a more pronounced reach with the rear limb extending past the fore-limb, as is expected in quadrupeds.

NPEX treatment also had a profound effect on molecular benefit in the animals 12 weeks post-stroke as detected by MRI T2 and T2 FLAIR images (FIG. 11). Dead/dying tissue was reduced by treatment and damaged tissue (green traces) were far reduced, involving mainly cortical tissue while largely preserving integrity of the ventricle compared to the control. Animals that received NPEX were able to survive larger infarct sizes (as much as 3.8 cm3 larger at 24 hours post-stroke) over the 12 week study than those that received control treatment. This is probably due to the anti-inflammatory properties of NPEX leading to decreased stroke severity and promoting better long-term outcomes including integrity of brain tissue, as well as behavioral and motor function.

Never before have EVs of any kind improved the outcome of stroke or for that matter any neural deficit in a large animal. Consistent with the small animal we show that NPEX effects the brain immediately, thus NPEX is fast acting in the pig and the mouse. We do not know of any other study that has suggested such and immediate effect of EVs on a stroked animal. Immediately NPEX improved survival and motor function (speed in which animals recover, time to stand, balance, etc). Molecular and phenotypic outcomes in a large animal species expand upon previously described rodent data, indicating that EVs exert a likely neuroprotective effect that is longer lasting. This effect is likely due in part to modulation of the secondary injury cascade by muting the immune response that occurs following stroke, as molecular and phenotypic differences are detectable as early as 24 hours post-stroke and a later effect on the enhanced M2 response, which is neuro-reparative. The longer lasting effects are observed throughout, with improved gait of the NPEX treated animals.

In summary, this is the first stroke study to suggest to mechanisms of action are consistent across animals and includes a study in a more complex brain with a structure that is similar to humans. Our in vitro human cell studies suggest neuroprotective action directly on the damaged neural cells, and the mouse and pig studies suggest protection through the immune system. The longer acting effects on neuro-reparative mechanisms in both species were observed and could be in part due to up regulation of Treg cells (mouse only to date). Importantly, the porcine brain unlike the mouse shares many homologies with the human brain including white matter ratio, presence of gyrencephalic cortex, cytoarchitecture, and size. Due to these similarities, the pig is considered a superior model system compared to rodents, and is likely more representative of anticipated benefits in human treatment of ischemic stroke with therapeutically produced neural progenitor derived EVs.

This is possible due to an ability to produce a large quantity of EVs from neural progenitor cultures, approximately 5 times greater than yields obtained from MSCs. The ability to generate EVs on a commercial scale consistently from cultures will require that EV-producing cells be dependent on a rigorous quality control process for handling the cells and purifying the resultant EVs.

Media is harvested from plates or flasks containing cultured cells. The harvested media was frozen at −20° C. before or after the primary filtration and thawed at 4° C. Filtration is completed in a sterile laminar flow hood to minimize contamination. The harvested media was filtered via dead end filtration through a 0.22 μm filter (EMD Millipore Stericup). This primary filtration removes any cellular debris and/or dead cells from the harvested media. This allows for the EVs and microvescicles to pass through to the filtrate for further purification.

Having undergone primary filtration, the media is ready for secondary filtration. This step utilizes a system including Centricon centrifuge units, which can process up to 70 ml culture medium per spin. This filtration process uses a 100 kDa filter to collect enriched EVs. During this process the media is forced through the filter discs via centrifugation at 4,000× g. Once the retentate reaches approximately 250 μL to 5 mL per unit, a buffer exchange is then performed using approximately 90% of the starting media volume of PBS+/+. The PBS+/+ is passed through the Centricon filters by centrifugation at 2,000× g until the retentate reaches 250 μL to 1 mL of volume per filter unit. At this point the retentate is collected for EV analysis. A sample is taken for immediate nanoparticle tracking analysis, and purified EVs from the same cell type were pooled, triturated, and aliquoted into DNase/RNase free tubes for storage at −20° C.

For larger volume ultrafiltrations, Amicon stirred-cell ultrafiltration units were used, connected 3 units in tandem. Each base unit held 400 ml medium, which was further expanded using a reservoir to increase throughput. This step utilizes a system including a pressure vessel, and three EMD Millipore Stirred Cell Units (Fisher Scientific, USA) working in sequence. This step utilizes dead end filtration as well with media passing through a 100 kDa filter disc (Fisher Scientific, USA). During this process the media is forced through the filter discs using 25 psi of positive pressure supplied by compressed nitrogen gas. Once the retentate reaches 50 ml/stirred cell unit the system is de-pressurized. A buffer exchange is then performed using 50% of the starting media volume of PBS+/+. The PBS+/+ is passed through the stirred cell filters until the retentate reaches 50 mL of volume per filter unit. At this point the retentate is collected for EV analysis. A sample is taken for immediate nanoparticle tracking analysis, and purified EVs from the same cell type were pooled, triturated, and aliquoted into DNase/RNase free tubes for storage at −20° C.

To determine hFGF2 content, cryopreserved samples of NPEX™, APEX, and MSCEX EVs were thawed at 4° C. and lysed with an equal volume of EV lysis buffer to release exosomal content. The lysate was analyzed for human FGF2 using a commercially available human FGF2 ELISA assay kit (Thermo Scientific—Catalog number KHG0021) following manufacturers protocol. FGF2 standards provided with the kit were used to generate a standard curve and quantitated FGF2 in the test sample. FGF2 was detected in NPEX™ sample at 490 pG/mL. No FGF2 could be detected either in APEX™ or in MSCEX™ samples.

To compare proteins unique to neural EVs versus MSC derived EVs, purified EV proteins were subjected to mass spectroscopy by Bioproximity, LLC.

Ultracentrifugation and ultrafiltration methods were used as previously described. Large volume purifications utilized the Amicon stirred-cell system to purify over 24 liters of cell culture medium in a single work week.

Fibroblast Growth Factor ELISA results

The protein human Fibroblast Growth Factor 2 (hFGF2) is added to hNP1™ culture media to maintain the proliferative state of hNP1™ cells. EVs produced by hNP1™ cells (NPEX) cultured in hFGF2 supplemented media may accumulate/contain any of this hFGF2 protein as part of the exosomal protein content. NPEX EVs, harvested from hNP1™ culture media collected from live hNP1™ cell cultures were tested for the presence of hFGF2. These purified and cryopreserved NPEX™ EVs contained detectable levels of hFGF2 after thaw. On the other hand, EVs from hAstroPro™ human astrocytes (APEX), and from mesenchymal stem cells (MSCEX), where the cell culture media is not supplemented with hFGF2, did not contain any detectable hFGF2. Taken together, these data indicate that proteins supplemented in the medium are taken up by the cells and are present in the enriched EV sample even in the absence of transfection or other techniques to overexpress proteins in the cells.

FIG. 12 is a Venn diagram showing the number of proteins unique to, and shared by NPEX, APEX, and MSCEX vesicles. A total of 2727 proteins were shared by all of these vesicles (Table 3). APEX and MSCEX shared 2786 proteins that were not in NPEX vesicles (Table 4). NPEX shared 467 proteins only with MSCEX (Table 5) and 426 proteins only with APEX vesicles (Table 6). MSCEX had 536 unique proteins that were not identified in APEX or NPEX vesicles (Table 7). APEX had 596 proteins that were not identified in NPEX or MSCEX vesicles (Table 8). NPEX had 1653 proteins that were not identified in APEX or MSCEX vesicles (Table 9).

