METHOD OF PRODUCING MICROPARTICLES

Abstract
The invention is based on the finding that microparticles can be produced by conditionally-immortalised cells. The conditionally-immortalised cells may be stem cells. The Examples show the successful harvest of microparticles from conditionally immortalised neural stem cells and CD34+ cells. Conditional immortalisation provides a constant supply of clonal cells that produce microparticles such as exosomes. The conditionally immortalised cells are useful as “producer cells” for microparticles such as exosomes, which are typically harvested or isolated from the conditionally-immortalised cells.
Description
FIELD OF THE INVENTION

This invention relates to microparticles produced by cells, their use and production thereof.


BACKGROUND OF THE INVENTION

Stem cells have the ability to self-renew and to differentiate into functionally different cell types. They have the potential to be a powerful tool in the growing field of Regenerative Medicine, in particular regenerative therapy requiring tissue replacement, regeneration or repair (Banerjee et al. 2011). However, there are drawbacks to the use of stem cells in therapy: there is a need for a consistent and substantial supply of stem cells with functional and phenotypic stability and the associated high costs and time delay caused by cell generation, storage, transport and handling; there is a requirement for immunological compatibility to avoid rejection of the stem cells by the recipient; and there are complex regulatory issues related to potential safety risks of tumour or ectopic tissue formation. Further, despite the therapeutic efficacy of stem cell transplantation, there is no convincing evidence for a direct long-term effect of the transplanted stem cells, for example through engraftment and differentiation into reparative or replacement cells.


Neural stem cells (NSCs) are self-renewing, multipotent stem cells that generate neurons, astrocytes and oligodendrocytes (Kornblum, 2007). The medical potential of neural stem cells is well-documented. Damaged central nervous system (CNS) tissue has very limited regenerative capacity so that loss of neurological function is often chronic and progressive. Neural stem cells (NSCs) have shown promising results in stem cell-based therapy of neurological injury or disease (Einstein et al. 2008). Implanting neural stem cells (NSCs) into the brains of post-stroke animals has been shown to be followed by significant recovery in motor and cognitive tests (Stroemer et al. 2009). It is not completely understood how NSCs are able to restore function in damaged tissues but it is now becoming increasingly recognised that NSCs have multimodal repairing properties, including site-appropriate cell differentiation, pro-angiogenic and neurotrophic activity and immunomodulation promoting tissue repair by the native immune system and other host cells (Miljan & Sinden, 2009, Horie et al., 2011). It is likely that many of these effects are dependent on transient signalling from implanted neural stem cells to the host milieu, for example NSCs transiently express proinflammatory markers when implanted in ischaemic muscle tissue damage which directs and amplifies the natural pro-angiogenic and regulatory immune response to promote healing and repair (Hicks et al., unpublished data). In chronic stroke brain, NSCs also have a substantial neurotrophic effect. For example, they promote the repopulation of the stoke-damaged striatal brain tissue with host brain derived doublecortin positive neroblasts (Hassani, O'Reilly, Pearse, Stroemer et al., PLoS One. 2012; 7(11)).


Furthermore, on the basis of a large body of NSC restorative effects in animal models with chronic stroke, a clinical trial using neural stem cells is being carried out by ReNeuron Limited (Surrey, UK), to trial the treatment of disabled stroke patients using its “CTX0E03” conditionally-immortalised cortex-derived neural stem cells (Clinicaltrials.gov Identifier: NCT01151124).


Mesenchymal stem cells (MSCs) are lineage-restricted stem cells which have the potential to differentiate into mesenchymal cell types only, namely of the adipocytic, chondrocytic and osteocytic lineages (Pittenger et al 1999; Ding et al. 2011). MSCs (also referred to as Mesenchymal Stromal Cells and Mesenchymal Progenitor Cells) are derived from a variety of sources including bone marrow, blood, adipose and other somatic tissues. The therapeutic potential of MSCs, however, is more directed towards the application of their pro-angiogenic and immune modulating properties as undifferentiated cells. Production of human MSCs is limited by the inability of these cells to expand in numbers stably beyond approximately 15-20 population doublings.


Mesenchymal stem cell-conditioned medium (MSC-CM) has a therapeutic efficacy similar to that of MSCs themselves, suggesting a paracrine mechanism of MSC-based therapy (Timmers et al. 2007). WO-A-2009/105044 discloses that particles known as exosomes, secreted by MSCs, comprise at least one biological property of the MSCs and suggests the use of these MSC particles in therapy, while Théry et al. 2011 provides a general review of exosomes and other similar secreted vesicles. Whereas some of the drawbacks of using stem cells directly as therapeutic agents are overcome by using the mesenchymal stem cell-derived exosomes (e.g. storage, transport and handling), the problem remains of providing a consistent and substantial supply of functionally and phenotypically stable stem cells to produce the exosomes. For therapeutic use, the exosomes preferably need to be produced on a large scale. In the absence of a stem cell line, replenishment of the cells through repeated derivation from a source of stem cells is required, which incurs recurring costs for testing and validation of each new batch. Furthermore, the diseases and disorders that can be treated by MSCs may be limited.


There remains a need for improved stem cell-based therapies.


SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that microparticles can advantageously be produced by conditionally-immortalised cells. The conditionally-immortalised cells may be stem cells. The Examples show the successful harvest of microparticles from conditionally immortalised neural stem cells and CD34+ cells. Conditional immortalisation provides a constant supply of clonal cells that produce microparticles such as exosomes. The conditionally immortalised cells are useful as “producer cells” for microparticles such as exosomes, which are typically harvested or isolated from the conditionally-immortalised cells.


A first aspect of the invention provides the use of a conditionally-immortalised cell to produce microparticles. The conditionally-immortalised cell is typically:

    • a mesenchymal stem cell, optionally selected from a bone marrow derived stem cell, an endometrial regenerative cell, a mesenchymal progenitor cell or a multipotent adult progenitor cell;
    • a neural stem cell, optionally selected from a neurosphere initiating stem cell, or an oligodendrocyte precursor cell;
    • a haematopoietic stem cell, optionally a CD34+ cell and/or isolated from umbilical cord blood, or optionally a CD34+/CXCR4+ cell; a non-haematopoietic umbilical cord blood stem cell;
    • a very small embryonic like stem cell (VSEL);
    • an induced pluripotent stem (iPS) cell;
    • a fibroblast; or
    • a dendritic cell.


The microparticle that is produced may be an exosome, microvesicle, membrane particle, membrane vesicle, exosome-like vesicle, ectosome-like vesicle, ectosome or exovesicle. Typically, the microparticle is an exosome. In one embodiment, the cell is a mesenchymal stem cell and the microparticle is an exosome.


Conditional immortalisation may be achieved by introducing an immortalisation factor which is inactive unless the cell is supplied with an activating agent. Such an immortalisation factor may be a gene such as c-mycER. The c-MycER gene product is a fusion protein comprising a c-Myc variant fused to the ligand-binding domain of a mutant estrogen receptor. The conditionally-immortalised cells are typically cultured in the presence of the activating agent. For c-MycER conditionally-immortalised cells, the activating agent is 4-hydroxytamoxifen (4-OHT). Accordingly, the microparticles are typically isolated from conditionally-immortalised cells that are in the immortalised state at the time of microparticle isolation.


It has also been found that it is possible to alter the production of microparticles by conditionally-immortalised cells by the addition of components to the culture medium, by culturing the stem cells under hypoxic conditions, or by co-culture with other cell types, thereby providing an improved method of producing stem cell microparticles.


A second aspect of the invention provides a method of producing a microparticle, comprising isolating a microparticle from a conditionally-immortalised cell-conditioned medium, optionally wherein the cell is as defined above. In certain embodiments of this aspect:

    • the cell-conditioned medium may comprise one or more components which induce the release of microparticles by the stem cells into the medium;
    • the cells may be cultured under hypoxic conditions;
    • the cells may be co-cultured with a different cell type;
    • the cells may be cultured in a multi-compartment bioreactor; and/or
    • the cells may be stem cells that are partially-differentiated.


A further aspect of the invention provides a method of producing a stem cell microparticle, typically a neural stem cell microparticle or a microparticle from another stem cell type (as detailed above). The method may comprise culturing the stem cells, typically conditionally-immortalised stem cells, in an environment that allows stem cell differentiation and collecting the microparticles that are produced by the cells. The microparticles may be isolated from partially-differentiated neural stem cells. The stem cells may be cultured under conditions that allow the efficient removal of metabolic waste. In one embodiment, an environment that allows stem cell differentiation is culture in a multi-compartment bioreactor, typically for a prolonged period of time (for example more than seven days). The method may comprise isolating a microparticle from a stem cell-conditioned medium. The stem cell-conditioned medium may comprise one or more additive components or agents which stimulate the release of microparticles by the stem cells into the medium. The one or more components may be selected from transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ) and/or tumour necrosis factor-alpha (TNF-α). The microparticles may be isolated from stem cell-conditioned medium wherein the stem cells were cultured under hypoxic conditions. The microparticles may be isolated from stem cell-conditioned medium produced by stem cells co-cultured with a different cell type, typically endothelial cells, in order to create the NSC niche environment.


A further aspect of the invention provides a microparticle obtainable by a method aspect of the invention.


Another aspect of the invention provides a method of screening for an agent that alters the production of a microparticle by a conditionally-immortalised cell, comprising contacting a conditionally-immortalised cell with a candidate agent and observing whether the rate of production of microparticles by the contacted conditionally-immortalised cell increases or decreases compared to a control.


A further aspect of the invention provides a kit for use in a method for producing a microparticle, comprising: (a) a medium; (b) a conditionally-immortalised cell; (c) optionally the one or more components of claim 19 or 20; (d) optionally the microparticle of claim 22 suitable for use as a control; (e) optionally a detection agent suitable for specific detection of the produced microparticles; and (f) instructions for producing the microparticle of any of claim 22 using the kit.


Further aspects and embodiments of the invention are defined below, and in the claims.


The invention also relates to the finding that neural stem cells contain microparticles that are therapeutically useful. The methods discussed herein with regard to the production of microparticles from neural stem cells may be applied to methods of producing microparticles from the other cell types described for the first aspect of the invention.


One aspect of the invention provides a neural stem cell microparticle. The microparticle may be an exosome, microvesicle, membrane particle, membrane vesicle, exosome-like vesicle, ectosome-like vesicle, ectosome or exovesicle. Typically, the microparticle is an exosome. The microparticle may be derived from a conditionally-immortalised neural stem cell that has been cultured in an environment that allows stem cell differentiation. The microparticle may be isolated from partially-differentiated neural stem cells. In one embodiment, an environment that allows stem cell differentiation is a multi-compartment bioreactor, typically where the cells are cultured for more than seven days. The microparticle may be derived from a neural stem cell line. In some embodiments, the neural stem cell line may be the “CTX0E03” cell line, the “STR0C05” cell line, the “HPC0A07” cell line or the neural stem cell line disclosed in Miljan et al Stem Cells Dev. 2009. In some embodiments, the microparticle is derived from a stem cell line that does not require serum to be maintained in culture. The microparticle may have a size of between 30 nm and 1000 nm, or between 30 and 200 nm, or between 30 and 100 nm, as determined by electron microscopy; and/or a density in sucrose of 1.1-1.2 g/ml. The microparticle may comprise RNA. The RNA may be mRNA, miRNA, and/or any other small RNA. The microparticle may comprise one, two, three or four of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532. The microparticle may comprise one or more lipids, typically selected from ceramide, cholesterol, sphingomyelin, phosphatidylserine, phosphatidylinositol, phosphatidylcholine. The microparticle may comprise one or more tetraspanins, typically CD63, CD81, CD9, CD53, CD82 and/or CD37. The microparticle may comprise one or more of TSG101, Alix, CD109, thy-1 and CD133. The microparticle may comprise at least 10 of the proteins present in Table 19 or Table 21. The microparticle may comprise at least one biological activity of a neural stem cell or a neural stem cell-conditioned medium. At least one biological activity may be a tissue regenerative activity. The microparticle of the invention is typically isolated or purified.


A further aspect of the invention provides a neural stem cell microparticle for use in therapy. The therapy may be regenerative therapy requiring tissue replacement, regeneration or repair, for example where the therapy requires angiogenesis, neurogenesis and/or neuroprotection. The therapy may be for a neurological disease, disorder or deficit. The therapy may improve functional and/or cognitive recovery. The therapy may be of stroke, peripheral arterial disease, neuropathy or any other disease or disorder that requires tissue regeneration, revascularisation or local anti-inflammatory action, including:

    • (i) Neurological disorder, disease or deficit, such as Parkinson's disease, Alzheimer's disease, Stroke, or ALS;
    • (ii) Lysosomal storage disorders;
    • (iii) Cardiovascular disorders, such as Myocardial Infarction, congestive heart failure, Peripheral Arterial Disease, diabetic ulcers, wound healing;
    • (iv) Diseases of the lung, including Idiopathic Pulmonary Fibrosis, Respiratory Distress Syndrome, Chronic Obstructive Pulmonary Disease, Idiopathic Pulmonary Hypertension, Cystic Fibrosis and Asthma;
    • (v) Metabolic or inflammatory disorders, such as Diabetes (I or II), rheumatoid arthritis, osteoarthritis, lupus, Crohn's disease, Inflammatory Bowel Disease, or Graft versus Host Disease;
    • (vi) Psychiatric disorders, such as Depression, Bipolar disorder, Schizophrenia or an Autistic syndrome disorder such as Autism, Asperger's syndrome or Rett Syndrome;
    • (vii) Blindness-causing diseases of the retina, such as Age-related macular degeneration, Stargardt disease, diabetic retinopathy, retinitis pigmentosa; and
    • (viii) Demyelinating diseases, such as multiple sclerosis, cerebral palsy, central pontine myelinolysis, tabes dorsalis, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukodystrophies, Guillain-Barre syndrome, Anti-MAG peripheral neuropathy and Charcot-Marie-Tooth disease.


In one embodiment, the microparticle is an exosome and therapy is of a disease or condition requiring tissue replacement, regeneration or repair. In another embodiment, the microparticle is a microvesicle and the therapy is of a disease requiring angiogenesis or a neurological disease, disorder or deficit.


The therapy may also be a prophylactic therapy to induce tolerance, typically immunotolerance, in a host that is subsequently, concurrently or simultaneously to receive the stem cells from which the microparticle is derived. The administration of one or more doses of microparticles of the invention to a patient, prior to or concurrent with administration of a stem cell therapy, can be used to reduce the risk of an adverse immune response, i.e. “rejection”, of the stem cell therapy.


A further aspect of the invention provides the use of a neural stem cell microparticle in the manufacture of a medicament for the treatment of a disease.


Another aspect of the invention provides a composition comprising a neural stem cell microparticle and a pharmaceutically acceptable excipient, carrier or diluent.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 depicts electron micrographs of CTX0E03 conditionally-immortalised neural stem cells producing microparticles. Panels A-E show intracellular multivesicular bodies (MVBs) containing exosomes between 30 nm and 50 nm in diameter and Panel F shows microvesicles >100 nm in diameter released from neural stem cells through a process of budding at the cell membrane.



FIG. 2 is an outline protocol for the identification, characterisation and production of microparticles from stem cells.



FIG. 3 shows Human angiogenesis ELISA strip optical density read out performed on CTX0E03 conditioned and un-conditioned medium.



FIG. 4A shows the amount of protein (measured by BCA assay) extracted from 15 ml of media containing microparticles purified from the Integra system compared to normal culture conditions (3 days T175). FIG. 4B shows the FACS detection (at 2 ug/ml, 1:250) of (i) CD63 in Integra cultured CTX0E03 exosomes (top left panel) and microvesicles (top right panel) and (ii) CD81 in Integra cultured CTX0E03 exosomes (bottom left panel) and microvesicles (bottom right panel).



FIG. 5 shows the amount of isolated total RNA measured at 260/280 nm extracted from 15 ml of media containing microparticles purified by filtration from the Integra system compared to normal culture conditions (3 days T175).



FIG. 6A shows the results of a wound closure/scratch assay representing the migration activity of normal human dermal fibroblasts (NHDF) cultured in CTX0E03 conditioned media or upon the addition of purified CTX0E03 exosomes. FIG. 6B shows the results of a scratch assay after 72 hours, comparing the effect of 10 μg CTX0E03 exosomes to basal conditions (without exosomes). FIG. 6C shows the % of healed areas for basal conditions, 2 μg/ml exosomes, 6 μg/ml exosomes, 20 μg/ml exosomes and an LSGS (low serum growth supplement) positive control. The top panel of FIG. 6C shows exosomes isolated from CTX0E03 cells cultured for 2 weeks in the Integra Celline system and the bottom panel of FIG. 6C shows exosomes isolated from CTX0E03 cells cultured for 6 weeks in the Integra Celline system. FIG. 6D compares CTX0E03 cells to a negative control (saline) in an in vivo injection wound healing assay.



FIG. 7 shows the quantity of purified exosomes obtained per culture medium from standard CTX0E03 (T175) cultures vs the Integra CELLine system at the 3 week time point.



FIG. 8A shows the concentration of exosomes harvested from two different flasks after 1 week, 2 weeks and 3 weeks of CTX0E03 Integra CELLine culture system. FIG. 8B shows the concentration of exosomes harvested from a single Integra CELLine flask during a 6 week continuous culture of CTX0E03 cells.



FIG. 9 shows the fold change of expression levels of various mRNA markers measured in CTX0E03 cells cultured for 3 weeks in the Integra CELLine system compared to standard (“control”) CTX0E03 (T175) cultures.



FIG. 10 shows the fold up and down regulation of various miRNAs in exosomes obtained from CTX0E03 cells cultured for 3 weeks in Integra bioreactor culture and microparticles obtained from standard CTX0E03 (T175) cultures, assessed against a baseline expression level in CTX0E03 cells in standard (T175) culture.



FIG. 11 depicts the miRNA profiles obtained from deep sequencing of miRNA from CTX0E03 cells (“CTX”), microvesicles (“MV”) and exosomes (“EXO”) cultured under standard (T175) conditions. FIGS. 11a and 11b show results from two cultures.



FIG. 12 shows the effect of hNSC microvesicles on angiogenesis of HUVECs. FIG. 12A is a photograph showing the clear increase in tube formation observed when microvesicles are added (right hand panels) compared to basal HUVECs. FIGS. 12B and 12C show the increase in total tube length provided by the hNSC microvesicles at various concentrations (0.05 μg, 0.1 μg, 0.3 μg—FIG. 12B; and 0.6 μg/ml—FIG. 12C).



FIG. 13 shows the effect of hNSC microvesicles on neurite outgrowth in PC-12 cells.



FIG. 14 is an electropherogram showing the total RNA content profile in CTX0E03 cells, exosomes and microvesicles as determined by Agilent RNA bioanalyser.



FIG. 15 is a schematic presentation of the percentage of coding genes fully overlapping exon, and non-coding transcripts located with intron or intergenic sequences (produced by running NGS BAM files against GENCODE sequence data set).



FIG. 16 depicts the top ranking preferentially shuttled novel miRNAs in exosomes and MV compared to CTX0E03 producer cells.



FIG. 17 shows the results of NanoSight analysis undertaken to determine the particle size and concentration of CTX0E03 exosomes (FIG. 17A) and microvesicles (FIG. 17B) cultured in the Integra Celline system for 1, 2, 3, 4, 5 and 6 weeks



FIG. 18 shows Venn diagrams comparing the proteomic data from CTX0E03 exosomes and microvesicles (18A and 18B), and comparing neural stem cell exosomes with mesenchymal stem cell exosomes (18C and 18D). FIG. 18A illustrates the number of unique proteins within CTX0E03 exosomes and microvesicles, isolated from week 2 Integra culture system. FIG. 18B compares the biological processes associated with the identified proteins within the CTX0E03 exosomes and microvesicles. FIG. 18C compares the CTX0E03 neural stem cell exosome proteome to a Mesenchymal Stem Cell exosome proteome, and FIG. 18D compares the biological processes associated with the identified proteins in the MSC derived exosomes with the neural stem cell derived exosomes.



FIG. 19 shows the 30 biological processes found to be associated with NSC derived exosomes and not mesenchymal stem cell exosomes.



FIG. 20 shows the successful conditional immortalisation of CD34+ cells. (A) is a diagram of qRT-PCR cycles showing the presence of c-mycERTAM mRNA in lentivirus infected human CD34+ progenitor cells derived from cord blood. (B) is a control sample showing the detection of the exosome marker Alix in immunoprecipitate. (C) shows Alix expression in exosomes immunoprecipitated from both CD34+ and CD34+cMycERTam cells.





DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that microparticles can advantageously be produced by conditionally-immortalised cells. The conditionally-immortalised cells may be stem cells. The Examples show the successful production of microparticles by conditionally immortalised neural stem cells and CD34+ cells. Conditional immortalisation provides a constant supply of clonal cells that produce microparticles such as exosomes. The conditionally immortalised cells are useful as “producer cells” for microparticles such as exosomes, which are typically harvested or isolated from the conditionally-immortalised cells.


The present inventors have also surprisingly identified microparticles in neural stem cells. These microparticles retain some of the functions of the neural stem cells from which they are derived and are typically therapeutically useful for the same treatments as the neural stem cells. The microparticles are advantageous over the corresponding stem cells because they are smaller and less complex, thereby being easier to produce, maintain, store and transport, and have the potential to avoid some of the regulatory issues that surround stem cells. The microparticles can be produced continuously, by isolation from conditioned media, for example in a bioreactor such as a multi-compartment bioreactor, which allows for large scale production and the provision of an “off-the-shelf” therapy. The multi-compartment bioreactor is typically a two-compartment bioreactor.


It has further been found that, surprisingly, culturing stem cells (of any type, not limited to neural stem cells) in an environment that allows the stem cells to begin to differentiate, increases dramatically the yield of microparticles produced.


The inventors have surprisingly observed that culturing stem cells (of any type, not limited to neural stem cells) in a multi-compartment bioreactor, results in partial differentiation of the stem cells, into stem cells in a more differentiated form. This differentiation in culture does not require the addition of an agent to induce differentiation. This differentiation typically requires a culture period of at least one week, at least two weeks or at least three weeks. The changes to the stem cells that occur in culture in a multi-compartment bioreactor are reflected by the microparticles produced by the cultured stem cells. Therefore, by culturing stem cells in a multi-compartment bioreactor, it is possible to induce differentiation of the cells. Accordingly, microparticles from partially differentiated stem cells can be produced by harvesting microparticles from stem cells cultured in a multi-compartment bioreactor, typically for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks or at least six weeks. Optionally, the cells, e.g. NSCs, have been cultured for no more than ten weeks. In one embodiment, the invention provides a method of producing microparticles by isolating the microparticles from partially-differentiated neural stem cells.


The inventors have also found that it is possible to induce the secretion of microparticles from stem cells. This finding, which also is not limited to neural stem cells and can be used for the production of microparticles from any stem cell, allows for an improved yield of microparticles to be obtained from a stem cell culture. Several agents have been identified that enhance the secretion of microparticles to different degrees, which has the further advantage of being able to control the amount of microparticles that are secreted. Culturing stem cells under hypoxic conditions also improves microparticle production. Further, it has been found that co-culturing a stem cell with a different cell type, in particular an endothelial cell type can beneficially alter the microparticles that are produced by the stem cell.


In a further embodiment, the invention provides microparticles, typically exosomes, produced by serum-free stem cells. Serum is required for the successful culture of many cell lines, but contains many contaminants including its own exosomes. As described below, the inventors have produced microparticles from stem cells that do not require serum for successful culture.


Microparticles

The invention relates to the production of microparticles from conditionally-immortalised cells


The invention provides, in one aspect, microparticles obtainable from a conditionally-immortalised neural stem cell, mesenchymal stem cell or haematopoietic stem cell. A neural stem cell microparticle is a microparticle that is produced by a neural stem cell; a mesenchymal stem cell microparticle is a microparticle that is produced by a mesenchymal stem cell; a haematopoietic stem cell microparticle is a microparticle that is produced by a haematopoietic stem cell. Typically, the microparticle is secreted by the stem cell. More typically, the microparticle is an exosome or a microvesicle. Microparticles from some stem cells (that are not conditionally immortalised), such as mesenchymal stem cells, are known in the art.


A “microparticle” is an extracellular vesicle of 30 to 1000 nm diameter that is released from a cell. It is limited by a lipid bilayer that encloses biological molecules. The term “microparticle” is known in the art and encompasses a number of different species of microparticle, including a membrane particle, membrane vesicle, microvesicle, exosome-like vesicle, exosome, ectosome-like vesicle, ectosome or exovesicle. The different types of microparticle are distinguished based on diameter, subcellular origin, their density in sucrose, shape, sedimentation rate, lipid composition, protein markers and mode of secretion (i.e. following a signal (inducible) or spontaneously (constitutive)). Four of the common microparticles and their distinguishing features are described in Table 1, below.









TABLE 1







Various Microparticles












Microparticle
Size
Shape
Markers
Lipids
Origin





Microvesicles
100-1000 nm  
Irregular
Integrins,
Phosphatidylserine
Plasma





selectins,

membrane





CD40 ligand


Exosome-like
20-50 nm
Irregular
TNFRI
No lipid rafts
MVB from


vesicles




other







organelles


Exosomes
30-100 nm;
Cup
Tetraspanins
Cholesterol,
Multivesicular



(<200 nm)
shaped
(e.g. CD63,
sphingomyelin,
endosomes





CD9),
ceramide, lipid





Alix,
rafts,





TSG101,
phosphatidylserine





ESCRT


Membrane
50-80 nm
Round
CD133,
Unknown
Plasma


particles


no CD63

membrane









Microparticles are thought to play a role in intercellular communication by acting as vehicles between a donor and recipient cell through direct and indirect mechanisms. Direct mechanisms include the uptake of the microparticle and its donor cell-derived components (such as proteins, lipids or nucleic acids) by the recipient cell, the components having a biological activity in the recipient cell. Indirect mechanisms include microvesicle-recipient cell surface interaction, and causing modulation of intracellular signalling of the recipient cell. Hence, microparticles may mediate the acquisition of one or more donor cell-derived properties by the recipient cell. It has been observed that, despite the efficacy of stem cell therapies in animal models, the stem cells do not appear to engraft into the host. Accordingly, the mechanism by which stem cell therapies are effective is not clear. Without wishing to be bound by theory, the inventors believe that the microparticles secreted by neural stem cells play a role in the therapeutic utility of these cells and are therefore therapeutically useful themselves.