TABLE 3
Protein codes found in NPEX, APEX, and MSCEX
1A0A023UFG1
2A0A024QZ30
3A0A024R0R4
4A0A024R1Q5
5A0A024R269
6A0A024R294
7A0A024R2A8
8A0A024R3H6
9A0A024R3W5
10A0A024R473
11A0A024R4U7
12A0A024R663
13A0A024R6C6
14A0A024R7F1
15A0A024R816
16A0A024R900
17A0A024R972
18A0A024R9G7
19A0A024RA85
20A0A024RAB6
21A0A024RB27
22A0A024RB49
23A0A024RBR1
24A0A024RC42
25A0A024RD01
26A0A024RDU6
27A0A087WTT9
28A0A087WTY7
29A0A087WU72
30A0A087WUP3
31A0A087WUR9
32A0A087WUZ8
33A0A087WV01
34A0A087WV58
35A0A087WVQ9
36A0A087WVY5
37A0A087WX41
38A0A087X1N7
39A0A087X208
40A0A087X270
41A0A088AWP7
42A0A090N7U3
43A0A090N8G0
44A0A090N8I2
45A0A090N8Z3
46A0A096LNH2
47A0A096LP10
48A0A096LPK6
49A0A0A0MQZ3
50A0A0A0MR11
51A0A0A0MRA3
52A0A0A0MRA8
53A0A0A0MT16
54A0A0A7NZX2
55A0A0B4J262
56A0A0C4DG44
57A0A0C4DGP4
58A0A0C4DGQ0
59A0A0C4DGR9
60A0A0C4DGV7
61A0A0C4DH10
62A0A0C4DH26
63A0A0C4DH32
64A0A0C4DH71
65A0A0D9SF05
66A0A0D9SF53
67A0A0D9SFF0
68A0A0E3SU01
69A0A0F7TC28
70A0A0G2JQ57
71A0A0J9YWL9
72A0A0J9YY17
73A0A0J9YY65
74A0A0K0K1J1
75A0A0K2FPC8
76A0A0R4J2G5
77A0A0S2Z3C0
78A0A0S2Z3H6
79A0A0S2Z3N2
80A0A0S2Z4F6
81A0A0S2Z4K6
82A0A0S2Z4S4
83A0A0U1RQF0
84A0A0U1RQK4
85A0A0U1RR05
86A0A0U1RRH6
87A0A0U4BW16
88A0A0U4DR30
89A0A0U5Q0I5
90A0A0X7YLB8
91A0A0X9T0I7
92A0A0X9TDD0
93A0A0X9UWM4
94A0A109PSY4
95A0A126GVT4
96A0A126GVV9
97A0A126GVY4
98A0A126GW97
99A0A126LAV0
100A0A126LAW8
101A0A140VJG3
102A0A140VJM5
103A0A140VJZ1
104A0A140VJZ4
105A0A140VK24
106A0A142K0N8
107A0N7J6
108A1A5C4
109A1L0U7
110A1YZK0
111A2IPH5
112A2J1M8
113A2MYD2
114A2N0U1
115A2RRE5
116A2RTY3
117A3KMF7
118A3KMG4
119A4D0R5
120A4D1F6
121A4D263
122A4D2F6
123A4FU99
124A5XEJ8
125A6NCJ1
126A6NDG6
127A6NHK2
128A6NMH8
129A8K1Z4
130A8K2Q7
131A8K2X9
132A8K4L4
133A8K580
134A8K5W7
135A8K646
136A8K674
137A8K6Q8
138A8K6R3
139A8K889
140A8K941
141A8K948
142A8KA19
143A8KAM8
144A8KAQ0
145A8MQ14
146A8MSG4
147A8MUM1
148A8MYJ1
149B0AZV0
150B0I1R4
151B0I1R7
152B0I1R8
153B0I1S0
154B1AH62
155B2R604
156B2R6E3
157B2R8C2
158B2R8R5
159B2RAG9
160B2RAK1
161B2RD27
162B2RDG9
163B2RDV2
164B2RP65
165B2ZZ86
166B3KMD3
167B3KMX8
168B3KNH6
169B3KNH8
170B3KNJ3
171B3KR88
172B3KS22
173B3KS78
174B3KT94
175B3KUL2
176B3KV04
177B3KVE3
178B3KVJ2
179B3KW39
180B3KX05
181B3KXG9
182B4DE59
183B4DEA8
184B4DEN3
185B4DGW2
186B4DGX3
187B4DH09
188B4DHQ3
189B4DHR1
190B4DHV2
191B4DI57
192B4DJ98
193B4DJM8
194B4DK16
195B4DK41
196B4DKE0
197B4DKG5
198B4DKL5
199B4DL04
200B4DL67
201B4DLV7
202B4DM31
203B4DMC6
204B4DMD7
205B4DMI9
206B4DMK9
207B4DMS8
208B4DMY4
209B4DN39
210B4DN66
211B4DN96
212B4DNS4
213B4DPB7
214B4DPF0
215B4DPS3
216B4DQF8
217B4DQJ9
218B4DRF2
219B4DRV4
220B4DRZ1
221B4DRZ5
222B4DSD8
223B4DSI2
224B4DSK2
225B4DTD5
226B4DTF5
227B4DTI4
228B4DTV0
229B4DUV1
230B4DVQ8
231B4DVY2
232B4DXJ3
233B4DXW6
234B4DXX8
235B4DYE2
236B4DYM1
237B4DYQ3
238B4DYV9
239B4DZ96
240B4DZD8
241B4DZK5
242B4DZL0
243B4DZX5
244B4DZY9
245B4E0C3
246B4E116
247B4E1Z8
248B4E2C9
249B4E310
250B4E358
251B4E396
252B4E3S1
253B5BUB5
254B5MC96
255B7SXT3
256B7WPN9
257B7Z1C7
258B7Z1Y1
259B7Z208
260B7Z225
261B7Z2P6
262B7Z395
263B7Z3F9
264B7Z3I3
265B7Z425
266B7Z4S3
267B7Z5R5
268B7Z5U1
269B7Z6G1
270B7Z6T9
271B7Z865
272B7ZAS5
273B7ZKX2
274B7ZKY2
275B7ZL41
276B7ZL68
277B7ZMC8
278B7ZVX0
279C0JYZ2
280C4P0A0
281C8CHS3
282C9IYI4
283C9IZK7
284C9J0E9
285C9J6F5
286C9J8V2
287C9J9C4
288C9JBI4
289C9JG87
290C9JKM5
291C9JKM9
292C9JSJ3
293C9JX31
294D3DPG0
295D3DQH8
296D3DTX7
297D3DUZ3
298D3DV53
299D3DWL0
300D3DX49
301D3VVE1
302D3VVP9
303D4YW75
304D6RCE4
305D6RCP5
306D6RCQ3
307D6RGX4
308D6RHD5
309D6RHJ5
310D6W573
311D9ZGF2
312E2QRN8
313E5RFF8
314E5RG49
315E7EMW7
316E7EQW5
317E7ESB3
318E7ETY2
319E7EUH7
320E7EX80
321E9PB28
322E9PDD2
323E9PFQ4
324E9PJ15
325E9PJG1
326E9PKH2
327E9PKQ0
328E9PS68
329F1LLU8
330F5GZB4
331F5H7Y6
332F6X3N5
333F8VBW7
334F8VPD4
335F8VQY3
336F8VY01
337F8VYK9
338F8W6H2
339F8W883
340F8WEI7
341G3V1C3
342G3V1Y7
343G3V2J9
344H0Y390
345H0Y3V5
346H0Y5A2
347H0Y720
348H0Y8I2
349H0Y935
350H0Y991
351H0Y9B6
352H0Y9P3
353H0Y9Z7
354H0YA40
355H0YAA7
356H0YDK7
357H0YDZ4
358H0YF10
359H0YFH1
360H0YHN7
361H0YJ21
362H0YJI8
363H0YM90
364H3BM60
365H3BMQ8
366H3BPI1
367H3BPK7
368H3BPS8
369H3BQK9
370H3BQQ2
371H3BQT0
372H3BRQ0
373H3BS19
374H3BS34
375H3BT13
376H3BV16
377H7BYH4
378H7BZ70
379H7BZ95
380H7BZL8
381H7BZY4
382H7C013
383H7C0I1
384H7C0Q9
385H7C132
386H7C1F8
387H7C1W1
388H7C3P6
389H7C446
390H7C4N3
391H7C4W1
392H7C557
393H7C5G5
394H9E7H3
395I3L174
396I3L1P4
397I3L3B3
398I3L3U6
399I6L965
400J3KNJ2
401J3KP25
402J3KQ96
403J3KRF1
404J3KTA9
405J3KTL8
406J3QQK7
407J3QQP2
408J3QR51
409J3QRM5
410J3QS68
411J3QS89
412J3QS93
413J3QSE7
414K7EIX3
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2323V9TNI8
2324W8YM35
2325X5DNI9
2326X5DP31
2327X5DPA6
2328X5DR62
2329X6R647
2330X6RE28
2331A0A024R3N3
2332A0A024R456
2333A0A024R462
2334A0A024R6P6
2335A0A024R7W9
2336A0A024R893
2337A0A024R9I0
2338A0A024R9Q1
2339A0A024RAA7
2340A0A024RC00
2341A0A087WTA8
2342A0A087WUA7
2343A0A087WVP1
2344A0A087WYF9
2345A0A087X163
2346A0A087X1V8
2347A0A0A0MRD2
2348A0A0A0MRS2
2349A0A0A0MSD0
2350A0A0C4DFM7
2351A0A0C4DG73
2352A0A0D9SFA9
2353A0A0E3DCV7
2354A0A0J9YVW2
2355A0A0J9YVW5
2356A0A0J9YX90
2357A0A0S2Z4B5
2358A0A0X9UWJ6
2359A0A126GVE6
2360A0A126LB13
2361A1L4H1
2362A6NEQ2
2363A6PVK5
2364A8K0K1
2365A8K0P8
2366A8K0R7
2367A8K2P0
2368A8K7T4
2369A8K874
2370A8K8X0
2371A8K964
2372A8MTJ3
2373B0AZS6
2374B3KM36
2375B3KNK5
2376B3KPZ8
2377B3KQU2
2378B3KWQ8
2379B3KX74
2380B4DEB0
2381B4DEG1
2382B4DFY0
2383B4DGQ7
2384B4DGQ8
2385B4DIP4
2386B4DJ30
2387B4DL30
2388B4DM84
2389B4DMV8
2390B4DP50
2391B4DPV2
2392B4DPY2
2393B4DRQ4
2394B4DU77
2395B4DW52
2396B4E0D8
2397B4E2L2
2398B4E2M7
2399B4E2Y4
2400B7Z1H7
2401B7Z2V6
2402B7Z4C0
2403B7Z6W4
2404B7ZLW8
2405B7ZMN7
2406C6GLW5
2407C9JH44
2408C9JR58
2409C9JW04
2410D6R9I8
2411D6REZ4
2412E1A689
2413E5RJI5
2414E9PAV3
2415E9PL83
2416E9PPG5
2417F1T0E5
2418F2Z2U4
2419F6QYI9
2420F8W0W6
2421F8W8H5
2422G3V471
2423H0Y645
2424H0YAB8
2425H0YB13
2426H0YJ11
2427H3BRX4
2428H3BT74
2429B3BUH4
2430H7BZH1
2431H7C149
2432H7C2W8
2433I6L894
2434I6L957
2435J3KQU2
2436J3QLS9
2437K0P793
2438K4DIA0
2439K4K7V6
2440K7EQ63
2441L0CQ38
2442M0QZR9
2443M0R261
2444M0R315
2445O00420
2446O14980
2447O15240
2448O43854
2449O75691
2450O95243
2451P00800
2452P01833
2453P05109
2454P0C869
2455P0CB47
2456P0CG39
2457P0DKL9
2458P12757
2459P26022
2460P28070
2461P35442
2462P35558
2463P43652
2464P54753
2465P55089
2466Q05CW1
2467Q09028
2468Q0VAS5
2469Q14455
2470Q16281
2471Q32MQ0
2472Q53HB3
2473Q53TR0
2474Q59EG0
2475Q59F22
2476Q59FG6
2477Q59H91
2478Q5BKT1
2479Q5FBY7
2480Q5T1R4
2481Q5TBK7
2482Q5TH58
2483Q658L2
2484Q658N2
2485Q684P5
2486Q69YM0
2487Q6IC83
2488Q6L9N1
2489Q6N097
2490Q6NUJ9
2491Q6NUN2
2492Q6NUP7
2493Q6NXR7
2494Q6P0Q1
2495Q6P2I0
2496Q6P5R7
2497Q6PJ72
2498Q6PK65
2499Q6QE20
2500Q6TCJ2
2501Q6TFL3
2502A0A075B6H0
2503A0A0A0MSY2
2504A0A0B4J1Z7
2505A0A0B4J2E5
2506A0A0J9YXN7
2507A0A0S2Z4J7
2508A4D2H0
2509A5PLN9
2510A8K2M0
2511A8K7H1
2512A8K9P0
2513B2R769
2514B2RA94
2515B3KMG3
2516B3KNF6
2517B3KSB9
2518B3KSG0
2519B3KSJ0
2520B3KU66
2521B4DJB9
2522B4DN12
2523B4DNP9