The microparticles and cells, e.g. stem cells, of the invention are isolated. The term “isolated” indicates that the microparticle, microparticle population, cell or cell population to which it refers is not within its natural environment. The microparticle, microparticle population, cell or cell population has been substantially separated from surrounding tissue. In some embodiments, the microparticle, microparticle population, cell or cell population is substantially separated from surrounding tissue if the sample contains at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% microparticles and/or stem cells. In other words, the sample is substantially separated from the surrounding tissue if the sample contains less than about 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of materials other than the microparticles and/or stem cells. Such percentage values refer to percentage by weight. The term encompasses cells or microparticles which have been removed from the organism from which they originated, and exist in culture. The term also encompasses cells or microparticles which have been removed from the organism from which they originated, and subsequently re-inserted into an organism. The organism which contains the re-inserted cells may be the same organism from which the cells were removed, or it may be a different organism.


Stem cells naturally produce microparticles by a variety of mechanisms, including budding of the plasma membrane (to form membrane vesicles and microvesicles) and as a result of the fusion of intracellular multivesicular bodies (which contain microparticles) with the cell membrane and the release of the microparticles into the extracellular compartment (to secrete exosomes and exosome-like vesicles).


In one embodiment, the neural stem cell that produces the microparticles of the invention can be a fetal, an embryonic, or an adult neural stem cell, such as has been described in U.S. Pat. No. 5,851,832, U.S. Pat. No. 6,777,233, U.S. Pat. No. 6,468,794, U.S. Pat. No. 5,753,506 and WO-A-2005121318. The fetal tissue may be human fetal cortex tissue. The cells can be selected as neural stem cells from the differentiation of induced pluripotent stem (iPS) cells, as has been described by Yuan et al. (2011) or a directly induced neural stem cell produced from somatic cells such as fibroblasts (for example by constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming as recently by Their et al, 2012). Human embryonic stem cells may be obtained by methods that preserve the viability of the donor embryo, as is known in the art (e.g. Klimanskaya et al., 2006, and Chung et al. 2008). Such non-destructive methods of obtaining human embryonic stem cell may be used to provide embryonic stem cells from which microparticles of the invention can be obtained. Alternatively, microparticles of the invention can be obtained from adult stem cells, iPS cells or directly-induced neural stem cells. Accordingly, microparticles of the invention can be produced by multiple methods that do not require the destruction of a human embryo or the use of a human embryo as a base material.


Typically, the cell population from which the microparticles are produced, is substantially pure. The term “substantially pure” as used herein, refers to a population of cells that is at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% pure, with respect to other cells that make up a total cell population. For example, with respect to stem cell, e.g. neural stem cell populations, this term means that there are at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% pure, stem cells compared to other cells that make up a total cell population. In other words, the term “substantially pure” refers to a population of stem cells of the present invention that contain fewer than about 25%, in some embodiments fewer than about 15%, and in some embodiments fewer than about 5%, of lineage committed cells in the original unamplified and isolated population prior to subsequent culturing and amplification.


A stem cell, e.g. neural stem cell, microparticle comprises at least one lipid bilayer which typically encloses a milieu comprising lipids, proteins and nucleic acids. The nucleic acids may be deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). RNA may be messenger RNA (mRNA), micro RNA (miRNA) or any miRNA precursors, such as pri-miRNA, pre-miRNA, and/or small nuclear RNA (snRNA).


A stem cell, e.g. neural stem cell, microparticle retains at least one biological function of the stem cell from which it is derived. Biological functions that may be retained include the ability to promote angiogenesis and/or neurogenesis, the ability to effect cognitive improvement in the brain of a patient that has suffered a stroke, or the ability to accelerate blood flow recovery in peripheral arterial disease. For example, CTX0E03 cells are known to inhibit T cell activation in a PBMC assay and, in one embodiment, the microparticles of the invention retain this ability to inhibit T cell activation in a PBMC assay. PBMC assays are well-known to the skilled person and kits for performing the assay are commercially available.


Example 8, Table 2 and FIG. 6 demonstrate that CTX0E03 stem cell exosomes retain the ability to close a wound in a “scratch” model of wound healing. The results in FIG. 6A show that the migration activity of normal human dermal fibroblasts (NHDF) cultured in CTX0E03 conditioned media is almost the same as the migration activity observed on the addition of purified exosomes. Accordingly, one biological function that microparticles of the invention may retain is the ability to stimulate migration activity of normal human dermal fibroblasts (NHDF).


Example 8 also shows that microvesicles of the invention are able to stimulate angiogenesis of primary HUVECs and to stimulate neurite outgrowth of PC-12 cells. Accordingly, a biological function that microparticles of the invention may retain is the ability to stimulate angiogenesis of primary HUVECs and/or to stimulate neurite outgrowth of PC-12 cells.


The proteomic analysis in Example 13 indicates that neural stem cell exosomes comprise biological functions associated with the production, packaging, function and degradation of genetic material. Accordingly, in one embodiment, exosomes of the invention retain these functions, typically one or more of RNA polymerase function, RNA degradation function, ribosome function and spliceosome function.


The microparticle has a diameter of 1000 nm or less. Typically, the microparticle of the invention will have a diameter of 200 nm or less, for example 100 nm or less. As noted in Table 1 above, microvesicles have a diameter of 100 nm to 1000 nm. Exosomes are typically defined as having a diameter of 30-100 nm, but more recent studies confirm that exosomes can also have a diameter between 100 nm and 200 nm, (e.g. Katsuda et al, Proteomics 2013 and Katsuda et al, Scientific Reports 2013). Accordingly, exosomes typically have a diameter between 30 nm and 150 nm. Membrane particles have a diameter of 50 nm to 80 nm and exosome-like particles have a diameter of 20 nm-50 nm. The diameter can be determined by any suitable technique, for example electron microscopy or dynamic light scattering. The term microparticle includes, but is not limited to: membrane particle, membrane vesicle, microvesicle, exosome-like vesicle, exosome, ectosome-like vesicle, ectosome or exovesicle.



FIG. 1 panels A-E show the presence in neural stem cells of MVB's containing exosomes between 30-50 nm in diameter, while panel F shows microvesicles >100 nm in diameter. Table 20 and FIG. 17 (below) show that typical neural stem cell exosomes were measured to have a diameter ranging from approximately 70 nm to approximately 150 nm, which is consistent with the size of exosomes (from mesenchymal stem cells) described in the art. Accordingly, exosomes of the invention typically have a diameter between 30 nm and 200 nm, more typically between 50 nm and 150 nm. As noted above, exosomes are typically positive for the Alix marker (UNIPROT Accession No. Q8WUM4).



FIG. 1F and Table 20 shows the observed size of typical neural stem cell microvesicles, with a mode diameter of approximately 150 nm-200 nm, or a median diameter of approximately 180 nm-350 nm. Accordingly, microvesicles of the invention typically have a diameter between 100 and 1000 nm, more typically between 150 nm and 350 nm.


Some microparticles of the invention express the CD133 surface marker. Other microparticles of the invention do not express the CD133 surface marker.


“Marker” refers to a biological molecule whose presence, concentration, activity, or phosphorylation state may be detected and used to identify the phenotype of a cell.


Exosomes are endosome-derived lipid microparticles of typically 30-100 nm diameter and sometimes between 100 nm and 200 nm diameter, that are released from the cell by exocytosis. Exosome release occurs constitutively or upon induction, in a regulated and functionally relevant manner. During their biogenesis, exosomes incorporate a wide range of cytosolic proteins (including chaperone proteins, integrins, cytoskeletal proteins and the tetraspanins) and genetic material. Consequently, exosomes are considered to be inter-cellular communication devices for the transfer of proteins, lipids and genetic material between cells, in the parent cell microenvironment and over considerable distance. Although the invention is not bound by this theory, it is possible that the exosomes are responsible for the efficacy of the neural stem cells. Therefore, exosomes from neural stem cells are themselves expected to be therapeutically efficacious.


Microparticles Designed to have Desired Functions


Microparticles retain at least some of the functions of the stem cells that produce them. Therefore, it is possible to design microparticles by manipulating the stem cell (which can be any stem cell type and is not limited to neural stem cells, although the neural stem cell microparticles of the invention are expressly included as an embodiment) to possess one or more desired functions, typically protein or miRNA. The manipulation will typically be genetic engineering, to introduce one or more exogenous coding, non-coding or regulatory nucleic acid sequences into the stem cell. For example, if an exosome containing VEGF and/or bFGF is desired, then the exosome-producing stem cell can be transformed or transfected to express (high levels of) VEGF and/or bFGF, which would then be incorporated into the microparticles produced by that stem cell. Similarly, iPS cells can be used to produce microparticles, and these cells can be designed to produce the proteins and nucleic acids (e.g. miRNA) that are required in the microparticles produced by the iPS cells. The invention therefore provides ad hoc microparticles, from any stem cell type, that contain a function that is not naturally present in the stem cell from which is produced, i.e. the microparticles (e.g. exosomes) contain one or more exogenous protein or nucleic acid sequences, are not naturally-occurring and are engineered. The use of conditionally-immortalised cells advantageously provides for the continuous production of these designed microparticles.


In one embodiment, isolated or purified microparticles are loaded with one or more exogenous nucleic acids, lipids, proteins, drugs or prodrugs which are intended to perform a desired function in a target cell. This does not require manipulation of the stem cell and the exogenous material can optionally be directly added to the microparticles. For example, exogenous nucleic acids can be introduced into the microparticles by electroporation. The microparticles can then be used as vehicles or carriers for the exogenous material. In one embodiment, microparticles that have been isolated from the cells that produced them are loaded with exogenous siRNA, typically by electroporation, to produce microparticles that can be deployed to silence one or more pathological genes. In this way, microparticles can be used as vehicles to deliver one or more agents, typically therapeutic or diagnostic agents, to a target cell. An example of this is a neural stem cell exosome comprising exogenous siRNA capable of silencing one or more pathological genes.


Microparticle Marker

The invention provides a population of isolated neural stem cell microparticles, wherein the population essentially comprises only microparticles of the invention, i.e. the microparticle population is pure. In many aspects, the microparticle population comprises at least about 80% (in other aspects at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%) of the microparticles of the invention.


The isolated neural stem cell microparticle of the invention is characterised in that it has a distinctive expression profile for certain markers and is distinguished from microparticles from other cell types. When a marker is described herein, its presence or absence may be used to distinguish the microparticle. For example, the term “may comprise” or “may express” also discloses the contrary embodiment wherein that marker is not present, e.g. the phrase “the microparticle may comprise one or more tetraspanins, typically CD63, CD81, CD9, CD53, CD82 and/or CD37” also describes the contrary embodiment wherein the microparticle may not comprise one or more tetraspanins, typically CD63, CD81, CD9, CD53, CD82 and/or CD37.


The stem cell, e.g. neural stem cell, microparticle of the invention is typically considered to carry a marker if at least about 70% of the microparticles of the population, e.g. 70% of the membrane particles, membrane vesicles, microvesicles, exosome-like vesicles, exosomes, ectosome-like vesicles, ectosomes or exovesicles show a detectable level of the marker. In other aspects, at least about 80%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or more of the population show a detectable level of the marker. In certain aspects, at least about 99% or 100% of the population show detectable level of the markers. Quantification of the marker may be detected through the use of a quantitative RT-PCR (qRT-PCR) or through fluorescence activated cell sorting (FACS). It should be appreciated that this list is provided by way of example only, and is not intended to be limiting. Typically, a neural stem cell microparticle of the invention is considered to carry a marker if at least about 90% of the microparticles of the population show a detectable level of the marker as detected by FACS.


The markers described herein are considered to be expressed by a cell of the population of the invention, if its expression level, measured by qRT-PCR has a crossing point (Cp) value below or equal to 35 (standard cut off on a qRT-PCR array). The Cp represents the point where the amplification curve crosses the detection threshold, and can also be reported as crossing threshold (ct).


In one embodiment, the invention relates to microparticles produced by a neural stem cell population characterised in that the cells of the population express one or more of the markers Nestin, Sox2, GFAP, βIII tubulin, DCX, GALC, TUBB3, GDNF and 100. In another embodiment, the microparticle is an exosome and the population of exosomes expresses one or more of DCX (doublecortin—an early neuronal marker), GFAP (Glial fibrillary acidic protein—an astrocyte marker), GALC, TUBB3, GDNF and 100.


The neural stem cell microparticles of the invention may express one or more protein markers at a level which is lower or higher than the level of expression of that marker in a mesenchymal stem cell microparticle of the same species. Protein markers that are expressed by the CTX0E03 cell microparticles are identified herein and below. In some embodiments, the microparticles may express a protein marker at a level relative to a tubulin or other such control protein(s). In some embodiments, the microparticles of the invention may express that protein at a level of at least +/−1.2 fold change relative to the control protein, typically at least +/−1.5 fold change relative to the control protein, at least +/−2 fold change relative to the control protein or at least +/−3 fold change relative to the control protein. In some embodiments, the microparticles may express a protein marker at a level of between 10−2 and 10−6 copies per cell relative to a tubulin or other control protein. In some embodiments, the microparticles of the invention may express that protein at a level of between 10−2 and 10−3 copies per cell relative to a tubulin or other control protein.


The neural stem cell microparticles of the invention may express one or more miRNAs (including miRNA precursors) at a level which is lower or higher than the level of expression of that miRNA (including miRNA precursors) in a mesenchymal stem cell microparticle of the same species. miRNA markers that are expressed by the CTX0E03 cell microparticles are identified below. In some embodiments, the microparticles of the invention may express the marker miRNA at a level of least +/−1.5 fold change, typically at least +/−2 fold change or at least +/−3 fold change (calculated according to the ΔΔct method, which is well-known) relative to U6B or 15a, or any other miRNA reference gene, also referred to as an internal control gene.


The neural stem cell microparticles of the invention may express one or more mRNAs at a level which is lower or higher than the level of expression of that mRNA in a mesenchymal stem cell microparticle of the same species. In some embodiments, the microparticles of the invention may express the marker mRNA at a level of least +/−1.5 fold change, typically at least +/−2 fold change or at least +/−3 fold change (calculated according to the ΔΔct method) relative to ATP5B or YWHAZ, or any other reference gene, also referred to as an internal control gene.


Exosomes of the invention typically express specific integrins, tetraspanins, MHC Class I and/or Class II antigens, CD antigens and cell-adhesion molecules on their surfaces, which may facilitate their uptake by specific cell types. Exosomes contain a variety of cytoskeletal proteins, GTPases, clathrin, chaperones, and metabolic enzymes (but mitochondrial, lysosomal and ER proteins are excluded, so the overall profile does not resemble the cytoplasm). They also contain mRNA splicing and translation factors. Finally, exosomes generally contain several proteins such as HSP70, HSP90, and annexins that are known to play signalling roles yet are not secreted by classical (ER-Golgi) mechanisms.


The lipid bilayer of an exosome is typically enriched with cholesterol, sphingomyelin and ceramide. Exosomes also express one or more tetraspanin marker proteins. Tetraspanins include CD81, CD63, CD9, CD53, CD82 and CD37. Exosomes can also include growth factors, cytokines and RNA, in particular miRNA. Exosomes typically express one or more of the markers TSG101, Alix, CD109, thy-1 and CD133. Alix (Uniprot accession No. Q8WUM4), TSG101 (Uniprot accession No. Q99816) and the tetraspanin proteins CD81 (Uniprot accession No. P60033) and CD9 (Uniprot accession No. P21926) are characteristic exosome markers.


Alix is an endosomal pathway marker. Exosomes are endosomal-derived and, accordingly, a microparticle positive for this marker is characterised as an exosome. Exosomes of the invention are typically positive for Alix. Microvesicles of the invention are typically negative for Alix.


Microparticle Proteome

Tables 18 and 20 list all proteins detected by mass spectrometry in exosomes and microvesicles, respectively, isolated from CTX0E03 cells cultured for two weeks in an Integra Celline multicompartment bioreactor. In one embodiment, exosomes of the invention comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% of the proteins listed in Table 18. Similarly, microvesicles of the invention typically comprise at least 70% at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% of the proteins listed in Table 20. In a further embodiment, the proteome of a microvesicle or exosome of the invention is least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% identical to the proteome provided in Table 18 (exosome) or Table 20 (microvesicle). When determining the protein content of a microparticle or exosome, mass spectrometry is typically used, for example the LC/MS/MS method described in Example 13.


Tables 19 and 21 show the 100 most abundant proteins detected by mass spectrometry in exosomes and microvesicles, respectively, isolated from CTX0E03 cells cultured for two weeks in an Integra Celline multicompartment bioreactor. Typically, an exosome of the invention comprises the first ten proteins listed in Table 19, more typically the first 20, the first 30, the first 40 or the first 50 proteins listed in Table 19. Similarly, a microparticle of the invention typically comprises the first ten proteins listed in Table 21, more typically the first 20, the first 30, the first 40 or the first 50 proteins listed in Table 21. In one embodiment, an exosome of the invention comprises all 100 proteins listed in Table 19. In one embodiment, a microvesicle of the invention comprises all 100 proteins listed in Table 21. Typically, the 100 most abundant proteins in an exosome or microvesicle of the invention contain at least 70 of the proteins identified in Table 19 (exosome) or Table 21 (microparticle). More typically, the 100 most abundant proteins in an exosome or microvesicle of the invention contain at least 80, at least 90, at least 95, 96, 97, 98 or 99, or all 100 of the proteins identified in Table 19 (exosome) or Table 21 (microparticle).


Microparticle miRNA Content


Example 12 (and the related FIG. 11) shows the results of deep sequencing of miRNA present in CTX0E03 cells, microvesicles and exosomes produced by these cells. This Example shows that, surprisingly, the number of different miRNA species present in the microparticles is greatly reduced compared to the number of different miRNA species present in the cells; the microparticles contain fewer than 120 different miRNAs whereas the cells contain between 450 and 700 miRNA species. The microparticles contain a majority of hsa-miR-1246.


The data in Example 12 also show that the microparticles are characterised by four main miRNA species, namely hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532. These four miRNAs are the only miRNAs present at a read count of greater than 1000 in the microparticles; these four miRNAs are present in massive excess compared to the other miRNAs in the microparticles. This is in contrast to the profile in the cells, which contain a much greater number of miRNAs present at high (read count greater than 1000) or very high (read count greater than 10,000) levels. Although not bound by theory, the inventors propose that hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532 are selectively trafficked (or otherwise incorporated) into the microparticles and are thought to play a role in the function of the microparticles.


Typically, in one embodiment microparticles, e.g. exosomes, of the invention contain one, two, three or all four of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532. Each of these miRNA markers is typically present at a read count (optionally determined using the deep sequence technique described in Example 12) of at least 1000 per microparticle. hsa-miR-1246 may optionally have a read count of at least 2000, 5000, 10,000, 20,000, or 25,000 per microparticle. Hsa-miR-4492 may optionally have a read count of at least 2000, 3000, 4000 or 5000 per microparticle. Hsa-miR-4532 may optionally have a read count of at least 2000 or 3000 per microparticle.


In one embodiment, each of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and/or hsa-miR-4532 is present in the microparticle, e.g. exosome, at a higher read count than is present in the cell that produced the microparticle. In particular, miR-1246 typically has a read count in the microparticle at least twice the read count in the cell, more typically at least 4, 5, 6, 7, or 8 times the read count in the cell, and optionally 10, 15 or 20 times the read count in the cell.


In one embodiment, microparticles of the invention contain hsa-let-7a-5p, has-miR-92b-3p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-10a-5p, hsa-100-5p and/or hsa-99b-5p at a lower read count than is present in the cell that produced the microparticle. Typically, each of these miRNAs has a read count of less than 1000 in the microparticles of the invention, more typically less than 100, for example less than 50. Optionally, microparticles of the invention contain hsa-let-7a-5p at a read count of less than 50 or less than 25.


In one embodiment, microparticles of the invention contain fewer than 150 types of miRNA (i.e. different miRNA species) when analysed by deep sequencing, typically fewer than 120 types of miRNA.


In one embodiment, hsa-miR-1246 is the most abundant miRNA in the microparticles of the invention (optionally determined using the deep sequence technique described in Example 12). Typically, at least 40% of the total count of miRNA in microparticles (e.g. microvesicles and exosomes) of the invention is hsa-miR-1246. Typically, at least 50% of the total count of miRNA in exosomes of the invention is hsa-miR-1246.


hsa-miR-4492 is typically the second-most abundant miRNA in the microparticles of the invention. Typically, at least 3% of the total count of miRNA in microparticles (e.g. microvesicles and exosomes) of the invention is hsa-miR-4492. More typically, at least 4% of the total count of miRNA in microparticles (e.g. microvesicles and exosomes) of the invention is hsa-miR-4492.


Typically, at least 2% of the total count of miRNA in microparticles (e.g. microvesicles and exosomes) of the invention is hsa-miR-4532.


Typically, at least 1% of the total count of miRNA in microparticles (e.g. microvesicles and exosomes) of the invention is hsa-miR-4488.


In one embodiment microparticles of the invention contain one or both of hsa-miR-4508, hsa-miR-4516 at a level at least 0.1% of the total miRNA content of the particle.


One or more of hsa-miR-3676-5p, hsa-miR-4485, hsa-miR-4497, hsa-miR-21-5p, hsa-miR-3195, hsa-miR-3648, hsa-miR-663b, hsa-miR-3656, hsa-miR-3687, hsa-miR-4466, hsa-miR-4792, hsa-miR-99b-5p and hsa-miR-1973 may be present in the microparticles of the invention.


Typically, each of hsa-let-7a-5p and hsa-100-5p is present at less than 1%, more typically less than 0.1% or less than 0.05% of the total miRNA count in microparticles of the invention.


In a typical exosome of the invention, at least 50% of the total count of miRNA is hsa-miR-1246, and less than 0.1% of the total miRNA count is hsa-let-7a-5p.


In one embodiment, at least 90% of the total count of miRNA in microparticles of the invention comprises hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532. Typically, at least 95% or 96% of the total count of miRNA in microparticles of the invention comprises hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532. Less than 10% of the total miRNA content of these microparticles is an miRNA that is not hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532.


Combinations of the miRNA embodiments discussed above are provided. For example, a microparticle of the invention typically contains each of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532 at a read count of at least 1000 and contains each of hsa-let-7a-5p, hsa-miR-92b-3p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-10a-5p, hsa-100-5p and hsa-99b-5p at a read count of less than 100. Typically, at least 90% or at least 95% of the total miRNA in these microparticles is hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532.


A microparticle (e.g. microvesicle or exosome) of the invention typically has hsa-miR-1246 as the most abundant miRNA and hsa-miR-4492 is the second-most abundant miRNA. In this embodiment, at least 40% of the total count of miRNA in microparticles (e.g. microvesicles and exosomes) of the invention is hsa-miR-1246 and at least 3% of the total count of miRNA in the microparticle is hsa-miR-4492. At least 2% of the total count of miRNA in these microparticles is hsa-miR-4532 and at least 1% of the total count of miRNA in these microparticles is hsa-miR-4488. Each of hsa-let-7a-5p and hsa-100-5p is present at less than 0.1% of the total miRNA count in these microparticles.


Plotting the deep sequencing results in the exosomes and microvesicles as relative fold change compared to the cells confirms that hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532 are significantly upregulated in the exosomes and microvesicles compared to the cells. This comparison also shows that miRNA hsa-miR-3195 is the miRNA that is most upregulated, in both exosomes and microvesicles. Although the absolute reads of hsa-miR-3195 are in the range of ˜40 for exosomes and microvesicles, there is no hsa-miR-3195 detected in the cells. Accordingly, hsa-miR-3195 is uniquely found in the exosomes and microvesicles of the invention and, in one embodiment, an exosome or microvesicle of the invention comprises hsa-miR-3195.


In one embodiment, microparticles of the invention comprise one or more of the following miRNA precursors:











AC079949.1



(SEQ ID NO: 738)



GGCCGCGCCCCGTTTCCCAGGACAAAGGGCACTCCGCACCGGAC







CCTGGTCCCAGCG;







AP000318.1



(SEQ ID NO: 739)



CCCACTCCCTGGCGCCGCTTGTGGAGGGCCCAAGTCCTTCTGAT







TGAGGCCCAACCCGTGGAAG;







AL161626.1



(SEQ ID NO: 740)



CGCCGGGACCGGGGTCCGGGGCGGAGTGCCCTTCCTCCTGGGAA







ACGGGGTGCGGC;







AC004943.1



(SEQ ID NO: 741)



GCTTCACGTCCCCACCGGCGGCGGCGGCGGTGGCAGTGGCGGCG







GCGGCGGCGGTGGCGGCGGCGGCGGCGGCGGCGGCTC;



and







AL121897.1



(SEQ ID NO: 742)



GCCGCCCCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGC







CCGCTTTCGGCTCGGGCCTCAGGTGAGTCGGAGGGGCCGGGCGC







C






In one embodiment, microparticles of the invention comprise one, two or three of the following mature miRNAs derived from the precursors listed above (as detailed in part D of Example 12):











(derived from AL161626.1-201)



(SEQ ID NO: 743)



ggcggagugcccuucuuccugg







(derived from AP000318.1-201)



(SEQ ID NO: 744)



ggagggcccaaguccuucugau







(derived from AC079949.1-201)



(SEQ ID NO: 745)



gaccaggguccggugcggagug






These 5 miRNA precursors and 3 mature miRNAs have not previously been isolated and each sequence is therefore also provided as a new sequence per se. Accordingly, in one aspect, the invention provides a composition comprising one or more of the miRNA precursors AC079949.1, AP000318.1, AL161626.1, AC004943.1 and AL121897.1. In another embodiment, the invention provides a composition comprising one or more of the mature miRNAs ggcggagugcccuucuuccugg (derived from AL161626.1-201), ggagggcccaaguccuucugau (derived from AP000318.1-201) and gaccaggguccggugcggagug (derived from AC079949.1-201). Optionally, the composition is a pharmaceutical composition comprising one or more of the miRNA precursors and/or one or more of the mature miRNAs and a pharmaceutically-acceptable carrier or diluent. As noted in Example 12, these miRNAs and precursors appear to be selectively shuttled into the exosomes and microvesicles and so may be at least partially responsible for the function of the microparticles.


Example 12 also shows that neural stem cell microparticles comprise a variety of non-coding RNA species. In one embodiment, microparticles of the invention comprise one or more of ribosomal RNA, small nucleolar RNA, small nuclear RNA, microRNA, large intergenic non-coding RNA and miscellaneous other RNA (e.g. RMRP, vault RNA, metazoan SRP and/or RNY).