2524B4DPX6
2525B4DQ18
2526B4DQL5
2527B4DTK1
2528B7Z1Z9
2529B7Z5P0
2530C7TPG7
2531C9J5J4
2532C9JYJ6
2533D3DPB7
2534D6RHV3
2535D7RF68
2536E0CX09
2537E1U340
2538E5RH59
2539E5RJV2
2540E7EPF0
2541E7ESP4
2542E7EW93
2543E9PQ73
2544F5GX09
2545F5GYN0
2546F5H4E5
2547F5H6Q4
2548F8VWA3
2549F8W1K8
2550G3V599
2551H0UI12
2552H0Y853
2553H0YAH0
2554H0YFK0
2555H3BPJ7
2556H3BPS9
2557H3BTH8
2558H7C0I7
2559H7C4L7
2560J3KS13
2561J3QS03
2562L0R5C4
2563O75603
2564P08588
2565P0CE72
2566P20742
2567P21453
2568P35251
2569P49720
2570Q14766
2571Q15333
2572Q15389
2573Q15631
2574Q17RW2
2575Q53RS3
2576Q53S27
2577Q53T09
2578Q5BN46
2579Q5RHP9
2580Q5T6X2
2581Q5TCI4
2582Q5VX52
2583Q6AHZ7
2584Q6IBJ0
2585Q6NWP9
2586Q6P5T1
2587Q6PIP7
2588Q6U2F8
2589Q6UXN8
2590Q6UXQ0
2591Q6Y288
2592Q6ZMS2
2593Q6ZMU0
2594Q6ZMY6
2595Q6ZNI2
2596Q6ZNS1
2597Q6ZQQ2
2598Q6ZQY1
2599Q6ZRK5
2600Q6ZRK6
2601Q6ZRM8
2602Q6ZU10
2603Q6ZV46
2604Q6ZWC0
2605Q6ZWE2
2606Q6ZWG9
2607Q70JA7
2608Q71F56
2609Q71U70
2610Q75RY1
2611Q765P7
2612Q7LGC8
2613Q7Z418
2614Q7Z527
2615Q7Z528
2616Q7Z5A3
2617Q7Z5L7
2618Q7Z5P4
2619Q7Z5W6
2620Q7Z5Y7
2621Q7Z7L8
2622Q7Z7M0
2623Q7Z7M9
2624Q86SQ6
2625Q86SV6
2626Q86TJ9
2627Q86UD1
2628Q86V85
2629Q86VD1
2630Q86VY4
2631Q86WI1
2632Q86XP0
2633Q86YP6
2634Q8IUZ8
2635Q8IV28
2636Q8IV92
2637QSIVE3
2638Q8IWD5
2639Q8IXM7
2640Q8IY33
2641Q8IYA7
2642Q8IYF3
2643Q8IYP2
2644Q8IZ52
2645Q8IZD9
2646Q8IZK6
2647Q8IZQ8
2648Q8N1G2
2649Q8N2C7
2650Q8N397
2651Q8N4T4
2652Q8N715
2653Q8N7P7
2654Q8N7S3
2655Q8N8V4
2656Q8N904
2657Q8N987
2658Q8N9V6
2659Q8NA33
2660Q8NAV8
2661Q8NAY8
2662Q8NB82
2663Q8NCD8
2664Q8ND61
2665Q8NE22
2666Q8NEZ4
2667Q8TA93
2668Q8TAS6
2669Q8TB82
2670Q8TD20
2671Q8TDY8
2672Q8TE73
2673Q8WUV3
2674Q8WXC6
2675Q8WXE0
2676Q92861
2677Q969F2
2678Q96AA8
2679Q96BY7
2680Q96JF0
2681Q96KN7
2682Q96KP6
2683Q96M27
2684Q96M95
2685Q96N28
2686Q96QE3
2687Q96QZ0
2688Q96S01
2689Q96T80
2690Q96TB4
2691Q99715
2692Q9BWG1
2693Q9C005
2694Q9H1K6
2695Q9H212
2696Q9H382
2697Q9H3R1
2698Q9H4L7
2699Q9HBR0
2700Q9HBU3
2701Q9HD29
2702Q9NQ33
2703Q9NQW1
2704Q9NR48
2705Q9NTB9
2706Q9NZ53
2707Q9P2B2
2708Q9UC91
2709Q9UDL0
2710Q9UHK0
2711Q9UL81
2712Q9ULI1
2713Q9ULL0
2714Q9ULL1
2715Q9ULM8
2716Q9UPU5
2717Q9UPU9
2718Q9Y3Q7
2719S4R3E2
2720U5XN63
2721U6FSN9
2722V9GYK6
2723V9H019
2724V9HW33
2725V9HW38
2726V9HW43
2727X2L7S8
TABLE 4
Proteins in both MSCEX and APEX
1A0A024R0P8
2A0A024R3G0
3A0A087WVV2
4A0A087WXZ2
5A0A0A0MR07
6A0A0A0MSZ4
7A0A0G2JIC2
8A0A0K0K1H9
9A0A0S2Z4G9
10A0A140VK05
11A0A140VK46
12A2RU30
13A2VCQ4
14A4PB67
15A8K3Y6
16A8KAQ8
17A8MXT8
18B1AKG0
19B2R7P8
20B2R8Z8
21B2RBJ8
22B3KMD9
23B3KQF9
24B3KQX9
25B3KVF9
26B4DDT3
27B4DE33
28B4DFP1
29B4DHX7
30A0A024RA75
31A0A024RDS3
32A0A087WV40
33A0A087WYY5
34A0A0C4DFX3
35A0A0J9YX34
36A1JUI8
37A1L4G8
38A6NHB5
39A7J1R1
40A8K2H9
41A8K3B0
42A8K4S1
43A8K5U9
44A8K7A4
45B1AKQ8
46B2R780
47B2RAQ9
48B3KNK9
49B3KVV5
50B3KXA5
51B4DH55
52B4DKZ3
53B4DL46
54B4DNX1
55B4DQF6
56B4DQQ9
57B4DRV9
58B4DS32
59B4DUQ1
60B4DUY7
61B4DVU1
62B4DWG4
63B4DWH5
64B4DZN3
65B4E047
66B4E3M5
67B7Z1I2
68B7Z2W3
69B7Z2Z8
70B7Z6U7
71B7ZMB3
72B8Y0L3
73B8ZZ80
74B9ZVT1
75C5HTY9
76C9J406
77C9J7H8
78C9J9E8
79C9JJV6
80C9JNG9
81C9JPM3
82D3DS95
83A0A087X1Q2
84A0A087X225
85A0A0A0MTC7
86A0A0S2Z430
87A8K2T9
88B4DE36
89B4DMR3
90B4E2A1
91B4E3S2
92B9EK61
93C9JEL4
94C9JXX4
95A0A087X0T3
96A0A0S2Z3J5
97B3KUJ8
98B4DM82
99B4DP93
100B4DS46
101B4DY39
102B7Z2Y4
103C9J673
104A0A024R8L6
105A0A024RBZ8
106A0A087WUI7
107A0A0B4J1Z4
108A0A0S2Z3L7
109A0A0S2Z4I7
110A0A0U1RQJ2
111A0A0U1RQK7
112A2A352
113A0A024R1N1
114A0A024R8F1
115A0A087WVD1
116A0A0C4DGF5
117A0A0C4DGS5
118A0A0S2Z542
119A0A140TA45
120A0A140VJW5
121A4FVC0
122A4QPE1
123A5PKV2
124A6NEM2
125A8K5S1
126A8K6S1
127A8K9T9
128B1AMW7
129B1Q3B3
130B2R577
131B2R7F8
132B2R7M3
133B2R7T2
134B2R8H4
135B3KN59
136B3KQT9
137B3KS79
138B3KSG1
139B3KSR8
140B3KTM9
141B3KTP2
142B3KTP9
143B3KW21
144B3KWF2
145B3KXY6
146B4DE00
147B4DF60
148B4DGH6
149B4DH24
150B4DI69
151B4DID5
152B4DK14
153B4DL98
154B4DLA1
155B4DLA3
156B4DNR7
157B4DPJ4
158B4DRT0
159B4DTX0
160B4E368
161B7Z1D9
162B7Z992
163B8ZZF3
164C9J4J8
165C9J4Z7
166C9JBE1
167C9JDW2
168CQJJP8
169CQJUM1
170C9JZG1
171C9JZW3
172C9K028
173D3DQB3
174D3DQI7
175D3DWY7
176D3DX70
177D3K0R1
178D6RAL9
179A0A024R0G8
180A0A024R1Y8
181A0A024RCZ4
182A0A087WTQ6
183A0A087X2E9
184A0A140VK29
185B1ALD0
186B2RBA0
187B3KWJ0
188B4DGD7
189B4DKI0
190B4DM67
191A6NE76
192A6NJA2
193A9LSU1
194B4DDL1
195B4DIV8
196B4DNH8
197B4DP22
198B4DR58
199B4DTN6
200B4DX99
201B4DYQ7
202B4E0S6
203B4E2M0
204B7Z2S7
205B7Z2V8
206B7Z864
207C9JPU9
208C9JZ53
209D6R972
210A0A024R0G0
211A0A024R4C5
212A0A024R7Z5
213A0A024RC72
214A0A087WUW5
215A0A087WVV1
216A0A087X1N8
217A0A087X232
218A0A0A0MT20
219A0A0N7A6P0
220A0A0U1RQV3
221A0A140TA54
222A0AV88
223A3KC71
224A4QPB0
225A7E2Y1
226A7LFP5
227A8K6I4
228B1ALC0
229B2R514
230B2R6L0
231B2R7J8
232B2RCM3
233B2ZZ90
234B3KM30
235B3KMW3
236B3KP89
237B3KR36
238B3KUS2
239B3KW84
240B4DFR3
241B4DG30
242A0A024R879
243A0A087WYV9
244A0A087X054
245A0A087X0S5
246A0A0D9SG88
247A0A0E3JSF5
248A0A0S2Z3W2
249A0A0S2Z6B4
250A0A0U1RRC9
251A0PJC9
252A1KY36
253A4UCT2
254A8K0G3
255A8K709
256A8K8Z1
257A8KAY3
258B2R8G9
259B2RBF8
260B3KWG6
261B3KY55
262B4DHT1
263B4DI54
264B4DKW1
265B4DL92
266B4DLZ2
267B4DMF7
268B4DPQ6
269B4DPS0
270B4DPU0
271B4DPU6
272B4DQ56
273B4E0N9
274B4E261
275B4E302
276B7Z273
277B7Z2M1
278B7Z4F6
279B7Z4W2
280B7Z856
281B7Z9M4
282B7ZAJ4
283B7ZB44
284B7ZLI7
285B7ZM04
286B9DI82
287B9EGI2
288C0JYZ1
289C1PHA2
290C4P0D4
291C9J315
292C9J9C1
293C9JKA9
294C9JN98
295D3DPK5
296D3DPU8
297D3DUK1
298A0A024R1A3
299A0A024RC58
300A0A0S2Z5A5
301A8K2R3
302A8K482
303A8K6V0
304B1ALD9
305B1B5R8
306B2RDG0
307B3KU23
308B3KUI4
309B4DGN8
310B4DIW7
311B4E3I6
312C9J539
313A0A087WX80
314A0A140VK07
315B3KUJ2
316B3KXN4
317B4DJF2
318B4DM34
319A0A087WZ85
320B2RCP7
321B3KNZ4
322A0A024R127
323A0A024R755
324A0A024RDT5
325A0A087WUV5
326A0A087WUZ3
327A0A0A0MSX9
328A0A0A7C7U2
329A0A0C4DG95
330A0A0C4DH07
331A0A0D9SG74
332A0A0J9YVZ6
333A0A0J9YWZ2
334A0A0S2Z3K0
335A0A0U1RRA4
336A0A140VJF9
337A4UCT0
338A8K7E0
339A8KA38
340A8KA74
341B1AH99
342B2RCA1
343B2RCJ7
344B2RDI7
345B3KN37
346B3KNG6
347B3KNX0
348B3KPI8
349B3KQH1
350B3KRC6
351B3KRN4
352B3KS36
353B3KS49
354B3KSB2
355B3KT90
356B3KTR9
357B3KUP0
358B3KY30
359B4DDH3
360B4DFM1
361B4DL06
362B4DM22
363B4DM79
364B4DQH6
365B4DUI5
366B4E0H8
367B7Z1H4
368B7Z213
369B7Z2N5
370B7Z5S9
371B7Z658
372B7Z7E5
373B9EIS5
374C9J1P7
375C9JC48
376C9JUP7
377C9K031
378D6R9L2
379D6RAF4
380D6RAX7
381D6RB21
382D6RBE9
383D6RBJ7
384D6RCL6
385D6RDP1
386D6RF19
387D6RF62
388D6RF77
389D6RF86
390D6RGK8
391D6RGV2
392D6RJ91
393D6RJ96
394D6RJA4
395D6RJI3
396D9ZGF4
397E2DRY6
398E5RGB0
399E5RGR6
400E5RGU3
401E5RH00
402E5RIV9
403E6Y3G0
404E7ERL0
405E7ETE2
406E7ETF9
407E7EX41
408E9PCM2
409E9PD92
410E9PFL9
411E9PGT6
412E9PJF7
413E9PKD5
414E9PKP4
415E9PKW6
416E9PMR6
417E9PNW4
418E9PQ82
419E9PRM1
420E9PS23
421E9PS78
422F5GX11
423F5GXD8
424F5GZN3
425F5H520
426F6MF51
427F8VUF6
428FSVYY9
429F8VZY9
430F8W7P5
431F8W943
432FSWBV3
433F8WCH0
434F8WD56
435FSWDR7
436F8WEQ7
437G3CIH8
438G3V1B5
439G3V1S6
440G3V210
441G3V281
442G3V4S5
443G3V5Z7
444G3XAM7
445G5E9W1
446H0Y409
447H0Y430
448H0Y465
449H0Y4A0
450H0Y5U1
451H0Y7G9
452H0Y7R7
453H0Y8M8
454H0Y9N2
455H0Y9Q9
456H0YAY4
457H0YCE1
458H0YCX6
459H0YCY8
460H0YD73
461H0YEL2
462H0YGK8
463H0YH82
464H0YI09
465H0YI26
466H0YJP3
467H0YM36
468H0YMB1
469H0YMP8
470H3BPX2
471H3BQU9
472H6UK83
473H6VRG2
474H7BXY6
475H7BZL4
476H7C063
477H7C064
478H7C0U5
479H7C1H6
480H7C1U0
481H7C2H5
482H7C410
483H7C5K4
484H7C5L4
485H7C5N8
486H7C5W6
487H7C5W8
488I1YAQ5
489I3L1Y9
490I3L392
491I3L3P5
492I3L425
493I3VM54
494J3KND3
495J3KNE3
496J3KPM9
497J3KRB5
498J3KRT5
499J3KRT8
500J3KSV6
501J3KTQ0
502J3QKR0
503J3QL20
504J3QQT0
505J3QQX2
506J3QR48
507J3QRS3
508J3QS32
509J3QS41
510J3QTA5
511J7HH10
512J7M3T8
513K7EJ01
514K7EJH8
515K7EKM4
516K7EKW4
517K7EL50
518K7ELI6
519K7EM20
520K7EP19
521K7EPB9
522K7ER62
523K7ERC6
524K7ESB6
525K7ESE8
526K7ESG6
527K7N7D6
528L7QJ95
529L8EAR0
530M0QZ17
531M0QZC5
532M0R0N8
533M0R0Q9
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2345H0Y5C0
2346H0Y5F3
2347H0Y5U0
2348H0Y8S0
2349H0Y987
2350H0Y998
2351H0Y9C2
2352H0YAM7
2353H0YAP9
2354H0YC35
2355H0YC94
2356H0YCR9
2357H0YEM3
2358H0YET2
2359H0YIH3
2360H0YJ03
2361H0YJD3
2362H0YKB3
2363H0YKS8
2364H0YL50
2365H0YM03
2366H0YM92
2367H3BPF6
2368H3BTD4
2369H3BTN5
2370H3BU78
2371H3BUH7
2372H7BY82
2373H7C1M6
2374H7C2H7
2375H7C2Q8
2376H7C2U0
2377H7C2Y0
2378H7C4X4
2379I3L4F9
2380I3L4N7
2381J3K000
2382K7EKL3
2383K7EKS1
2384K7EM90
2385K7EMN2
2386K7EPL0
2387K7ERF7
2388M0R0U4
2389M1VKI9
2390O94985
2391O95617
2392P30419
2393P98174
2394Q00839
2395Q05BW8
2396Q05DA4
2397Q05DS5
2398Q14650
2399Q1RMZ5
2400Q2EF79
2401Q2VP91
2402Q2YD88
2403Q3B7A7
2404Q4G0Q4
2405Q4LE57
2406Q4VC00
2407Q504V9
2408Q508I7
2409Q53F09
2410Q53FI4
2411Q53FK2
2412Q53FU5
2413Q53GU8
2414Q53GW3
2415Q53HE2
2416Q53HU7
2417Q53S41
2418Q58FF2
2419Q59EG5
2420Q59EZ1
2421Q59F71
2422Q59FD9
2423Q59GH5
2424Q59GK2
2425Q59GZ8
2426Q59HA3
2427Q59HB3
2428Q59HB5
2429Q5FC05
2430Q5HYA2
2431Q5JPH6
2432Q5JQ13
2433Q5QNY5
2434Q5T0F3
2435Q5TA02
2436Q5TFM2
2437Q60FE5
2438Q658J0
2439Q68CW0
2440Q68CX6
2441Q6AI13
2442Q6EHZ3
2443Q6PEG1
2444Q6PK56
2445Q6PKD2
2446Q6PQ81
2447Q6S4P3
2448Q6U2L6
2449Q6U8A4
2450Q6UQL6
2451Q6UUU9
2452Q6W6M8
2453Q6XYD2
2454Q6ZMY0
2455Q6ZMY3
2456Q6ZN49
2457Q6ZNB8
2458Q6ZNL4
2459Q6ZNU3
2460Q6ZP35
2461Q6ZP53
2462Q6ZR44
2463Q6ZRZ8
2464Q6ZS01
2465Q6ZS03
2466Q6ZS14
2467Q6ZSD7
2468Q6ZSL4
2469Q6ZT31
2470Q6ZT94
2471Q6ZTY7
2472Q6ZVC6
2473Q71RC2
2474Q71S06
2475Q71SW6
2476Q71UD4
2477Q71UM7
2478Q75MN1
2479Q75MY0
2480Q75N18
2481Q75N89
2482Q7KZ71
2483Q7L4M3
2484Q7L4N0
2485Q7L8K0
2486Q7RTQ9
2487Q7RU04
2488Q7Z2F6
2489Q7Z2X7
2490Q7Z355
2491Q7Z3G7
2492Q7Z3T9
2493Q7Z427
2494Q7Z487
2495Q7Z497
2496Q7Z4Z1
2497Q7Z6M3
2498Q7Z738
2499Q7Z757
2500Q7Z7K9
2501Q7Z7R0
2502Q86TQ3
2503Q86TX4
2504Q86U12
2505Q86U75
2506Q86U79
2507Q86UP3
2508Q86V48
2509Q86W61
2510Q86WD0
2511Q86XU5
2512Q86YN0
2513Q8IVF4
2514Q8IWC8
2515Q8IWL5
2516Q8IXX0
2517Q8IY44
2518Q8IY97
2519Q8IY98
2520Q8IYQ9
2521Q8IZD4
2522Q8N1I1
2523Q8N1I8
2524Q8N274
2525Q8N294
2526QSN4Z1
2527Q8N505
2528Q8N5L9
2529Q8N6NS
2530Q8N6P3
2531Q8N7Y3
2532Q8N7Z5
2533Q8N9C4
2534Q8N9F8
2535Q8N9K4
2536Q8N9Q8
2537Q8NA68
2538Q8NB89
2539Q8NBH6
2540Q8NCJ3
2541Q8NE02
2542Q8NF03
2543Q8NF19
2544Q8NF24
2545Q8NF60
2546Q8NG20
2547Q8NH73
2548Q8NI27
2549Q8TAS1
2550Q8TAT5
2551Q8TB01
2552Q8TB95
2553Q8TBN2
2554Q8TDG6
2555Q8TDS4
2556Q8TEP3
2557Q8TES4
2558Q8WTY5
2559Q8WU03
2560Q8WUI6
2561Q8WVX2
2562Q8WW96
2563Q8WWB2
2564Q8WX69
2565Q8WZ56
2566Q92468
2567Q92547
2568Q92681
2569Q92945
2570Q93063
2571Q93093
2572Q969I5
2573Q96AA2
2574Q96AQ0
2575Q96AR9
2576Q96AX1
2577Q96B07
2578Q96B60
2579Q96BA4
2580Q96BG6
2581Q96C61
2582Q96CD8
2583Q96CV8
2584Q96D30
2585Q96DQ5
2586Q96DZ4
2587Q96EB3
2588Q96G38
2589Q96GF5
2590Q96GW1
2591Q96HC2
2592Q96HF4
2593Q96HI1
2594Q96HN5
2595Q96IE3
2596096JJ7
2597Q96K48
2598Q96K89
2599Q96KC0
2600Q96LJ7
2601Q96LN7
2602Q96LR2
2603Q96MN8
2604Q96N76
2605Q96NB3
2606Q96NX2
2607Q96PA3
2608Q96PJ0
2609Q96PV0
2610Q96Q06
2611Q96QU5
2612Q96RL8
2613Q96RP4
2614Q96RS2
2615Q96RZ7
2616Q96SE4
2617Q99435
2618Q99529
2619Q99538
2620Q99666
2621Q9BR56
2622Q9BR60
2623Q9BS14
2624Q9BS75
2625Q9BSD0
2626Q9BTL0
2627Q9BVS9
2628Q9BW34
2629Q9BXG2
2630Q9BXV5
2631Q9BZ93
2632Q9BZQ0
2633Q9H049
2634Q9H0M4
2635Q9B2E0
2636Q9H2E1
2637Q9H369
2638Q9H3B0
2639Q9H3R3
2640Q9H3U3
2641Q9H511
2642Q9H5S1
2643Q9H5T0
2644Q9H717
2645Q9H7J0
2646Q9H8J8
2647Q9HAP0
2648Q9HAP1
2649Q9HB74
2650Q9HBB2
2651Q9HBP0
2652Q9HC77
2653Q9HCI6
2654Q9NP01
2655Q9NPA9
2656Q9NPM2
2657Q9NQG5
2658Q9NRE2
2659Q9NRY4
2660Q9NS89
2661Q9NSH2
2662Q9NSK3
2663Q9NSM5
2664Q9NT11
2665Q9NTC4
2666Q9NTU6
2667Q9NV59
2668Q9NVF8
2669Q9NVY6
2670Q9NW05
2671Q9NW43
2672Q9NWD6
2673Q9NWP5
2674Q9NWW3
2675Q9P0V0
2676Q9P1C5
2677Q9P1G4
2678Q9P1N9
2679Q9P1Y0
2680Q9P262
2681Q9P2G7
2682Q9UBC7
2683Q9UD42
2684Q9UD69
2685Q9UDE8
2686Q9UDY3
2687Q9UDZ8
2688Q9UE33
2689Q9UE89
2690Q9UES0
2691Q9UFZ4
2692Q9UG85
2693Q9UGU0
2694Q9UIU0
2695Q9UJ56
2696Q9UJM0
2697Q9UJM1
2698Q9UJN9
2699Q9UJZ2
2700Q9UJZ7
2701Q9UM89
2702Q9UMB3
2703Q9UMN4
2704Q9UNF3
2705Q9UNU2
2706Q9UPH5
2707Q9UQC1
2708Q9UQS6
2709Q9Y267
2710Q9Y2Q2
2711Q9Y4R1
2712Q9Y546
2713Q9Y5E1
2714Q9Y623
2715Q9Y6D3
2716Q9Y6N6
2717Q9Y6U3
2718R4GMY1
2719R4GNC2
2720R4GNC7
2721R4RWV3
2722S4R328
2723S4R3E9
2724S4R3G7
2725S4R451
2726S5FMV1
2727U3KPX5
2728U3KQA9
2729U3KQE2
2730U3KQI3
2731U3KQK2
2732U3KQK8
2733U3KQP1
2734U3PXP0
2735U3REJ1
2736V5QSK8
2737V9GYK3
2738V9GYL0
2739V9GYV7
2740V9GZ17
2741V9GZ54
2742V9GZ76
2743V9GZR9
2744V9H0H3
2745V9H1C1
2746V9H1D9
2747V9HVZ6
2748V9HVZ7
2749V9HW24
2750V9HW31
2751V9HW35
2752V9HW55
2753V9HW63
2754V9HW80
2755V9HW89
2756V9HW90
2757V9HW95
2758V9HWA6
2759V9HWA9
2760V9HWC0
2761V9HWC1
2762V9HWC6
2763V9HWC7
2764V9HWE9
2765V9HWF4
2766V9HWF5
2767V9HWG9
2768V9HWH6
2769V9HWH9
2770W5X314
2771W6I206
2772W6MEN4
2773W8QEH3
2774W8QRJ0
2775X5D2F4
2776X5D2Z4
2777X5D784
2778X5D7K9
2779X5D7R7
2780X5DQS5
2781X5DQV1
2782X5DR74
2783X6R3R3
2784X6R6Z1
2785X6RJP6
2786X6RLX0
TABLE 5
Proteins in both NPEX and MSCEX
1A0A024RBF5
2A0A087WTU5
3A0A087WTY5
4A0A087WZY3
5A0A0A0MSD7
6A0A0U1RQQ9
7A8K457
8B0YIW5
9B2R6M6
10B2RWN5
11B3KM34
12B3KS32
13B4DK21
14B4DLA8
15B4DM04
16B4DYP3
17B7Z220
18B7Z3F8
19A0A0A0MRE5
20A8K5A6
21B2RWN8
22B3KQE6
23B4DIY9
24F2Z2Y4
25I7GY12
26K7EIZ7
27A1DRY3
28A8K2S5
29B4DJ85
30D3DSZ2
31D6RHJ6
32E5RK22
33A0A090N7W7
34A0A024R9U8
35A0A087WTQ1
36A0A0A0MTS5
37A0A0J9YWF7
38A0A0S2Z5A6
39A0A140G945
40A0PJA6
41A8MYA2
42B3KY01
43B4DDZ2
44B4DJ87
45B4DN40
46B4DPI7
47B4DQJ6
48B7WNR7
49B7ZAV4
50C9J4J0
51D1CS68
52E9PR32
53H0Y4V9
54H0YE86
55H3BM13
56H7C124
57J3KNF5
58J3KTE8
59J3QQZ1
60K7EMU5
61A0A087X1K6
62B9EK65
63D2CPJ9
64A8K9G8
65E9PDY4
66E9PKB7
67F5GWB9
68H0Y5A3
69H0YDX7
70H0YGC5
71I3L2H2
72K7ENM8
73A0A024R2Q7
74A0A024RAC9
75A0A087X043
76A1L497
77A8K5C2
78B2R7A1
79B3KML1
80B4DPN0
81B4DPP6
82B4DSG6
83B4DW61
84B4DY21
85B4E1C1
86B7Z3E5
87B7Z8R2
88B7ZM83
89C9JUF9
90A0A0S2SW46
91A0A140G961
92A5YM44
93A8K3I0
94B2RCM2
95B2RD90
96B2RDH6
97B2RUU6
98B3KPC9
99B4DKV6
100C7FEB0
101E7EUN2
102F2Z2C0
103G3F4G3
104H3BUZ9
105I3L2G4
106J3KST4