Example 4 shows miRNAs present in microparticles produced by the CTX0E03 cells and having a Cp below 35 as determined by a qRT-PCR array. Typically, in one embodiment microparticles of the invention contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 or more, or all, of the following miRNAs (identified according by name according to Ambros et al and accessible at www.mirbase.org):

















hsa-let-7a



hsa-let-7b



hsa-let-7c



hsa-let-7d



hsa-let-7e



hsa-let-7f



hsa-let-7g



hsa-let-7i



hsa-miR-100



hsa-miR-101



hsa-miR-103a



hsa-miR-106b



hsa-miR-10a



hsa-miR-10b



hsa-miR-124



hsa-miR-125a-5p



hsa-miR-125b



hsa-miR-126



hsa-miR-127-5p



hsa-miR-128



hsa-miR-129-5p



hsa-miR-130a



hsa-miR-132



hsa-miR-134



hsa-miR-137



hsa-miR-141



hsa-miR-146b-5p



hsa-miR-150



hsa-miR-155



hsa-miR-15a



hsa-miR-15b



hsa-miR-16



hsa-miR-17



hsa-miR-181a



hsa-miR-182



hsa-miR-183



hsa-miR-185



hsa-miR-18a



hsa-miR-18b



hsa-miR-192



hsa-miR-194



hsa-miR-195



hsa-miR-196a



hsa-miR-205



hsa-miR-20a



hsa-miR-20b



hsa-miR-21



hsa-miR-210



hsa-miR-214



hsa-miR-218



hsa-miR-219-5p



hsa-miR-22



hsa-miR-222



hsa-miR-23b



hsa-miR-24



hsa-miR-26a



hsa-miR-301a



hsa-miR-302a



hsa-miR-302c



hsa-miR-33a



hsa-miR-345



hsa-miR-375



hsa-miR-378



hsa-miR-424



hsa-miR-7



hsa-miR-9



hsa-miR-92a



hsa-miR-93



hsa-miR-96



hsa-miR-99a










In one embodiment, the CTX0E03 microparticles contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more of the following miRNAs (which are selected from the list above):

















hsa-let-7g



hsa-miR-101



hsa-miR-10a



hsa-miR-10b



hsa-miR-126



hsa-miR-128



hsa-miR-129-5p



hsa-miR-130a



hsa-miR-134



hsa-miR-137



hsa-miR-155



hsa-miR-15a



hsa-miR-15b



hsa-miR-16



hsa-miR-17



hsa-miR-182



hsa-miR-183



hsa-miR-185



hsa-miR-18b



hsa-miR-192



hsa-miR-194



hsa-miR-195



hsa-miR-20a



hsa-miR-20b



hsa-miR-210



hsa-miR-218



hsa-miR-301a



hsa-miR-302a



hsa-miR-302c



hsa-miR-345



hsa-miR-375



hsa-miR-378



hsa-miR-7



hsa-miR-9



hsa-miR-93



hsa-miR-96



hsa-miR-99a










Proteins Detected by a Dot-Blot

Example 5 shows proteins present in microparticles produced by the CTX0E03 cells, as detected by a dot-blot. Typically, microparticles of the invention contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the following proteins:

















EDA-A2



Galectin-3



IGFBP-2



IGFBP-rp1/IGFBP-7



IL-1a



LECT2



MCP-1



SPARC



TIMP-1



Thrombospondin-1



VEGF










Galectin-3 and Thrombospondin-1 are also identified as present in exosomes and microvesicles in Example 13. TIMP-1 is identified in Example 13 as being present in exosomes.


Example 5 also shows that the microparticles produced by the CTX0E03 cells may also express 1, 2, 3, 4 or 5 of the following proteins:

















EGF-R/ErbB1



MDC



Endostatin



Follistatin



Csk










EGF-R and Csk are also identified as present in exosomes and microvesicles in Example 13.


Galectin-3, SPARC, TIMP-1, Thrombospondin-1, VEGF, MDC and Endostatin are known to be modulate angiogenesis. Accordingly, microparticles containing one or more of these proteins are useful in treating diseases or disorders requiring modulation of angiogenesis.


IL-1a, LECT2, MCP-1 and Csk are known to modulate inflammation. Accordingly, microparticles containing one or more of these proteins are useful in treating diseases or disorders requiring modulation of inflammation.


Microparticles containing one or more of (i) Galectin-3, SPARC, TIMP-1, Thrombospondin-1, VEGF, MDC and Endostatin, and one or more of (ii) IL-1a, LECT2, MCP-1 and Csk, may be useful for treating diseases or disorders requiring modulation of angiogenesis and inflammation.


Neural Stem Cells in Multi-Compartment Bioreactor Culture

As shown in Example 10 and FIG. 9 below, after multi-compartment bioreactor culture for three weeks, neural stem cells express a number of markers at significantly higher levels than neural stem cells cultured according to standard procedure in a standard single-compartment T175 flask. In one embodiment, microparticles of the invention are isolated from NSCs that have been cultured, typically in a multi-compartment bioreactor, for at least two weeks, typically at least three weeks, at least four weeks, at least five weeks or at least six weeks. Optionally, the NSCs have been cultured for no more than ten weeks, e.g. between 2 and 10 weeks, between 3 and 10 weeks, between 4 and 10 weeks, between 5 and 10 weeks or between 6 and 10 weeks. Microparticles may also be isolated from the other cell types described herein, including mesenchymal stem cells and haematopoietic stem cells, that have been cultured for these periods in a bioreactor.


CTX0E03 neural stem cells cultured for three weeks in a multi-compartment bioreactor express DCX, GALC, GFAP, TUBB3, GDNF and IDO at a higher level than neural stem cells cultured in a standard single-compartment T175 cell culture. Accordingly neural stem cells that have been cultured in a multi-compartment bioreactor, typically for a week or more, ten days or more, two weeks or more, or at least three weeks, four weeks, five weeks or more, may express one or more of DCX, GALC, GFAP, TUBB3, GDNF and IDO. Cells cultured in a two-compartment bioreactor typically show increased expression of one or more of DCX, GALC, GFAP, TUBB3, GDNF and IDO compared to the stem cells cultured under standard conditions. The expression level of these markers in the multi-compartment bioreactor-cultured cells is typically significantly higher than in the cells cultured in a standard single-compartment T175 culture flask. Typically, a stem cell cultured in a multi-compartment bioreactor expresses one or more of DCX1, GALC, GFAP, TUBB3, GDNF or IDO at a level least 2 fold higher than in CTX0E03 cells cultured in a T-175 flask according to standard culture procedure. In one embodiment, microparticles, typically exosomes, are obtained from neural stem cells that show increased expression of one or more of DCX, GALC, GFAP, TUBB3, GDNF and IDO compared to the stem cells cultured under standard conditions. For example, microparticles can be obtained from freshly filtered conditioned medium collected from Integra CeLLine bioreactor cultured neural stem cells.


The upregulated markers include DCX (doublecortin—an early neuronal marker), GFAP (Glial fibrillary acidic protein—an astrocyte marker), GALC, TUBB3, GDNF and IDO. CTX0E03 cells are able to differentiate into 3 different cell types: neurons, astrocytes and oligodendrocytes. The high levels of DCX and GFAP after three weeks in a multi-compartment bioreactor indicates that the cultured stem cells have partially differentiated and have entered the neuronal (DCX+ cells) and/or astrocytic (GFAP+ cells) lineage. Accordingly, in one embodiment the invention provides a microparticle produced by a neural stem cell population that expresses (i) one or more markers associated with a neuronal lineage, typically DCX and/or (ii) one or more markers associated with an astrocytic lineage, typically GFAP. In another embodiment, the invention provides neural stem cell microparticles, typically exosomes, that express (i) one or more markers associated with a neuronal lineage, typically DCX and/or (ii) one or more markers associated with an astrocytic lineage, typically GFAP. These cells, or the microparticles (typically exosomes) derived from these cells, express DCX and/or GFAP at a higher level than the corresponding stem cells in standard (T-175) culture. Typically, these cells or microparticles express DCX and/or GFAP at a level at least 2 fold more than the stem cells, more typically at least 2.5 fold more than the corresponding stem cells in standard culture, at least 5 fold more than the corresponding stem cells in standard culture, at least 7.5 fold more than the corresponding stem cells in standard culture or at least 10 fold more than the corresponding stem cells in standard culture. For expression of DCX, the fold change in the cells or microparticles compared to the corresponding stem cells in standard (T-175) culture can optionally be at least 20 fold, at least 50 fold, at least 100 fold, at least 500 fold or at least 1000 fold more than the standard stem cells.


The term “bioreactor” is to be given its usual meaning in the art, i.e. an apparatus used to carry out a bioprocess. The bioreactors described herein are suitable for use in cell culture, e.g. stem cell culture. Simple bioreactors for cell culture are single compartment flasks, such as the commonly-used T-175 flask (e.g. the BD Falcon™ 175 cm2 Cell Culture Flask, 750 ml, tissue-culture treated polystyrene, straight neck, blue plug-seal screw cap, BD product code 353028). Bioreactors can have multiple compartments, as is known in the art. These multi-compartment bioreactors typically contain at least two compartments separated by one or more membranes or barriers that separate the compartment containing the cells from one or more compartments containing gas and/or culture medium. Multi-compartment bioreactors are well-known in the art. An example of a multi-compartment bioreactor is the Integra CeLLine bioreactor, which contains a medium compartment and a cell compartment separated by means of a 10 kDa semi-permeable membrane; this membrane allows a continuous diffusion of nutrients into the cell compartment with a concurrent removal of any inhibitory waste product. The individual accessibility of the compartments allows to supply cells with fresh medium without mechanically interfering with the culture. A silicone membrane forms the cell compartment base and provides an optimal oxygen supply and control of carbon dioxide levels by providing a short diffusion pathway to the cell compartment. Any multi-compartment bioreactor may be used according to the invention.


Example 11, Table 3 and FIG. 10 show that the miRNA content of exosomes produced by neural stem cells that have been cultured in a multi-compartment bioreactor, for three weeks, is different from the miRNA content of stem cells cultured in standard T-175 flasks and from microparticles produced by the neural stem cells cultured in a single-compartment T175 culture flask for three weeks. In one embodiment, the invention provides a microparticle, typically an exosome, wherein at least two, three, four, five, six or seven miRNAs are up or down regulated compared to in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation (see Example 11). The Fold Regulation of each miRNA is optionally at least two-fold up or down.


It can be seen from FIG. 6C and Example 8 that exosomes isolated from NSCs show particularly surprising efficacy when the NSCs have been cultured for several weeks. Accordingly, in one embodiment, exosomes of the invention are isolated from NSCs that have been cultured, typically in a multi-compartment bioreactor, for at least two weeks, typically at least three weeks, at least four weeks, at least five weeks or at least six weeks. Optionally, the NSCs have been cultured for no more than ten weeks, e.g. between 2 and 10 weeks, between 3 and 10 weeks, between 4 and 10 weeks, between 5 and 10 weeks or between 6 and 10 weeks.


In one embodiment, neural stem cell exosomes of the invention express one, two, three, four, five, six or seven of the following miRNAs at a higher level than is expressed in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation (where an asterisk indicates an miRNA where at least a two-fold regulation increase is preferred):

















hsa-miR-146b-5p*



hsa-let-7c*



hsa-miR-99a*



hsa-miR-132*



hsa-miR-378*



hsa-miR-181a*



hsa-let-7b*










In one embodiment, neural stem cell exosomes of the invention express one, two, three, four, five, six, seven, eight, nine, ten or more of the following miRNAs at a lower level than is expressed in the corresponding stem cells cultured in standard T-175 flasks, as calculated by


Fold Regulation (where an asterisk indicates an miRNA where at least a two-fold regulation decrease is preferred):

















hsa-miR-7*



hsa-miR-106b*



hsa-miR-101*



hsa-miR-302a*



hsa-miR-301a*



hsa-miR-183*



hsa-miR-219-5p*



hsa-miR-18a*



hsa-miR-15a*



hsa-miR-182*



hsa-miR-33a*



hsa-miR-96*



hsa-miR-18b*










In a further embodiment, NSC exosomes of the invention comprise (i) an increased level of at least one, two, three, four, five, six or seven of the miRNAs indicated above as being increased in exosomes compared to the corresponding cells in standard culture and (ii) a decreased level of at least one, two, three, four, five, six, seven, eight, nine, ten or more or more of the miRNAs indicated above as being decreased in exosomes compared to the corresponding cells in standard culture. For example, a neural stem cell exosome may contain a fold-regulation increase in three or more or more of the miRNAs indicated above as being increased in exosomes compared to the corresponding cells in standard culture and a fold-regulation decrease in three or more of the miRNAs indicated above as being decreased in exosomes compared to the corresponding cells in standard culture. In another exemplary embodiment, a neural stem cell exosome may contain a fold-regulation increase in five or more of the miRNAs indicated above as being increased in exosomes compared to the corresponding cells in standard culture and a fold-regulation decrease in five or more of the miRNAs indicated above as being decreased in exosomes compared to the corresponding cells in standard culture.


The term “expressed” is used to describe the presence of a marker within a cell or microparticle. In order to be considered as being expressed, a marker must be present at a detectable level. By “detectable level” is meant that the marker can be detected using one of the standard laboratory methodologies such as qRT-PCR, or qPCR, blotting, Mass Spectrometry or FACS analysis. A gene is considered to be expressed by a cell or microparticle of the population of the invention if expression can be reasonably detected at a crossing point (cp) values below or equal 35. The terms “express” and “expression” have corresponding meanings. At an expression level below this cp value, a marker is considered not to be expressed. The comparison between the expression level of a marker in a stem cell or microparticle of the invention, and the expression level of the same marker in another cell or microparticle, such as for example an mesenchymal stem cell, may preferably be conducted by comparing the two cell/microparticle types that have been isolated from the same species. Preferably this species is a mammal, and more preferably this species is human. Such comparison may conveniently be conducted using a reverse transcriptase polymerase chain reaction (RT-PCR) experiment.