107L0R875
108M0QY24
109M0QY93
110M0R167
111A0A140T9N1
112B3KV66
113B2RN10
114B4DX19
115A0A087WTM7
116A0A087WVZ9
117A0A087WZI7
118A0A0C4DGZ0
119A0A0F7T737
120A0A126GW30
121A8K5R3
122B1AA16
123B2RD71
124B4DQ03
125B4DRU3
126B4E1H3
127B4E3L4
128B5A930
129G1UI21
130H7C5E8
131I3L0S6
132K7ELE6
133K7ERU1
134O00303
135O15232
136O75144
137O75643
138O95757
139O95816
140P00390
141P04406
142P05455
143P06576
144P11908
145P12277
146P13010
147P13797
148P13798
149P14625
150P17787
151P18206
152P20592
153P20618
154P21399
155P23381
156F24534
157P25787
158P30520
159P30566
160P35052
161P37837
162P40925
163P43490
164P43699
165P48723
166P49721
167P49916
168P50991
169P55290
170P60842
171P61158
172P61970
173P78357
174P78371
175Q02809
176Q04446
177Q05BW9
178Q05CU5
179Q05DQ6
180Q08431
181Q13191
182Q14397
183Q14974
184Q16555
185Q4G0U7
186Q4LE70
187Q4QQP8
188Q56A80
189Q58F05
190Q59GL5
191Q59H14
192Q5SRE5
193Q5VTG7
194Q6P1A2
195Q6P668
196B4DVZ8
197E9PN76
198A0A0A0MS56
199A8K228
200B4DHL3
201A0A024R0V9
202A0A024R2F7
203B2RB99
204B3KVJ8
205B4DL71
206B7Z5Q5
207F8WCB0
208G3V470
209H0YGP2
210H7BXX0
211P11310
212P13639
213P22234
214P41250
215P52209
216P98095
217Q00610
218Q04760
219Q1JQ81
220Q4LE34
221Q4ZG60
222Q562P0
223Q5SZP4
224Q5T7Q5
225Q6NR85
226Q6PKB2
227A0A024RDI0
228B3KVT0
229B3KW08
230B5BU64
231F8VTS6
232Q17RV3
233Q4LE35
234Q53ZD9
235H7BZ78
236P20700
237P23528
238P28066
239P43686
240P51665
241P63241
242Q12765
243Q14666
244Q5T9B7
245A0A024R0F1
246A6NNK5
247A7Y9J9
248B2RCN5
249B3KNG8
250B3KW67
251B4DJ43
252B7ZBJ6
253E5RGB1
254E9PNV8
255G5E9C5
256H0YD68
257O15020
258A0A024R3W8
259A0A024R4E7
260A0A024R7L2
261A0A024R8K8
262A0A024R8L5
263A0A024R8U4
264A0A024R9F1
265A0A087WVC6
266A0A087WW67
267A0A087WYA1
268A0A0C4DFY7
269A0A0G2JNI0
270A0A0J9YY10
271A0A126GVS6
272A2RTX5
273A6NF36
274A8K324
275A8K4N3
276A8K8J5
277A8K8R2
278B3KQ44
279B3KWP3
280B3KXW2
281B4DEL6
282B4DH82
283B4DJT2
284B4DLC6
285B4DNG2
286B4DS99
287B4DWK2
288B4DYK9
289B4DYX8
290B4E303
291B4E367
292B7Z1P9
293B7Z671
294C9JDR5
295C9JVG0
296D3DTQ7
297D3DXG8
298D4PHA4
299E3W983
300E5G743
301B5RG96
302E5RGT3
303E7EVQ3
304E9JVC4
305E9PH60
306E9PK12
307F5GXY0
308F5GY37
309F5H2D1
310F5H8J8
311F8UU18
312F8VWL0
313F8W7F7
314G3V5M4
315H0Y3H6
316H0Y6A5
317H0YBF7
318H0YF13
319H0YGG5
320H0YL42
321H3BRY5
322H7C463
323I6NLS4
324J3QKX5
325O00410
326O14943
327O15067
328O95996
329O96005
330P05386
331P06744
332P08069
333P12956
334P22102
335P29401
336P30153
337P32119
338P42785
339P50747
340P53396
341P62314
342Q06323
343Q06AH7
344Q08629
345Q13393
346Q13616
347Q14563
348Q15828
349Q1KLZ0
350Q29RF7
351Q2I0A9
352Q32Q12
353Q495G0
354Q53R19
355Q59EI5
356Q59ET3
357Q59GA8
358Q5SYQ9
359Q5XUU0
360Q6FH47
361Q6IFB0
362Q6LAP8
363Q6W4X9
364Q6XYC2
365Q6ZMN7
366Q6ZS81
367Q6ZTQ4
368Q6ZWK7
369Q7L2H7
370Q7Z3Y4
371Q7Z3Z0
372Q7Z442
373Q7Z4C2
374Q7Z534
375Q7Z5C1
376Q7Z6C1
377Q86T01
378Q86T64
379Q86VU1
380Q86X45
381Q86XV5
382Q86YS3
383Q8IVC0
384Q81VV2
385Q8IWJ2
386Q8IXV0
387Q8IYT4
388Q8IZ41
389Q8IZC6
390Q8N303
391Q8N3N3
392Q8N3T6
393Q8N696
394Q8N8W4
395Q8NAD5
396Q8NBX0
397Q8NFN8
398Q8NG11
399Q8TA92
400Q8TAK2
401Q8TCE1
402Q8TDJ6
403Q8TDW7
404Q8TEU7
405Q8WUM4
406Q8WVV7
407Q8WVW5
408Q8WY27
409Q92484
410Q92820
411Q92863
412Q92905
413Q92973
414Q96AA1
415Q96HI4
416Q96I12
417Q96KR9
418Q96LC1
419Q96P44
420Q96RY7
421Q96SM3
422Q96ST3
423Q99436
424Q99598
425Q99707
426Q99832
427Q9BT36
428Q9BT78
429Q9BTY9
430Q9BWV3
431Q9BX63
432Q9BXS5
433Q9BXT5
434Q9C0C2
435Q9H0G2
436Q9H1W6
437Q9H3E1
438Q9H3Q7
439Q9HC03
440Q9NR71
441Q9NU86
442Q9NWN9
443Q9NYC9
444Q9P0S0
445Q9UCB0
446Q9UCS3
447Q9UE54
448Q9UF55
449Q9UGA0
450Q9UHV7
451Q9UIW2
452Q9UKP4
453Q9UKU9
454Q9ULC4
455Q9ULJ3
456Q9UNA2
457Q9UNM6
458Q9UPV8
459Q9Y266
460S4R3N1
461S4R3S7
462S4R400
463T1S9D5
464V9HWF8
465W4VSQ3
466W8SJH0
467X6R6S3
TABLE 6
Proteins in NPEX and APEX
1A0A024R3G7
2A0A024R8E2
3A0A087WTU9
4A0A087X191
5A0A0A0MSS2
6A0A0A0MTL9
7A0A0A6YYJ5
8A0A0J9YY72
9A4FTX9
10A5D906
11A5HML1
12B4DDH4
13B4DDI8
14B4DFH6
15B4DJQ7
16B4DV85
17B4DXB2
18B7Z7R2
19B7Z8P5
20B3KSE9
21B4DIA1
22B4DMS3
23B4E2P9
24B4E3I5
25B7Z380
26B7Z403
27B7Z625
28B7Z8W6
29B7ZLP5
30C9J2C0
31C9JER5
32E7EUT5
33E9PFH7
34E9PKG1
35G1UI17
36H0Y871
37H0YME5
38H3BMM5
39H9KV48
40J3QS51
41K7EIJ8
42K7ELF8
43K7ELK0
44A0A140CTX8
45A8K3Y5
46B4DFM5
47B4DGH0
48B4DWX3
49B4DXM1
50B7Z780
51C9JMY1
52A0A024R9W5
53A0A068F7M9
54A0A0A0MQZ2
55A0A0K0Q2Z1
56A8K719
57A8KAC4
58B2R6V9
59B3KW36
60B4DEN5
61B4DJX6
62B4DMD5
63B4DMN1
64B4DPD5
65B4DUJ5
66B4DZR3
67B7ZLC9
68B7ZLX9
69F5H6J0
70F8VV04
71H0YEZ5
72H7C1I7
73J3KS22
74K7ESP4
75A0A126GW47
76B3KVV6
77B4DEQ2
78B4DL70
79B4E3H4
80B9EK46
81C9JJE5
82C9K0S6
83A8K7H3
84A9UEZ6
85B3KNM0
86D4YW74
87F8W888
88G3V200
89G3V3A7
90H3BT58
91A0A024RAM0
92A0A0G2JS52
93A0A0U1ZI18
94A8K972
95A8K9U4
96A8KA50
97B2R6U8
98B3KMK2
99B4DV51
100B4DVV1
101B4DYQ4
102B4DZP8
103B5BUC0
104B7Z1E1
105B7Z7U9
106A0A024RA21
107A0A087X201
108A2MYD1
109A8K6R5
110A8K9I9
111B2R7E8
112B3KXS5
113B4DIX9
114C5J0G2
115C9J5X1
116E9PGN7
117F5H7S7
118H7BY57
119I3L2J8
120J3KR44
121A0A140VK43
122A8K2P6
123B7Z6W9
124A0A087WXD4
125B3KT18
126A0A024R333
127A0A024R637
128A0A059U7G5
129A0A0C4DGM5
130A0A0S2Z4F3
131A6NHN2
132A8K291
133B2R4S5
134B4DDN3
135B4DDS9
136B4DFK1
137B4DM68
138B4DNJ2
139B4DRC6
140B4DZV8
141B4DZW2
142B5A928
143D3DWL9
144E5RFX4
145E7ENU7
146F8WBE0
147H0YCC0
148H7BYM0
149I3NI20
150J3KP74
151J3KRC3
152O60391
153O60486
154O60760
155O75322
156O76061
157P02549
158P11216
159P20908
160P27105
161P42694
162P42702
163P48426
164P54578
165P59998
166P60660
167P61812
168Q01995
169Q0VAP0
170Q14571
171Q15181
172Q15670
173Q1WWK5
174Q1WWK6
175Q4LE40
176Q53EU0
177Q53GP3
178Q53HI6
179Q562U0
180Q580X3
181Q59EA4
182Q59ES1
183Q59EY7
184Q59G44
185Q59G78
186Q59GJ6
187Q5GIA7
188Q5H9N4
189Q5T765
190Q5TDF0
191Q5VT06
192Q5W0B7
193Q5XJ04
194Q6IN74
195Q6NSB2
196F8W9Z1
197H0YHD9
198F6TR53
199H0Y796
200A7E2C0
201B0I1R1
202A0A0G2JNC5
203B3KNW3
204B4DGV1
205D6RJD3
206E9PMI5
207H0YD65
208O43491
209O95782
210P16035
211P19320
212Q05D08
213Q12771
214Q2QD09
215Q2VPJ6
216Q59HC8
217Q5R210
218Q5VZB5
219Q68D26
220A0A024R566
221A0A024R899
222A0A087WTK0
223A0A087WWY8
224A0AQK0K1L8
225A0A0U1RRM0
226A0JLR0
227A4F4K4
228A4FVC3
229A6NLU0
230A8K1G1
231B4DEX9
232B4DF07
233B4DHL2
234B4DJ31
235B4DRK5
236B7Z6W5
237B7ZAG8
238C9JCJ5
239C9JQU8
240E5RIA9
241E9PI86
242F5H5K0
243F8W1A4
244H0Y831
245H0YF06
246H0YLN7
247H3BRP2
248H7BYJ9
249J3KR05
250J3KTP9
251K7EJ78
252A6MW40
253A6NIW5
254B3KM58
255B3KXP1
256B4DFC4
257B4DS57
258B4DVN5
259B7Z670
260J3QSF4
261O43242
262O75554
263P02771
264P07476
265P13521
266P52951
267Q53FB6
268Q53G25
269Q53GZ5
270Q53H60
271Q53HR1
272Q53SW3
273Q59FX3
274Q59GW6
275Q59H85
276Q5GJ68
277Q5U0Q1
278Q5VWT9
279Q68DF0
280Q6ICB4
281A0A0S2Z455
282B4DHJ3
283B4E354
284B5BTY4
285O75952
286A0A024R5W0
287A8K607
288B3KTA8
289Q16798
290B7Z7A4
291G3V504
292H0YCA1
293B3KS69
294F8W062
295A0A024QZ45
296A0A0A0MTE8
297A0A0A1TTP8
298A4FU65
299A8K8J2
300A8K9A5
301B3KQQ7
302B4DI59
303B4DNH7
304B4DVM1
305B4E2K5
306B4E3M8
307B7Z6H0
308B8XXQ3
309D6RBD3
310D6W632
311E5RI06
312E7EPB6
313E7ERH2
314E7ERU0
315E7ETU9
316E9PMI6
317F5GX94
318F5GYF1
319F8VRQ0
320F8VRZ4
321G3V2J8
322G3V595
323H0YAN8
324H0YCE8
325H0YJK0
326M0QXS6
327P08697
328P61604
329Q13415
330Q1L857
331Q4FEB4
332Q53FV3
333Q53G79
334Q5SPU2
335Q6IS14
336P29317
337B4DHN0
338A0A024QZL4
339A0A024R235
340A0A024R8H6
341A0A087WZX0
342A0A0G2JNJ8
343B4DFL3
344B4DVM7
345I6L9I5
346P84243
347Q0EFC9
348Q13787
349Q15102
350Q6PK50
351Q6PKT5
352Q6UVJ0
353Q6ZP82
354Q7Z3P6
355Q7Z5Q5
356Q7Z5V0
357Q7Z5V6
358Q86UF4
359Q86Y65
360Q8IY52
361Q8IYA8
36208IZ02
363Q8N754
364Q8N948
365Q8N959
366Q8N995
367Q8NEZ3
368Q8NHP6
369Q8NI77
370Q8TA90
371Q8TCL2
372Q8TE54
373Q8WU08
374Q8WXI3
375Q8WY81
376Q96AP5
377Q96DF0
378Q96M29
379Q9BZ26
380Q9H197
381Q9H3W5
382Q9H4B7
383Q9H5H4
384Q9H7K8
385Q9H853
386Q9NQY1
387Q9NS29
388Q9P019
389Q9P2Y4
390Q9UBV4
391Q9UEH4
392Q9UHV4
393Q9UKN7
394Q9UKV8
395Q9UKX3
396Q9UNP4
397Q9UQH3
398Q9UQM3
399Q9Y2D4
400Q9Y2I4
401Q9Y2J7
402Q9Y5T6
403Q9Y617
404S4R2Z6
405S4R3K3
406S4R457
407V5N4G2
408V9HW13
409V9HW39
410V9HW77
411V9HWD6
412X5D2V8
413Q7Z407
414Q86UP2
415Q8N3Y0
416Q8TB46
417Q8TBN9
418Q92736
419Q96QD5
420Q99698
421Q9H281
422Q9HCU0
423Q9UC36
424Q9UML6
425X5D9D6
426X6R3L3
TABLE 7
Proteins unique to MSCEX
1A0A024QYT5
2A0A024QZN8
3A0A024R059
4A0A024R1C3
5A0A024R241
6A0A024R2M8
7A0A024R407
8A0A024R4B7
9A0A024R4W0
10A0A024R5D9
11A0A024R5G9
12A0A024R5Z8
13A0A024R6C0
14A0A024R6F8
15A0A024R6I1
16A0A024R6L8
17A0A024R6X4
18A0A024R8B3
19A0A024R8V7
20A0A024R930
21A0A024R9X6
22A0A024RB85
23A0A024RCP3
24A0A068B0Y9
25A0A068BI38
26A0A087WSZ1
27A0A087WTU7
28A0A087WW54
29A0A087WXB0
30A0A087WY10
31A0A087WZV0
32A0A087X0V8
33A0A088QF11
34A0A0A0MRF6
35A0A0A0MRS7
36A0A0A0MRZ9
37A0A0A0MTE1
38A0A0A1TE42
39A0A0A6YY96
40A0A0B4J1S7
41A0A0C4DFX6
42A0A0C4ZN31
43A0A0G2JMD2
44A0A0G2JNH7
45A0A0G2JNU3
46A0A0G2JQU7
47A0A0J9YY34
48A0A0K0KS85
49A0A0S2Z3G9
50A0A0S2Z645
51A0A0U1RQC7
52A0A0U1RRH9
53A0A140F1N4
54A0A140T9S2
55A0A140VK13
56A0AVL2
57A0PJ76
58A0PJM7
59A1KZ92
60A1L3V6
61A212N5
62A2RRC9
63A2VDK1
64A6NFQ2
65A6NFU0
66A7MAY2
67A8K0Z6
68A8K2M5
69A8K454
70A8K5X8
71A8K6R9
72A8K9U6
73A8MYV6
74A9YTQ3
75B0QYK4
76B1AHR3
77B1AJW0
78B1AUU8
79B2R6H3
80B3R6X6
81B2R6Y1
82B2R702
83B2R928
84B2R969
85B2RB72
86B2RC85
87B2RE36
88B2RWN6
89B3KQ04
90B3KR61
91B3KRU1
92B3KS75
93B3KSD8
94B3KTT6
95B3KU51
96B3KUR8
97B3KV69
98B3KW35
99B3KW47
100B3KXX8
101B4DDE5
102B4DE87
103B4DER4
104B4DEV8
105B4DFA5
106B4DFX9
107B4DMR9
108B4DIA7
109B4DIH7
110B4DNL5
111B4DNW7
112B4DPQ0
113B4DQI9
114B4DQQ0
115B4DR29
116B4DSP1
117B4DST5
118B4DTZ9
119B4DU14
120B4DWE2
121B4DWG5
122B4DWQ4
123B4DXC4
124B4DYB8
125B4DYX7
126B4DZK1
127B4DZP2
128B4E0I8
129B4E164
130B4E1Z4
131B4E295
132B4E3R6
133B4E3S9
134B5MCN7
135B7Z2D6
136B7Z2E6
137B7Z3I0
138B7Z3K9
139B7Z427
140B7Z4H0
141B7Z549
142B7Z5X3
143B7Z8B9
144B7Z9H7
145B7Z9J0
146B7ZAB8
147B7ZAL5
148B7ZL25
149B7ZMF3
150B8ZZH7
151C4B4C6
152C9J4M6
153C9J613
154C9JGT3
155C9JGV6
156C9JLB7
157C9JQI2
158C9JSI2
159C9JT28
160C9JYI9
161D2KTB5
162D2XBF0
163D3DV75
164D3DVD8
165D6R9U7
166D6RA00
167D6RF53
168D6RGY2
169D6W633
170E3W994
171E5KMI6
172E5KR05
173E5KRK5
174E5KTI5
175E5RGN8
176E5RHI0
177E7ENN3
178E7EUY3
179E7EWC2
180E7EX20
181E9PKTI
182E9PQI5
183E9PR16
184F2Z2C8
185F2Z2K5
186F5GX59
187F5GYJ8
188F5H065
189F5H226
190F5H432
191F5H4B6
192F6TLX2
193F8VU88
194F8VXG7
195F8VXL3
196F8VY04
197F8W0U9
198F8W9U4
199F8W9W0
200F8WCF6
201G1UI33
202G5E971
203G5ELZ6
204H0Y468
205H0Y5R6
206H0Y6I0
207H0Y8G5
208H0YAN3
209H0YBI2
210H0YC83
211H0YCJ2
212H0YEL4
213H0YH65
214H0YL91
215H0YM70
216H3BP20
217H3BSC0
218H3BSE1
219H3BSM2
220H3BTW3
221H3BV41
222H7BYJ1
223H7BYX7
224H7C074
225H7C1W8
226I3L3E8
227I3L3P7
228J3KN38
229J3KNC6
230J3KPK1
231J3KQ09
232J3KTB8
233J3QL70
234K0I859
235K7EIS7
236K7EKQ3
237K7EL65
238K7ELC2
239K7ELW0
240K7EMJ5
241K7EMR1
242K7EPJ9
243K7ESE0
244L0R6V3
245L8E853
246M0R1K3
247M0R361
248O00469
249O00562
250O00592
251O14530
252O14879
253O15013
254O15042
255O15131
256O43505
257O43820
258O60245
259O60266
260O60294
261O60296
262O60343
263O60522
264O60543
265O60603
266O75161
267O75339
268O75390
269O75578
270O94844
271O94854
272O94925
273O95165
274O95177
275O95361
276P00450
277P00488
278P00519
279P02538
280P02786
281P04054
282P04075
283P04818
284P05091
285P05231
286P05937
287P06493
288P09622
289P09960
290P0DMV8
291P10398
292P13667
293P13807
294P15311
295P18065
296P22670
297P23019
298P28065
299P28482
300P29074
301P30532
302P31327
303P34932
304P35916
305P40189
306P42898
307P46782
308P47755
309P48058
310P48643
311P49863
312P50748
313P51828
314P61978
315P62701
316P63208
317P68104
318P78477
319Q10570
320Q10713
321Q12770
322Q12905
323Q12913
324Q13241
325Q13315
326Q13765
327Q13885
328Q14019
329Q14112
330Q14584
331Q14678
332Q14691
333Q14980
334Q149N5
335Q14CN4
336Q15274
337Q15431
338Q15751
339Q15878
340Q1L838
341Q1RMC9
342Q1W6H9
343Q20BJ8
344Q2I096
345Q2TA76
346Q38SD2
347Q3KP44
348Q3KRB0
349Q3ZCN5
350Q3ZCT4
351Q495V9
352Q4G0L2
353Q4G192
354Q4QRK8
355Q4VNC1
356Q4W5F5
357Q53GQ2
358Q53HH4
359Q53QE9
360Q53Y51
361Q58P21
362Q59E87
363Q59G22
364Q59G34
365Q59GT7
366Q59GW5
367Q59GX6
368Q59HG1
369Q5CCK6
370Q5EBN2
371Q5F0I5
372Q5I0G3
373Q5J9B1
374Q5NV87
375Q5STU8
376Q5SVJ3
377Q5T764
378Q5TEJ7
379Q5U608
380Q5VU34
381Q5VUJ6
382Q5YYI1
383Q5VYS4
384Q5VZ00
385Q5VZB4
386Q5VZL5
387Q68CZ1
388Q68DA4
389Q68DB7
390Q6EKJ0
391Q6FI27
392Q6NT96
393Q6P0N9
394Q6P1K8
395Q6P2S1
396Q6P4R9
397Q6P6D5
398Q6PD62
399Q6UWZ7
400Q6UXN9
401Q70IA8
402Q71U36
403Q75MN6
404Q7L6B3
405Q7Z3Z1
406Q7Z4W1
407Q7Z6M0
408Q86T35
409Q86U18
410Q86UU0
411Q86UW9
412Q86V60
413Q86XL3
414Q86YL5
415Q8IVH2
416Q8IWP9
417Q8IYA6
418Q8N304
419Q8N3P5
420Q8N4P6
421Q8N5U0
422Q8N7F5
423Q8NBB8
424Q8NE35
425Q8NES3
426Q8NFP9
427Q8NFQ8
428Q8NGS8
429Q8TAA3
430Q8TC08
431Q8TEQ6
432Q8WVC6
433Q8WVJ9
434Q8WWJ6
435Q8WWL7
436Q8WXI9
437Q8WYG9
438Q8WYJ0
439Q92530
440Q92539
441Q92823
442Q969U7
443Q96A69
444Q96B09
445Q96B65
446Q96CU9
447Q96CZ8
448Q96F45
449Q96FJ0
450Q96GE4
451Q96HY6
452Q96JB2
453Q96JM4
454Q96MM8
455Q96N57
456Q96PX1
457Q96QF4
458Q96QI5
459Q96S69
460Q99539
461Q99574
462Q9BPU6
463Q9BQN1
464Q9BQS2
465Q9BS61
466Q9BSD7
467Q9BW71
468Q9BWD1
469Q9BWT3
470Q9BXB5
471Q9BXT8
472Q9BYF1
473Q9C091
474Q9H0Q0
475Q9H2E5
476Q9H2I8
477Q9H2Z3
478Q9H7U7
479Q9H7W5
480Q9H8N6
481Q9H8Q1
482Q9HA17
483Q9HAR0
484Q9HD74
485Q9NP17
486Q9NPU7
487Q9NR45
488Q9NRD1
489Q9NT26
490Q9NTG1
491Q9NW02
492Q9NX58
493Q9NX91
494Q9NZF5
495Q9P283
496Q9P291
497Q9P2M7
498Q9P2N1
499Q9UBG9
500Q9UBL0
501Q9UC78
502Q9UEH6
503Q9UF08
504Q9UFA7
505Q9UIA9
506Q9UJD0
507Q9UJT0
508Q9UL70
509Q9ULJ7
510Q9ULW6
511Q9UME3
512Q9UNM1
513Q9UNS1
514Q9UPN4
515Q9UPV0
516Q9Y216
517Q9Y4L1
518Q9Y613
519Q9Y696
520Q9Y6K1
521Q9Y6S7
522Q9Y6U9
523R4GMT0
524S4R2X0
525S4R314
526S4R3C8
527U3KQK0
528U5YBI9
529V9GY77
530V9GYE3
531V9HW26
532V9HWC4
533V9HWH0
534X5D7Q2
535X5D9D2
536X6RL08
TABLE 8
Proteins unique to APEX
1A0A087WXN9
2A0A0A0MTD1
3A0A0C4DFS8
4A0A0J9YX41
5A0A140VJI6
6A2VED2
7B0YJ74
8A0A0B4J1X7
9A0A5A9
10A8K1P5
11B3KMN2
12B5A934
13B7Z9C0
14A0A087X1U1
15A8K0G7
16B7ZLC4
17A0A140VKF7
18B4DY95
19A0A024R1K7
20A0A024RCS3
21A0A0G2JPN1