As used herein, the term “significant expression” or its equivalent terms “positive” and “+” when used in regard to a marker shall be taken to mean that, in a cell or microparticle population, more than 20%, preferably more than, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 98%, 99% or even all of the cells of the cells/microparticles express said marker.


As used herein, “negative” or “−” as used with respect to markers shall be taken to mean that, in a cell or microparticle population, less than 20%, 10%, preferably less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or none of the cells/microparticles express said marker.


Expression of microparticle surface markers may be determined, for example, by means of flow cytometry and/or FACS for a specific cell surface marker using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art) to determine whether the signal for a specific microparticle surface marker is greater than a background signal. The background signal is defined as the signal intensity generated by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker. For a marker to be considered positive the specific signal observed is typically more than 20%, preferably stronger than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000%, 10000% or above, greater relative to the background signal intensity. Alternative methods for analysing expression of microparticle surface markers of interest include visual analysis by electron microscopy using antibodies against cell-surface markers of interest.


“Fluorescence activated cell sorting (FACS)” is a method of cell purification based on the use of fluorescent labelled antibodies. The antibodies are directed to a marker on the cell surface, and therefore bind to the cells of interest. The cells are then separated based upon the fluorescent emission peak of the cells.


Microparticle markers (including surface and intracellular proteins) can also be analysed by various methods known to one skilled in the art to assay protein expression, including but not limited to gel electrophoresis followed by western blotting with suitable antibodies, immunoprecipitation followed by electrophoretic analysis, and/or electron microscopy as described above, with microparticle permeabilisation for intraparticle markers. For example, expression of one or more tetraspanins may be assayed using one or more of the above methods or any other method known to one skilled in the art. RNA levels may also be analysed to assess marker expression, for example qRT-PCR.


Microparticle Function

As noted above, a stem cell microparticle retains at least one biological function of the stem cell from which it is derived. Biological functions that may be retained include the ability to promote angiogenesis, tissue regeneration, tissue repair, and/or neurogenesis, the ability to effect cognitive improvement in the brain of a patient that has suffered a stroke, or the ability to accelerate blood flow recovery in peripheral arterial disease.


For example, CTX0E03 cells are known to inhibit T cell activation in a PBMC assay and, in one embodiment, the microparticles of the invention retain this ability to inhibit T cell activation in a PBMC assay. PBMC assays are well-known to the skilled person and kits for performing the assay are commercially available.


Example 8, Table 2 and FIG. 6 demonstrate that CTX0E03 stem cell exosomes retain the ability to close a wound in a “scratch” model of wound healing. The results show that the migration activity of normal human dermal fibroblasts (NHDF) cultured in CTX0E03 conditioned media is almost the same as the migration activity observed on the addition of purified exosomes. Accordingly, one biological function that microparticles of the invention may retain is the ability to stimulate migration activity of normal human dermal fibroblasts (NHDF). NHDF migration assays are known in the art. Stimulation of NHDF migration may be determined using an in vitro scratch (wound closure) assay, for example the assay of Example 8(A). Wound closure is calculated as the area covered by NHDF cells in relation to the initial wound area as determined at 0 hours. Stimulation of NHDF migration in this assay is typically defined as an increase in wound closure, typically a wound closure at least 1.2× greater, more typically at least 1.5× greater, than the wound closure under basal conditions (without the microparticles) after 24 hours. After 48 hours, the wound closure is typically at least 1.2× greater or 1.5× greater, more typically at least 2× greater, than the wound closure under basal conditions (without the microparticles). Stimulation of NHDF migration may also be defined as causing a wound closure of 100%, as determined by the scratch assay, at least 24 hours before 100% wound closure is observed under basal conditions.


Example 8 also shows that microvesicles of the invention are able to stimulate angiogenesis of primary HUVECs and to stimulate neurite outgrowth of PC-12 cells. Accordingly, a biological function that microparticles of the invention may retain is the ability to stimulate angiogenesis of primary HUVECs and/or to stimulate neurite outgrowth of PC-12 cells. Angiogenesis and neurite outgrowth assays are known in the art. Stimulation of angiogenesis of primary HUVECs may be determined using a 24 hour angiogenesis assay using an ibidi p-slide and Wimtube detection and analysis of tube length and bifurcation points, for example the assay of Example 8(B). Stimulation of angiogenesis in this assay is typically defined as an increase compared to basal angiogenesis, e.g. >100% basal angiogenesis, typically at least 110%, at least 120% or at least 140% basal angiogenesis (i.e. at least 1.1×, at least 1.2× or at least 1.4× the basal level of angiogenesis). Stimulation of neurite outgrowth may be determined by detecting outgrowth of PC-12 cells through a 1 μm insert, for example the assay of Example 8(C). Stimulation of neurite outgrowth in this assay is typically defined as an increase in neurite outgrowth compared to basal conditions (without microparticles), or an increase in neurite outgrowth when the microparticle is combined with NGF compared to the addition of NGF alone, as quantified by a spectrophotometer.


The proteomic analysis in Example 13 indicates that neural stem cell exosomes comprise biological functions associated with the production, packaging, function and degradation of genetic material. Accordingly, in one embodiment, exosomes of the invention retain these functions, typically one or more of RNA polymerase function, RNA degradation function, ribosome function and spliceosome function.


Immunogenicity

The (allogeneic) neural stem cell microparticles of the invention typically either do not trigger an immune response in vitro or in vivo or trigger an immune response which is substantially weaker than that which would be expected to be triggered upon injection of an allogeneic stem cell population into a patient. In certain aspects of the invention, the neural stem cell microparticles are considered not to trigger an immune response if at least about 70% of the microparticles do not trigger an immune response. In some embodiments, at least about 80%, at least about 90% or at least about 95%, 99% or more of the microparticles do not trigger an immune response. Preferably the microparticles of the invention do not trigger an antibody mediated immune response or do not trigger a humoral immune response. More preferably the microparticles of the invention do not trigger either an antibody mediated response or a humoral immune response in vitro. More preferably still, the microparticles of the invention do not trigger a mixed lymphocyte immune response. It will be understood by one skilled in the art that the ability of the cells of the invention to trigger an immune response can be tested in a variety of ways.


CTX0E03 cells transplanted in a rodent model of limb ischemia have been previously demonstrated a faster and transient up-regulation of host genes involved in angiogenesis, such as CCL11, CCL2, CXCL1, CXCL5, IGF1, IL1β, IL6, HGF, HIF1α, bFGF, VEGFA, and VEGFC, compared to vehicle treated controls. hNSC treatment transiently elevates host innate immune and angiogenic responses and accelerates tissue regeneration.


The CTX0E03 cell line has been previously demonstrated, using a human PBMC assay, not to be immunogenic. Accordingly, microparticles produced by CTX0E03 cells are also expected to be non-immunogenic. The lack of immunogenicity allows the microparticles to avoid clearance by the host/patient immune system and thereby exert their therapeutic effect without a deleterious immune and inflammatory response.


Stem Cells

The stem cell that produces the microparticle may be a stem cell line, i.e. a culture of stably dividing stem cells. A stem cell line can to be grown in large quantities using a single, defined source. Immortalisation may arise from a spontaneous event or may be achieved by introducing exogenous genetic information into the stem cell which encodes immortalisation factors, resulting in unlimited cell growth of the stem cell under suitable culture conditions. Such exogenous genetic factors may include the gene “myc”, which encodes the transcription factor Myc. The exogenous genetic information may be introduced into the stem cell through a variety of suitable means, such as transfection or transduction. For transduction, a genetically engineered viral vehicle may be used, such as one derived from retroviruses, for example lentivirus.


Additional advantages are provided by using a conditionally immortalised stem cell line, in which the expression of the immortalisation factor can be regulated without adversely affecting the production of therapeutically effective microparticles. This may be achieved by introducing an immortalisation factor which is inactive unless the cell is supplied with an activating agent. Such an immortalisation factor may be a gene such as c-mycER. The c-MycER gene product is a fusion protein comprising a c-Myc variant fused to the ligand-binding domain of a mutant estrogen receptor. C-MycER only drives cell proliferation in the presence of the synthetic steroid 4-hydroxytamoxifen (4-OHT) (Littlewood et al. 1995). This approach allows for controlled expansion of e.g. neural stem cells in vitro, while avoiding undesired in vivo effects on host cell proliferation (e.g. tumour formation) due to the presence of c-Myc or the gene encoding it in microparticles derived from the neural stem cell line. A suitable c-mycER conditionally immortalized neural stem cell is described in U.S. Pat. No. 7,416,888. The use of a conditionally immortalised neural stem cell line therefore provides an improvement over existing stem cell microparticle isolation and production. Other methods of conditional immortalisation are known in the art.


Preferred conditionally-immortalised cell lines include the CTX0E03, STR0C05 and HPC0A07 neural stem cell lines, which have been deposited at the European Collection of Animal Cultures (ECACC), Vaccine Research and Production laboratories, Public Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, with Accession No. 04091601 (CTX0E03); Accession No. 04110301 (STR0C05); and Accession No. 04092302 (HPC0A07). The derivation and provenance of these cells is described in EP1645626 B1. The advantages of these cells are retained by microparticles produced by these cells.


The cells of the CTX0E03 cell line may be cultured in the following culture conditions:

    • Human Serum Albumin 0.03%
    • Transferrin, Human 5 μg/ml
    • Putrescine Dihydrochloride 16.2 μg/ml
    • Insulin Human recombinant 5 μ/ml
    • Progesterone 60 ng/ml
    • L-Glutamine 2 mM
    • Sodium Selenite (selenium) 40 ng/ml


Plus basic Fibroblast Growth Factor (10 ng/ml), epidermal growth factor (20 ng/ml) and 4-hydroxytamoxifen 100 nM for cell expansion. The cells can be differentiated by removal of the 4-hydroxytamoxifen. Typically, the cells can either be cultured at 5% CO2/37° C. or under hypoxic conditions of 5%, 4%, 3%, 2% or 1% O2. These cell lines do not require serum to be cultured successfully. Serum is required for the successful culture of many cell lines, but contains many contaminants including its own exosomes. A further advantage of the CTX0E03, STR0C05 or HPC0A07 neural stem cell lines, or any other cell line that does not require serum, is that the contamination by serum is avoided.


The cells of the CTX0E03 cell line (and microparticles derived from these cells) are multipotent cells originally derived from 12 week human fetal cortex. The isolation, manufacture and protocols for the CTX0E03 cell line is described in detail by Sinden, et al. (U.S. Pat. No. 7,416,888 and EP1645626 B1). The CTX0E03 cells are not “embryonic stem cells”, i.e. they are not pluripotent cells derived from the inner cell mass of a blastocyst; isolation of the original cells did not result in the destruction of an embryo.


The CTX0E03 cells (and microparticles derived from these cells) are angiogenic and so are useful in treating diseases requiring angiogenesis, such as Peripheral Arterial Disease. The cells (and microparticles derived from these cells) are also neurogenic and are therefore useful in treating diseases requiring neurogenesis, such as the ischaemia (stroke) damaged brain. CTX0E03 is a clonal cell line that contains a single copy of the c-mycER transgene that was delivered by retroviral infection and is conditionally regulated by 4-OHT (4-hydroxytamoxifen). The C-mycER transgene expresses a fusion protein that stimulates cell proliferation in the presence of 4-OHT and therefore allows controlled expansion when cultured in the presence of 4-OHT. This cell line is clonal, expands rapidly in culture (doubling time 50-60 hours) and has a normal human karyotype (46 XY). It is genetically stable and can be grown in large numbers. The cells are safe and non-tumorigenic. In the absence of growth factors and 4-OHT, the cells undergo growth arrest and differentiate into neurons and astrocytes. Once implanted into an ischemia-damaged brain, these cells migrate only to areas of tissue damage.


The development of the CTX0E03 cell line has allowed the scale-up of a consistent product for clinical use. Production of cells from banked materials allows for the generation of cells in quantities for commercial application (Hodges et al, 2007).


Pollock et al 2006 describes that transplantation of CTX0E03 in a rat model of stroke (MCAo) caused statistically significant improvements in both sensorimotor function and gross motor asymmetry at 6-12 weeks post-grafting. These data indicate that CTX0E03 has the appropriate biological and manufacturing characteristics necessary for development as a therapeutic cell line.


Stevanato et al 2009 confirms that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.


Smith et al 2012 describe preclinical efficacy testing of CTX0E03 in a rat model of stroke (transient middle cerebral artery occlusion). The results indicate that CTX0E03 implants robustly recover behavioral dysfunction over a 3 month time frame and that this effect is specific to their site of implantation. Lesion topology is potentially an important factor in the recovery, with a stroke confined to the striatum showing a better outcome compared to a larger area of damage.


Neural retinal stem cell lines (for example as described in U.S. Pat. No. 7,514,259) may also be used according to the invention.


The term “culture medium” or “medium” is recognized in the art, and refers generally to any substance or preparation used for the cultivation of living cells. The term “medium”, as used in reference to a cell culture, includes the components of the environment surrounding the cells. Media may be solid, liquid, gaseous or a mixture of phases and materials. Media include liquid growth media as well as liquid media that do not sustain cell growth. Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices. Exemplary gaseous media include the gaseous phase to which cells growing on a petri dish or other solid or semisolid support are exposed. The term “medium” also refers to material that is intended for use in a cell culture, even if it has not yet been contacted with cells. In other words, a nutrient rich liquid prepared for bacterial culture is a medium. Similarly, a powder mixture that when mixed with water or other liquid becomes suitable for cell culture may be termed a “powdered medium”. “Defined medium” refers to media that are made of chemically defined (usually purified) components. “Defined media” do not contain poorly characterized biological extracts such as yeast extract and beef broth. “Rich medium” includes media that are designed to support growth of most or all viable forms of a particular species. Rich media often include complex biological extracts. A “medium suitable for growth of a high density culture” is any medium that allows a cell culture to reach an OD600 of 3 or greater when other conditions (such as temperature and oxygen transfer rate) permit such growth. The term “basal medium” refers to a medium which promotes the growth of many types of microorganisms which do not require any special nutrient supplements. Most basal media generally comprise of four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. A basal medium generally serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. In one aspect, the growth medium may be a complex medium with the necessary growth factors to support the growth and expansion of the cells of the invention while maintaining their self-renewal capability. Examples of basal media include, but are not limited to, Eagles Basal Medium, Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, Medium 199, Nutrient Mixtures Ham's F-10 and Ham's F-12, McCoy's 5A, Dulbecco's MEM/F-I 2, RPMI 1640, and Iscove's Modified Dulbecco's Medium (IMDM).


Pharmaceutical Compositions

The stem cell microparticle of the invention is useful in therapy and can therefore be formulated as a pharmaceutical composition. A pharmaceutically acceptable composition typically includes at least one pharmaceutically acceptable carrier, diluent, vehicle and/or excipient in addition to the microparticles of the invention. An example of a suitable carrier is Ringer's Lactate solution. A thorough discussion of such components is provided in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.


The phrase “pharmaceutically acceptable” is employed herein to refer 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 problem or complication, commensurate with a reasonable benefit/risk ratio.


The composition, if desired, can also contain minor amounts of pH buffering agents. The carrier may comprise storage media such as Hypothermosol®, commercially available from BioLife Solutions Inc., USA. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E W Martin. Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic microparticle preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. In a preferred embodiment, the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.


The pharmaceutical composition of the invention may be in a variety of forms. These include, for example, semi-solid, and liquid dosage forms, such as lyophilized preparations, liquid solutions or suspensions, injectable and infusible solutions. The pharmaceutical composition is preferably injectable. A particular advantage of the microparticles of the invention is their improved robustness compared to the stem cells from which they are obtained; the microparticles can therefore be subjected to formulation, such as lyophilisation, that would not be suitable for stem cells.


It is preferred that the methods, medicaments and compositions of the invention are used for treating or repairing damaged tissue, and/or for the treatment, modulation, prophylaxis, and/or amelioration of one or more symptoms associated with tissue disorders. Particularly preferred is the use of the methods, medicaments, compositions and microparticles of the invention in regenerative therapy, typically the treatment of stroke, peripheral arterial disease or blindness-causing diseases of the retina.


Pharmaceutical compositions will generally be in aqueous form. Compositions may include a preservative and/or an antioxidant.


To control tonicity, the pharmaceutical composition can comprise a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride and calcium chloride.


Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included at a concentration in the 5-20 mM range. The pH of a composition will generally be between 5 and 8, and more typically between 6 and 8 e.g. between 6.5 and 7.5, or between 7.0 and 7.8.


The composition is preferably sterile. The composition is preferably gluten free. The composition is preferably non-pyrogenic.


In a typical embodiment, the microparticles are suspended in a composition comprising 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), Na+, K+, Ca2+, Mg2+, Cl, H2P04, HEPES, lactobionate, sucrose, mannitol, glucose, dextron-40, adenosine and glutathione. Typically, the composition will not include a dipolar aprotic solvent, e.g. DMSO. Suitable compositions are available commercially, e.g. HypoThermasol®-FRS. Such compositions are advantageous as they allow the microparticles to be stored at 4° C. to 25° C. for extended periods (hours to days) or preserved at cryothermic temperatures, i.e. temperatures below −20° C. The microparticles may then be administered in this composition after thawing.


The pharmaceutical composition can be administered by any appropriate route, which will be apparent to the skilled person depending on the disease or condition to be treated. Typical routes of administration include intravenous, intra-arterial, intramuscular, subcutaneous, intracranial, intranasal or intraperitoneal. For treatment of a disorder of the brain, one option is to administer the microparticles intra-cerebrally, typically to the site of damage or disease.


The microparticles will be administered at a therapeutically or prophylactically-effective dose, which will be apparent to the skilled person. Due to the low or non-existent immunogenicity of the microparticles, it is possible to administer repeat doses without inducing a deleterious immune response.


Therapeutic Uses

The microparticles of the invention are useful in the treatment or prophylaxis of disease. Accordingly, the invention includes a method of treating or preventing a disease or disorder in a patient using a microparticle of the invention. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.


As noted above, the compositions comprising miRNAs of the invention are also useful in these therapies, and references to therapeutic uses of microparticles herein therefore applies equally to the compositions comprising miRNAs.


Therapeutically useful microparticles of the invention have regenerative activity. A microparticle having regenerative activity is a microparticle that is capable of activating or enhancing regenerative processes, or inhibiting or reducing degenerative processes. Regenerative processes lead to renewal, restoration, repair and/or growth of cells and tissues. Degenerative processes lead to a loss of cell or tissue integrity and/or function. This may be particularly useful in treating damaged or disturbed cells or tissues, such as those resulting from Stroke, psychiatric disorders, myocardial infarction, Amyotrophic lateral sclerosis and Peripheral arterial disease.


The microparticles of the invention are useful in tissue regeneration. “Tissue regeneration” is the process of increasing the number of cells in a tissue following a trauma. The trauma can be anything which causes the cell number to diminish. For example, an accident, an autoimmune disorder or a disease state could constitute trauma. Tissue regeneration increases the cell number within the tissue and enables connections between cells of the tissue to be re-established, and the functionality of the tissue to be regained.


The therapy may be regenerative therapy requiring tissue replacement, regeneration or repair. The therapy may be for a neurological disease, disorder or deficit. The therapy may improve functional and/or cognitive recovery. The therapy may be of stroke, peripheral arterial disease, neuropathy or any other disease or disorder that requires tissue regeneration, revascularisation or local anti-inflammatory action, including:

    • (i) Neurological disorder, disease or deficit, such as Parkinson's disease, Alzheimer's disease, Stroke, or ALS;
    • (ii) Lysosomal storage disorders;
    • (iii) Cardiovascular disorders, such as Myocardial Infarction, congestive heart failure, Peripheral Arterial Disease, diabetic ulcers, wound healing;
    • (iv) Diseases of the lung, including Idiopathic Pulmonary Fibrosis, Respiratory Distress Syndrome, Chronic Obstructive Pulmonary Disease, Idiopathic Pulmonary Hypertension, Cystic Fibrosis and Asthma;
    • (v) Metabolic or inflammatory disorders, such as Diabetes (I or II), rheumatoid arthritis, osteoarthritis, lupus, Crohn's disease, Inflammatory Bowel Disease, or Graft versus Host Disease;
    • (vi) Psychiatric disorders, such as Depression, Bipolar disorder, Schizophrenia or an Autistic syndrome disorder such as Autism, Asperger's syndrome or Rett Syndrome;
    • (vii) Blindness-causing diseases of the retina, such as Age-related macular degeneration, Stargardt disease, diabetic retinopathy, retinitis pigmentosa; and
    • (viii) Demyelinating diseases, such as multiple sclerosis, cerebral palsy, central pontine myelinolysis, tabes dorsalis, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukodystrophies, Guillain-Barre syndrome, Anti-MAG peripheral neuropathy and Charcot-Marie-Tooth disease.


In one embodiment, the microparticle and compositions containing them are not used for immune modulation. In one embodiment, the therapy is not related to immunomodulation.


The invention also provides a method for treating or preventing a disease or condition comprising administering an effective amount of the microparticle of the invention, thereby treating or preventing the disease. Typically, the disease or condition is as identified above.


The microparticles of the invention can be used to treat the same diseases as the stem cells from which they are obtained. Neural stem cells are known to be useful in the treatment of diseases including: Stroke, brain damage such as motor, sensory and/or cognitive deficit, psychiatric disorders, myocardial infarction, Amyotrophic lateral sclerosis, limb ischaemia, peripheral arterial disease. Accordingly, the microparticles of the invention are also useful in the treatment of Stroke, brain damage such as motor, sensory and/or cognitive deficit, psychiatric disorders, myocardial infarction, Amyotrophic lateral sclerosis, limb ischaemia, peripheral arterial disease.



FIG. 6 and Example 8 demonstrate that exosomes obtained from neural stem cells stimulate wound healing. Accordingly, in one embodiment, exosomes of the invention are used to treat a disease or condition requiring tissue replacement, regeneration or repair. Such conditions include diabetic ulcers and wound healing. FIG. 6C shows that exosomes isolated from NSCs cultured for 6 weeks are more efficacious than exosomes isolated from NSCs cultured for 2 weeks. Accordingly, in one embodiment, exosomes isolated from NSCs (typically CTX0E03 cells) that have been cultured (typically in a multi-compartment bioreactor) for at least 2 weeks, more typically at least 4 weeks or at least 6 weeks, are used to treat a disease or condition requiring tissue replacement, regeneration or repair. Optionally, the NSCs have been cultured for no more than ten weeks, e.g. between 2 and 10 weeks, between 3 and 10 weeks, between 4 and 10 weeks, between 5 and 10 weeks or between 6 and 10 weeks.


The observed increased efficacy of exosomes isolated from NSCs (CTX0E03 cells) that have been cultured (in a multi-compartment bioreactor) for 6 weeks correlates with the observed reduction in size of the exosomes to around 70 nm diameter, which also occurred after culturing the cells for 6 weeks. Accordingly, in one embodiment, exosomes isolated from NSCs (typically CTX0E03 cells) that have been cultured (typically in a multi-compartment bioreactor) for at least 6 weeks are used to treat a disease or condition requiring tissue replacement, regeneration or repair. As noted above, optionally the NSCs have been cultured for no more than ten weeks, e.g. between 6 and 10 weeks. In another embodiment, exosomes isolated from NSCs (typically CTX0E03 cells) having a diameter less than 100 nm, typically less than 80 nm, for example around 70 nm diameter, are used to treat a disease or condition requiring tissue replacement, regeneration or repair.


As shown in FIG. 12 and discussed in Example 8, microvesicles obtained from neural stem cells stimulate angiogenesis. Accordingly, in one embodiment, microvesicles of the invention are used to treat a disease or condition requiring angiogenesis, typically a disease or disorder that is treated by tissue regeneration and/or revascularisation. Microvesicles of the invention can be used in the treatment of cardiovascular disorders, such as Myocardial Infarction, congestive heart failure, Peripheral Arterial Disease, diabetic ulcers and wound healing. The stimulation of angiogenesis is also therapeutically useful in the treatment of ischaemia, in particular cardiac ischaemia and limb ischaemia. FIG. 12 shows that microvesicles harvested from NSCs cultured for at least 3 weeks are more efficacious than microvesicles isolated from NSCs cultured for 1 or 2 weeks. Accordingly, in one embodiment, microvesicles isolated from NSCs (typically CTX0E03 cells) that have been cultured (typically in a multi-compartment bioreactor) for at least 3 weeks, more typically at least 4 weeks or at least 6 weeks, are used to treat a disease or condition requiring angiogenesis. Optionally, the NSCs have been cultured for no more than ten weeks, e.g. between 3 and 10 weeks, between 4 and 10 weeks, between 5 and 10 weeks or between 6 and 10 weeks.


As shown in FIG. 13 and discussed in Example 8, microvesicles obtained from neural stem cells stimulate neurite outgrowth. Accordingly, in one embodiment, microvesicles of the invention are used to treat a neurological disease, disorder or deficit, such as Parkinson's disease, Alzheimer's disease, Stroke, neuropathy or ALS.


In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a particular disease in an amount sufficient to eliminate or reduce the risk or delay the outset of the disease. In therapeutic applications, compositions or medicaments are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a therapeutically- or pharmaceutically-effective dose. In both prophylactic and therapeutic regimes, agents are typically administered in several dosages until a sufficient response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to fade.


The microparticles of the invention may optionally be combined with a stem cell to provide a combination therapy. The stem cell is optionally the stem cell from which the microparticle is derived, e.g. if the microparticle is an exosome from a CTX0E03 cell, then the stem cell for use in combination therapy may be a CTX0E03 cell. A stem cell and microparticle can optionally be (i) administered together in a single pharmaceutical composition, (ii) administered contemporaneously or simultaneously but separately, or (iii) administered separately and sequentially, e.g. stem cell followed by microparticle, or microparticle followed by stem cell. When the stem cell and microparticle are administered separately and sequentially, the duration between the administration of the cell and microparticle may be one hour, one day, one week, two weeks or more.


In one embodiment, a prophylactic therapy induces tolerance, typically immunotolerance, in a host that is to receive the stem cells from which the microparticle is derived. In one embodiment, the administration of one or more doses of microparticles of the invention to a patient, prior to administration of a stem cell therapy, can be used to reduce the risk of an adverse immune response, i.e. “rejection”, of the stem cell therapy. In another embodiment, tolerance to the stem cells can be increased by administering stem cells together with microparticles of the invention, as discussed above.


Effective doses of the compositions of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human.