22A0A140VJP0
23B3KR94
24B3KSB6
25B4DET1
26B4DG15
27B4DJI2
28B7Z919
29C9J3L8
30A0A087X298
31B4DG65
32A0A0C4DH13
33B4DSP4
34B4DSR4
35C9J826
36A0A024RBC0
37A0A087X120
38A0A0C4DGC5
39A0A0U1WUY1
40A1L196
41A9CQZ2
42B3KX52
43A0A087X0D8
44B2RCJ5
45B4DN90
46B4DNZ9
47B4DZG1
48B7Z6I1
49C9J1D9
50C9J1K7
51A0AV47
52B3KNC3
53A0A024R120
54A0A087WUL9
55B3KMX4
56B3KNL6
57B3KPZ7
58B3KWB6
59B3KWC4
60D1CS35
61D2CPK5
62D3DUJ0
63D6REL5
64E5RHP0
65E9PCP0
66E9PJL7
67E9PNX2
68F5H0Y9
69F6QTA4
70F8W026
71G3V5R6
72G5E9J0
73H0Y757
74H0Y9N5
75H0YDE4
76H0YGV8
77H3BTQ9
78H7BZZ8
79H7C0D9
80J3KPD9
81J3KTL9
82K7EL58
83K7EMM9
84K7EQR9
85A2RRR7
86B4DFQ9
87B4DWW6
88A8MT18
89A0A024R461
90B4DZ23
91B7ZMJ6
92E5RJD0
93I7HAS0
94A0A075B6I0
95A0A087WU14
96A0A0U1ZBR1
97A0A140TA43
98A0A140VK41
99B2RA91
100B3KNB4
101B4DMA2
102B4DTA5
103B4DUF8
104A0A0C4DGH5
105A6NEP9
106ABK0K0
107B3KMS6
108B3KY88
109B7Z4X0
110E7ETB4
111E9PGC0
112F5H0M4
113F5H5D3
114H0Y463
115H7C080
116I3L0G6
117I3L1K1
118K7EJX8
119K7EK57
120A0A024R283
121A0A0C4DGB8
122A0A140VK27
123B4DY04
124B7ZM05
125K7ENN8
126B2R8P5
127H0Y873
128A0A024R542
129A0A024R7E1
130A0A0M4FNU3
131B4DUC5
132B4DZ03
133A0A024R957
134A0A0A0MRY9
135A0A140VK76
136B0YJ32
137B3KPK8
138B4DEY4
139B4DMJ7
140B4DNX8
141B4DQY2
142B4DR52
143B4DR67
144B4DTA9
145B4E058
146B7ZAW7
147B7ZLB7
148E9PNS9
149H7BXG6
150H7C520
151I3L466
152A0A0A0MRM2
153A4FU77
154A0A024QZ75
155A0A024R674
156A0A0A0MRH0
157A8K4M1
158B3KTQ2
159B4DJ21
160B4DMJ5
161B4DV55
162D3DSV6
163D6R9D5
164E9PP50
165H0Y2M6
166H0YCV9
167H0YJ91
168H7C5H1
169J3QRV6
170L8E9P0
171M0R120
172O00512
173O00763
174O14524
175O15078
176O15090
177O15244
178O43570
179O60291
180O75094
181O75600
182P00352
183P02749
184P05546
185P08910
186P10916
187P17927
188P27169
189P29400
190P35499
191P35749
192P39023
193P40939
194P46059
195P50225
196P54756
197P61964
198P84085
199Q003V9
200Q01831
201Q0VAI6
202Q12873
203Q12980
204Q13349
205Q14145
206Q14469
207Q15238
208Q15697
209Q15762
210Q16820
211Q2TSD0
212Q32Q67
213Q3SY52
214Q53G92
215Q53GF5
216Q53HR5
217Q53SE2
218Q58DX5
219Q59EF3
220Q59HA9
221Q5GJ67
222Q5TEU4
223Q68DP4
224Q69YH5
225Q6FHK0
226Q6FHK9
227Q6FHU3
228Q6FI03
229Q6IFE0
230Q6JH03
231Q6NUI8
232O15123
233O43301
234O43424
235O95239
236P02533
237P55283
238Q15142
239Q1HP67
240Q562N6
241Q5H9B5
242Q5HY54
243Q5HYL7
244Q5VUA0
245Q6IAX6
246Q6P1L4
247Q6P5Q4
248B3KXC1
249B3KP29
250B7Z7C2
251A0A024RDT3
252A8K990
253E5RGT9
254E9PMW9
255G3V174
256H7BXS7
257H7C402
258H9A532
259H3BP35
260A0A024RDR3
261B4DEW9
262C9J3M5
263I0CMK4
264K7ENA0
265B3KNV9
266B4DUK7
267K7EPF6
268A0A024QZF7
269A0A024R5V2
270M0QY71
271O75843
272O95905
273O96011
274P07585
275Q0VD99
276Q15374
277Q5VX85
278Q6IC98
279P30405
280Q5VIR6
281A0A0C4DFQ8
282B1AMW3
283A0A087WUF5
284A0A024R6K8
285B2R9P0
286A0A087WSV7
287A0A0K0KR34
288A6NFY4
289A0A024RD47
290B2RTX2
291E9PFF2
292F2Z2G2
293F5GWA7
294F5GXC7
295F5GYF8
296F5H3Q5
297H9C875
298B7Z2E2
299J3KQ72
300A0A024R909
301F5GXR6
302J3KS93
303A0A126GVW7
304B4E266
305A0A024R2H7
306A0A097CK88
307A0A140T999
308A8K8H7
309B4DND2
310B4DQM1
311B4DZ35
312E7ERI8
313F5GZQ4
314F8WFC4
315H0Y9E9
316H7C1T2
317K7EPY4
318M0R2D3
319O76074
320P15085
321Q3YA63
322Q5MNZ6
323Q6IPX8
324A0A087X2E5
325A0A0A0MQR7
326A0A0C4DGY8
327A0A0D9SF28
328A0A0S2Z333
329A0A0S2Z366
330A4D202
331A8K731
332B3KX01
333B3KX41
334B4DEM5
335B4DK22
336B4E337
337B7Z4N1
338C9J8U1
339D6R9E4
340E7BP61
341E7EWZ1
342E9KL23
343E9PJK2
344F5GXY2
345F5H032
346G3V454
347H0YF19
348H0YGH1
349H3BRL9
350K0A7K7
351A0A0G2JN61
352A0A0M4FEM1
353A0A0U1RQL8
354A8K6X5
355B3KW51
356B4DQG4
357B4DW50
358B4DZY4
359B4E1S5
360B4E1T9
361B4E3A4
362B7Z1Y2
363C9JUE0
364D6RDI0
365F8VUX9
366J9UPX7
367K7ENE1
368P13637
369P28827
370P31150
371P31946
372Q12769
373Q13841
374Q20BG7
375Q2F838
376Q58FF6
377Q658X2
378Q659A9
379Q6FGX9
380A0A024R3D4
381A0A024RAM6
382A0A0G2JP38
383A0A140VJV9
384A0A140VKF6
385B4DLH5
386D6RHE5
387O014777
388Q6IBD2
389A0A024R684
390A8K5F3
391B3KU50
392B4DUZ8
393K7EQ48
394O75665
395Q59EK9
396A0A024R7D2
397A3KFK1
398G3V0E6
399A0A024QYZ8
400A0A024RAM1
401A0A087WWI0
402A0A0A0MQR2
403A4D0S8
404A4F051
405A4QMW8
406A8K4Q4
407C9JVG3
408D6RC52
409E7ENJ7
410E9PHK0
411E9PQV8
412F5GZI5
413F5GZK2
414F5H2D0
415F5H6I7
416F6U236
417F8WAS2
418F8WAX8
419F8WCJ1
420H3BTW5
421H3BUA3
422K7EJH1
423M5EDK8
424O00160
425O15054
426P48444
427Q14258
428Q14434
429Q14690
430Q549N0
431Q6FH62
432A0A024RB92
433A0A087WW76
434B4DI80
435B4DTQ1
436B4E0B2
437D6RE83
438D9HTE9
439E9PMM8
440E9PNE6
441F2Z393
442F5H0P4
443H0Y476
444H0YCK3
445J3KRJ6
446J3QS44
447O75077
448P06727
449P20062
450Q09472
451Q14643
452Q6PKD3
453Q6QWC0
454Q6RBX8
455Q6UW88
456Q6UX71
457Q6ZMU5
458Q6ZP56
459Q6ZPD6
460Q6ZR36
461Q6ZU35
462Q6ZVM7
463Q709C8
464Q7M4R4
465Q7Z2V5
466Q7Z606
467Q7Z612
468Q7Z668
469Q7Z7H5
470Q7Z7P9
471Q86SG7
472QS6TE2
473Q86TI0
474Q86UE3
475Q86X82
476Q86XM6
477Q86XQ3
478Q86YC4
479Q8IUN7
480Q8IUQ0
481Q8IV08
482Q8IWJ6
483Q8IXA4
484Q8IYT1
485Q8N1M4
486Q8N1Y9
487Q8N4M9
488Q8NC18
489Q8NCS7
490Q8NGA0
491Q8TBG4
492Q8TCC9
493Q8WLP1
494Q8WV67
495Q8WVV4
496Q8WYB5
497Q92574
498Q92615
499Q92932
500Q92990
501Q969V5
502Q96FW1
503Q96G03
504Q96HX7
505Q96KR4
506Q96MY1
507Q99062
508Q99440
509Q99973
510Q99989
511Q9BT21
512Q9BT27
513Q9BVJ8
514Q9BWT2
515Q9BZF9
516Q9C0A1
517Q9H295
518Q9H2M9
519Q9H4N8
520Q9H6A9
521Q9H6L3
522Q9H6Z9
523Q9H769
524Q9H995
525Q9HBM3
526Q9NPL4
527Q9NS28
528Q9NUJ7
529Q9NWV8
530Q9NX81
531Q9NX98
532Q9NXP1
533Q9NYK1
534Q9NZ52
535Q9P0C1
536Q9P0P1
537Q9P2H3
538Q9UBK7
539Q9UDV6
540Q9UDX4
541Q9UFP2
542Q9UFU2
543Q9UG36
544Q9UG64
545Q9UGU8
546Q9UH61
547Q9UHC1
548Q9UHC9
549Q9ULH1
550Q9UNX4
551Q9UPS8
552Q9Y287
553Q9Y4G6
554S4R3L5
555T1WFC1
556U3KPS2
557U5YEI7
558V9GYM3
559V9HW42
560V9HWC2
561V9HWC3
562V9HWG1
563W6JLH6
564W8SBA0
565Q6ZNY8
566Q6ZP01
567Q7Z5U6
568Q86TA0
569Q86VI3
570Q86WN1
571Q86YG0
572Q8IWK6
573Q8IXR5
574Q8N111
575Q8N976
576Q8NG48
577Q8TCU4
578Q8WYK0
579Q99994
580Q9BRL4
581Q9BS12
582Q9BTS8
583Q9C0K3
584Q9H583
585Q9HBG7
586Q9P2H0
587Q9UF83
588Q9UHP3
589Q9UIT6
590Q9UJ65
591Q9UN47
592Q9UNY4
593U3KQU8
594X5D5A9
595X5D7Z6
596X5DNR2
TABLE 9
Proteins unique to NPEX
1A0A024DAK3
2A0A024QYX2
3A0A024QZJ1
4A0A024QZM2
5A0A024QZW5
6A0A024QZX5
7A0A024R095
8A0A024R0A0
9A0A024R233
10A0A024R2G9
11A0A024R2K1
12AQA024R301
13A0A024R4A0
14A0A024R4D1
15A0A024R4F1
16A0A024R4Z0
17A0A024R4Z5
18A0A024R5M9
19A0A024R5U3
20A0A024R5V6
21A0A024R5Y2
22A0A024R6G3
23A0A024R701
24A0A024R7B6
25A0A024R7L5
26A0A024R7T7
27A0A024R884
28A0A024R8F4
29A0A024R8I4
30A0A024R936
31A0A024R944
32A0A024RAF7
33A0A024RAS2
34A0A024RB01
35A0A024RBE5
36A0A024RBT9
37A0A024RCB7
38A0A024RCE1
39A0A024RCI0
40A0A024RD04
41A0A024RD09
42A0A024RD36
43A0A024RD77
44A0A024RDM3
45A0A024RDV8
46A0A024RDW5
47A0A075B6S4
48A0A075B734
49A0A087WSW7
50A0A087WT58
51A0A087WTH0
52A0A087WTK9
53A0A087WU78
54A0A087WUK8
55A0A087WUM0
56A0A087WVI0
57A0A087WW59
58A0A087WWE7
59A0A087WXK5
60A0A087WXM9
61A0A087WXX8
62A0A087WYC6
63A0A087WYK9
64A0A087WYW3
65A0A087WZ40
66A0A087WZC4
67A0A087WZR8
68A0A087WZZ7
69A0A087X052
70A0A087X0D6
71A0A087X176
72A0A087X1Z6
73A0A087X250
74A0A087X2B0
75A0A090N8H8
76A0A097IQZ3
77A0A0A0MQS0
78A0A0A0MRP0
79A0A0A0MRT2
80A0A0A0MRV0
81A0A0A0MRX4
82A0A0A0MS45
83A0A0A0MS53
84A0A0A0MS59
85A0A0A0MS84
86A0A0A0MSA7
87A0A0A0MSU4
88A0A0A0MT74
89A0A0A0MTC4
90A0A0A0MTD9
91A0A0A0MTQ1
92A0A0A0MTQ8
93A0A0A0N0L2
94A0A0A0N0M1
95A0A0B4JIR2
96A0A0B4J210
97A0A0B4J212
98A0A0B4J223
99A0A0B4J2C3
100A0A0C4DFX7
101A0A0C4DG17
102A0A0C4DG82
103A0A0C4DGA6
104A0A0C4DGI2
105A0A0C7DW92
106A0A0D9SF54
107A0A0D9SF63
108A0A0D9SG17
109A0A0E3JG42
110A0A0G2JHI3
111A0A0G2JI86
112A0A0G2JM47
113A0A0G2JNH0
114A0A0G2JP14
115A0A0G2JP37
116A0A0G2JRM9
117A0A0G2JRN3
118A0A0J9YVY3
119A0A0J9YVZ3
120A0A0J9YWL0
121A0A0J9YWY2
122A0A0K1JS24
123A0A0K2GMW5
124A0A0S2Z3P7
125A0A0S2Z451
126A0A0S2Z489
127A0A0S2Z4F1
128A0A0S2Z4H6
129A0A0S2Z4I9
130A0A0S2Z4R1
131A0A0S2Z4Y4
132A0A0S2Z4Y6
133A0A0S2Z563
134A0A0S2Z5B0
135A0A0S2Z5Z7
136A0A0S2Z618
137A0A0U1RQH4
138A0A0U1RQP0
139A0A0UIRQR9
140A0A0U1RRM1
141A0A126GVG1
142A0A126GWA2
143A0A126LAY8
144A0A126LB32
145A0A140HDC1
146A0A140T8X2
147A0A140T9Y3
148A0A140TA40
149A0A140TA77
150A0A140VJC9
151A0A140VJI0
152A0A140VJL3
153A0A140VJN8
154A0A140VJP5
155A0A140VJU3
156A0A140VK35
157A0A141PNN4
158A0A146IHP0
159A0A158T700
160A0JLR2
161A0PJY9
162A1A512
163A1L3A3
164A2A2M0
165A2A368
166A2ADX3
167A2NX49
168A2PYH4
169A2RUH7
170A2VCK2
171A4D126
172A4D1B7
173A4D1P7
174A4D1R1
175A4FU69
176A4FUA2
177A4QPE5
178A6H8W6
179A6NC48
180A6NC78
181A6NED2
182A6NP31
183A6NGH7
184A6NGQ3
185A6NHN7
186A6NHT5
187A6NM62
188A6NN40
189A6NP61
190A6Q8J1
191A6XAA7
192A7E294
193A7E2X7
194A7J1R0
195A8E631
196A8K0E1
197A8K146
198A8K1W3
199A8K3A3
200A8K3L7
201A8K3X2
202A8K4F0
203A8K4I1
204A8K525
205A8K594
206A8K5E6
207A8K5H7
208A8K5J8
209A8K6K4
210A8K6V3
211A8K7K0
212A8K855
213A8K8K1
214A8K8T9
215A8MX12
216A9QM74
217A9UK01
218B0QY51
219B0QY53
220B0QYP5
221B1AKL4
222B1AKN6
223B1AKN8
224B1ALU6
225B1B5Q8
226B2R5U7
227B2R694
228B2R6V2
229B2R6X2
230B2R734
231B2R736
232B2R7D2
233B2R7I3
234B2R7S8
235B2R7W6
236B2R7Z4
237B2R892
238B2R9P8
239B2R9S6
240B2RA29
241B2RAN2
242B2RB27
243B2RC06
244B2RCB8
245B2RCD2
246B2RCG9
247B2RD40
248B2RDD7
249B2RDT9
250B2RNB2
251B2RNT9
252B2RUU1
253B3FR89
254B3KM41
255B3KM42
256B3KMB1
257B3KMB8
258B3KMD2
259B3KMJ7
260B3KMZ6
261B3KN57
262B3KP18
263B3KPA6
264B3KPD7
265B3KPK9
266B3KPM6
267B3KQ65
268B3KQZ8
269B3KR52
270B3KRY3
271B3KS09
272B3KS20
273B3KS48
274B3KS82
275B3KSV0
276B3KSW4
277B3KSZ3
278B3KTD8
279B3KTM6
280B3KTR4
281B3KU97
282B3KUG5
283B3KUL9
284B3KUN1
285B3KUR9
286B3KUU1
287B3KV11
288B3KVH9
289B3KVJI
290B3KVN4
291B3KVP2
292B3KVU9
293B3KW07
294B3KW31
295B3KW52
296B3KWI5
297B3KWS8
298B3KX72
299B3KXH9
300B3KXW5
301B3KXX5
302B3KY97
303B4DE05
304B4DE48
305B4DE80
306B4DEI6
307B4DEX7
308B4DF38
309B4DFB6
310B4DFF1
311B4DPN8
312B4DG42
313B4DGF4
314B4DHN5
315B4DHX3
316B4DI94
317B4DIB9
318B4DIC2
319B4DJ53
320B4DKC2
321B4DKX2
322B4DL63
323B4DLP0
324B4DM77
325B4DMQ1
326B4DND4
327B4DP10
328B4DP52
329B4DQ24
330B4DQ80
331B4DQD4
332B4DRG0
333B4DRG2
334B4DRT4
335B4DS66
336B4DSA0
337B4DSC7
338B4DSC8
339B4DT06
340B4DTQ9
341B4DUB2
342B4DUY8
343B4DV73
344B4DVA7
345B4DWE9
346B4DWF9
347B4DWY2
348B4DWZ8
349B4DX03
350B4DXP2
351B4DXR7
352B3DXR8
353B4DY09
354B4DYD2
355B4DYE6
356B4DZ04
357B4DZ99
358B4DZC2
359B4PZC3
360B4DZC9
361B4DZD2
362B4DZS8
363B4E0B7
364B4E0E1
365B4E0J9
366B4E0Q4
367B4E143
368B4E173
369B4E282
370B4E2E2
371B4E2F7
372B4E2W8
373B4E2X3
374B4E3A8
375B4E3M6
376B5A954
377B5BU72
378B5MBY4
379B5TYJ1
380B7ZIG4
381B7Z282
382B7Z2F7
383B7Z2X2
384B7Z2Z2
385B7Z321
386B7Z3A3
387B7Z5H2
388B7Z5N6
389B7Z6E2
390B7Z6K2
391B7Z6P1
392B7Z6R5
393B7Z6T0
394B7Z6X5
395B7Z7R8
396B7Z7W2
397B7Z942
398B7Z970
399B7Z971
400B7Z9C6
401B7ZAR1
402B7ZAV2
403B7ZB07
404B7ZB16
405B7ZBD5
406B7ZBH1
407B7ZL21
408B7ZLC8
409B7ZMI2
410B7ZMM1
411B8ZZA5
412B9A6J2
413B9EG68
414B9EG95
415B9TWZ8
416C4IXU6
417C6ZGQ9
418C7S316
419C9J0Q5
420C9J268
421C9J408
422C9J524
423C9J712
424C9J7G0
425C9J8V3
426C9JBE8
427C9JCQ9
428C9JEN8
429C9JH19
430C9JSK8
431C9JYQ2
432D3DPF8
433D3DS02
434D3DSB5
435D3DVJ3
436D3DVS8
437D3DVT0
438D3DXI8
439D3DXI9
440D3TTY9
441D4IH22
442D5KMU6
443D6R921
444D6R936
445D6R938
446D6R9C2
447D6R9E3
448D6R9V7
449D6R9W4
450D6RB55
451D6RC76
452D6RD46
453D6RD74
454D6RE68
455D6RFH3
456D6RGK7
457D6RIC7
458D6RTK6
459D7NTU0
460E0Z3H0
461E2RYF6
462E5RFZ0
463E5RG94
464E5RHK2
465E5RJ52
466E6Y365
467E6Y3F9
468E7EME3
469E7EMS9
470E7ENT8
471E7EP60
472E7EPN9
473E7ERS3
474E7ESK9
475E7ET15
476E7ETR9
477E7EU35
478E7EUC7
479E7EV71
480E7EV99
481E7EVX8
482E9PB18
483E9PB90
484E9PCY5
485E9PDF5
486E9PEW0
487E9PF18
488E9PG59
489E9PGK7
490E9PGW9
491E9PIT3
492E9PK85
493E9PL71
494E9PLE2
495E9PLF1
496E9PMF9
497E9PMH3
498E9PMP7
499E9PNL2
500E9PPT0
501E9PQ63
502E9PQ78
503E9PQR7
504E9PS38
505E9PSI1
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1555Q9UDG3
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1590Q9UMV8
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1594Q9UNS2
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1597Q9UQP3
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1599Q9UQR9
1600Q9Y224
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1602Q9Y240
1603Q9Y264
1604Q9Y2B0
1605Q9Y2E5
1606Q9Y2G5
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1608Q9Y2M5
1609Q9Y2S0
1610Q9Y2T6
1611Q9Y2X7
1612Q9Y3I1
1613Q9Y3R5
1614Q9Y3X0
1615Q9Y423
1616Q9Y450
1617Q9Y467
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1622Q9Y4M4
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1631Q9Y6V7
1632R4GNE8
1633R4GNI9
1634S4R2Z0
1635S4R354
1636S4R3N6
1637S4R3P9
1638T2FFJ4
1639U3KQ69
1640U3KQL2
1641U5TP13
1642V9GYP9
1643V9HVU7
1644V9HW54
1645V9HWB9
1646W8JD10
1647X5D2S9
1648X5DNF2
1649X6R3I0
1650X6R772
1651X6R8W7
1652X6R922
1653X6RLN4

Therefore, the disclosed EVs are unique based on the source of cells from which they are derived. Moreover, these proteins can be used as a signature to identify the EVs.

This is the first documentation that we are aware of on a scale that requires the Amicon stirred-cell ultrafiltration units, allowing filtration and EV enrichment from 24 liters of media within one week, an amount of media that could not logistically be purified by ultracentrifuge, and would require intense manpower and multiple centrifuges using the smaller Centricon/Amicon centrifugal filter units.

The inclusion of hFGF2 from cell culture media in NPEX™, combined with the ability of NPEX EVs to distribute to targets within the CNS (as demonstrated in biodistribution section), suggests that these EVs can potentially deliver hFGF2 across the blood brain barrier to target CNS tissue. These results suggest that a potential method for delivering large molecules including proteins (hFGF2 in the example above) to targets in vivo, including the CNS, using extracellular vesicles (NPEX™ by supplementing the EV source cell (hNP1™ in example above) culture media with the molecule of interest.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

NEURAL CELL EXTRACELLULAR VESICLES (2025)
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