The CTX0E03 cell line has been shown to be effective in treating stroke, peripheral arterial disease, brain damage such as motor, sensory and/or cognitive deficit, and psychiatric disorders. The cells are currently being tested in a clinical trial for treatment of disabled stroke patients (Clinicaltrials.gov Identifier: NCT01151124). WO-A-2012/004611 describes the use of the CTX0E03 cells in treating psychiatric disorders including unipolar and bipolar depression, schizophrenia, obsessive compulsive disorder, autism and autistic syndrome disorders. Accordingly, microparticles produced by CTX0E03 cells are also able to treat stroke, peripheral arterial disease, blindness-causing diseases of the retina (such as retinitis pigmentosa), brain damage such as motor, sensory and/or cognitive deficit, and psychiatric disorders.


As used herein, the terms “treat”, “treatment”, “treating” and “therapy” when used directly in reference to a patient or subject shall be taken to mean the amelioration of one or more symptoms associated with a disorder, or the prevention or prophylaxis of a disorder or one or more symptoms associated with a disorder. The disorders to be treated include, but are not limited to, a degenerative disorder, a disorder involving tissue destruction, a neoplastic disorder, an inflammatory disorder, an autoimmune disease or an immunologically mediated disease including rejection of transplanted organs and tissues. Amelioration or prevention of symptoms results from the administration of the microparticles of the invention, or of a pharmaceutical composition comprising these microparticles, to a subject in need of said treatment.


Tracing Administered Cells and Microparticles In Vivo

The present invention provides a distinct marker profile for microparticles produced by neural stem cells. It is therefore possible to detect the presence of these microparticles in vivo, by testing a sample obtained from a patient and determining whether the marker profile in the sample matches that of the microparticles. If the sample profile matches the profile of the microparticles described herein, then this confirms the presence of the microparticles. This can be used to detect not only the presence and/or biodistribution of the microparticles themselves, but also the presence of stem cells producing the microparticles. This is particularly useful when detecting whether a stem cell administered in vivo has engrafted into the host tissue, and/or has migrated, for example in ADME(T) studies.


Detection of the microparticles in vivo can be used to monitor the course of a treatment wherein microparticles or stem cells are administered to a patient. Determining the presence, absence or amount of microparticles or cells producing microparticles of the invention in a patient allows the dosage regime to be altered accordingly, e.g. to increase or decrease the dose as required to provide an effective amount of microparticles or stem cells in vivo.


Methods of Producing Microparticles

Microparticles are isolated from cell conditioned media, typically stem cell conditioned media. The “conditioned medium” (CM) may be a growth medium for stem cells, which has been used to culture a mass culture of stem cells for at least about 12 hours, at least about 24 hours, at least about 48 hours or least about 72 hours, typically up to 168 hours (7 days), removed and sterilized by any suitable means, preferably by filtration, prior to use, if required.


Alternatively, microparticles may be harvested from a two-compartment bioreactor which allows the cell culture, and hence the conditioned media, to be maintained for longer periods of time, for example at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or more. The system maintains the cells and secreted microparticles within a small cell compartment (approximately 15 ml) which is separated from a larger reservoir of medium by a 10 kDa semi-permeable membrane. This allows the efficient removal of metabolic waste products while effectively maintaining an extremely high cell density to maximize microparticle production. Example 9, and FIGS. 7 and 8, demonstrate that use of a two-compartment bioreactor results in a much higher yield of microparticles than is obtained when a standard cell culture flask (T175 flask) is used.


The microparticles may be separated from other media components based on molecular weight, size, shape, hydrodynamic radius, composition, charge, substrate-ligand interaction, absorbance or scattering of electromagnetic waves, or biological activity. In one embodiment, the conditioned media is filtered using a filter of appropriate size to separate the desired microparticle, for example a 100K MWCO filter. Optionally, the stem cell-conditioned medium is concentrated prior to the isolation of the microparticles by subjecting the concentrated NSC-conditioned medium to size exclusion chromatography. The UV absorbant fractions can then be selected for isolation of the microparticles of interest.


Different microparticles can be isolated from the media by using different isolation techniques and parameters. For example, exosomes have a vesicle density of 1.13-1.19 g/mL and can be isolated by differential centrifugation and sucrose gradient ultracentrifugation at 100,000-200,000 g. Microvesicles can be isolated by filtration (100K MWCO) and differential centrifugation at 18,000-20,000 g. Membrane particles have a density of 1.04-01.07 g/ml and Exosome-like vesicles have a density of 1.1 g/ml.


A typical production method comprises: culturing stem cells to produce conditioned media; removing cell debris by centrifugation at 1500 rpm; isolating microvesicles (<1000 kDa) by ultrafiltration through a 100K MWCO filter or isolating exosomes (30-100 nm) by ultracentrifugation at 120,000 g; followed by quantification using a BCA protein assay.


Conditionally Immortalised Stem Cells as Producer Cells for Microparticles

In one aspect of the invention, conditionally immortalised stem cells are used to produce microparticles such as microvesicles and/or exosomes. Conditional immortalisation provides a constant supply of clonal cells that produce microparticles such as exosomes. These conditionally immortalised stem cells are typically neural stem cells, but may be a stem cell of any type, for example a haematopoietic stem cell or a mesenchymal stem cell. The conditionally immortalised stem cell may be a CD34+ cell. A method of producing stem cell microparticles is therefore provided, comprising the steps of culturing conditionally-immortalised stem cells and harvesting the microparticles that are produced by the cells. Conditional immortalisation of stem cells is known in the art, as described above; in one embodiment, conditional immortalisation is achieved by introducing c-mycER, which drives cell proliferation only in the presence of the synthetic steroid 4-hydroxytamoxifen (4-OHT). For the avoidance of doubt, this method is not limited to the use of neural stem cells.


The Examples demonstrate the production of exosomes from conditionally-immortalised neural stem cells and CD34+ cord blood cells. This exemplification can readily be applied to other cell types, including mesenchymal stem cells.


When the stem cell used to produce microparticles is a neural stem cell, it may be any of the neural stem cells described herein, for example the CTX0E03 conditionally-immortalised cell line which is clonal, standardised, shows clear safety in vitro and in vivo and can be manufactured to scale thereby providing a unique resource for stable exosome production. Alternatively, the neural stem cells may be neural retinal stem cell lines, optionally as described in U.S. Pat. No. 7,514,259.


Microparticles may also be produced from conditionally-immortalised neurosphere initiating stem cells (NS-IC) that are CD45−, CD34−. These cells can initiate neurosphere culture. A neurosphere is an aggregate or cluster of cells which includes neural stem cells and primitive progenitors. NS-IC cells are known in the art, for example as described in EP 1900811 B1 (StemCells Inc.). NS-IC cells are typically AC133+.


In another embodiment, the conditionally immortalised stem cells are oligodendrocyte precursor cells (OPCs). OPCs are precursors to oligodendrocytes and are typically also able to differentiate into astrocytes and, optionally, neurons. OPCs typically express the proteoglycan PDGFRα. OPCs are described, for example, in WO-A-2010/075500 (StemCells Inc.); these OPCs are PDGFRα+/CD105 and optionally CD133+.


When the stem cell used to produce microparticles is a mesenchymal stem cell, it may optionally be a conditionally-immortalised adipose-derived stem cell (“ADSC”) or a conditionally-immortalised version of the mesenchymal stem cells described in WO-A-2009/105044; these cells are CD29+, CD44+, CD49a+/e+, CD105+, CD166+, CD34−, CD45−.


In a further embodiment, microparticles are produced by conditionally-immortalised human bone marrow derived stem cells expressing CD73, CD90 and CD105 and not expressing CD14, CD19, CD34, CD45 and HLA-DR, wherein at least 50% of the cells of the cell population express glial fibrillary acidic protein (GFAP) and secrete BDNF. Such cells are described in EP-A-2620493 (Brainstorm Cell Therapeutics).


In one embodiment, the conditionally immortalised mesenchymal stem cell is an endometrial regenerative cell (“ERC”). ERCs are known in the art and are typically isolated from menstrual blood. ERCs typically express CD9, CD29, CD41a, CD44, CD59, CD73, CD90 and CD105.


The production of exosomes from non-conditionally-immortalised ERCs is described, for example, in US patent publication number US2013/0195899 (Medistem Inc.).


A further embodiment provides microparticles from conditionally immortalised multipotent adult progenitor cells (“MAPCs”), which are typically derived from bone marrow. MAPCs are known in the art and typically express Oct3 and telomerase. MAPCS are being developed by Athersys Inc. as the “Multistem” product. In one embodiment, MAPCs are human multipotent non-embryonic, non-germ cells that can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages, express telomerase, and optionally express Oct-3. These MAPCs have typically undergone at least 10-40 cell doublings in culture (see, for example, U.S. Pat. No. 8,147,824; Athersys, Inc.). In another embodiment the MAPCs are a human cell population that express CD90 and CD49c and one or more of sox-9, sox-11, hox-A5, and MSX-1, and optionally express telomerase. These cells typically show that marker profile after having undergone 22 cell doublings in culture.


Another embodiment of mesenchymal stem cells that can be conditionally-immortalised according to the invention are mesenchymal progenitor cells (MPCs). MPCs express SDF-1 (stroma derived factor 1—a potent stroma derived CXCα chemokine). MPCs can typically differentiate into bone, fat and cartilage. MPCs may typically be isolated by selection for SDF-1 expression, for example using an SDF-1 antibody. MPCs are typically isolated from bone marrow. MPCs are typically positive for at least one marker selected from the group consisting of STRO-Ibright VCAM-Ibright, THY-Ibright, CD146bright and STRO-2bright. Optionally, the MPCs carry at least two markers selected from the group of surface markers specific for MPCs consisting of STRO-Ibri, LFA-3, THY-I, VCAM-I, ICAM-I, PECAM-I, P-selectin, L-selectin, CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD29, CDI 8, CD61, beta-1 integrin, 6-19, thrombomodulin, CDIO, CD13, SCF, PDGF-R, EGF-R, IGFI-R, NGF-R, FGF-R, Leptin-R, RANKL and CD 146 or any combination of these markers. The survival and/or proliferation of the MPC, or their progeny, may be enhanced by exposure to SDF-I or an analog thereof (see, for example, WO2006032075, Angioblast Inc. [now Mesoblast, Inc.]). MPCs are being developed commercially by Mesoblast under the name “Revascor”.


Other conditionally-immortalised stem cells from which microparticles, such as exosomes, can be isolated include haematopoietic stem cells, typically CD34+ haematopoietic stem cells, optionally isolated from umbilical cord blood (often referred to as “cord blood cells”). Example 15 and FIG. 20A-C demonstrates that Human CD34+ progenitor cells derived from cord blood were successfully conditionally immortalized with a c-mycERTAM lentivirus, and produce exosomes expressing the characteristic marker Alix.


Umbilical cord blood also includes non-haematopoietic stem cells, which can also be conditionally immortalised and used to produce microparticles.


Hematopoietic stem cells are also described in U.S. Pat. No. 7,794,705 (Amorcyte Inc.). These cells are CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity and are able to form hematopoietic colonies in vitro.


Microparticles may also be produced by conditionally-immortalised very small embryonic like stem cells (VSELs) derived from a human, which are typically CD133+ CXCR4+ CD34+ Lin CD45 (for example as described by WO2010039241, NeoStem Inc.). The VSELs are optionally Oct-4+, SSEA+, Nanog+, or express Oct-4 protein in nuclei and SSEA antigens on the surface.


The conditionally-immortalised stem cell may be a Multipotent Adult Progenitor Cell (“MAPC”). MAPCs are known in the art (see, for example, Reading et al. J Immunol. 2013 May 1; 190(9):4542-52).


Induced pluripotent stem (iPS) cells may also be conditionally-immortalised and used to produce exosomes. iPS cells are known in the art, as reviewed by Malik and Rao Methods Mol Biol, 2013; 997-23-33.


Conditionally Immortalised Differentiated (Non-Stem) Cells as Producer Cells for Microparticles

Conditional immortalisation is not limited to stem cells. Accordingly, in a further aspect of the invention, conditionally immortalised differentiated (i.e. non-stem) cells are used to produce microparticles such as microvesicles and/or exosomes.


Fibroblasts may be conditionally-immortalised and used to produce microparticles such as exosomes. Typically, the fibroblast is a human dermal fibroblast.


Dendritic cells may also be conditionally-immortalised and used to produce microparticles such as exosomes.


Methods of Inducing Microparticle Secretion

The inventors have found that it is possible to increase the production of microparticles by cells, typically stem cells. This finding, which is not limited to neural stem cells and can be used for the production of microparticles from any cell (typically stem cell), allows for an improved yield of microparticles to be obtained from a stem cell culture. This improved yield is particularly advantageous when combined with the continuous supply of microparticles provided by conditionally-immortalised cells.


A first technique to increase the production of microparticles by the (stem) cells is to treat the stem cells with one or more of TGF-β, IFN-γ or TNF-α, typically at between 1 and 25 ng/ml e.g. 10 ng/ml, for between 12 to 96 hours prior to the removal of conditioned media.


As explained in Example 2 below, the frequency of the occurrence of multivesicular bodies (MVBs) was observed to be altered by the presence of TGF-β, IFN-γ or TNF-α (10 ng/ml). The frequency was highest in the presence of TGF-β, followed by IFN-γ, followed by TNF-α. Therefore, adding one or more of TGF-β, IFN-γ or TNF-α to the stem cell culture medium will stimulate the production of microparticles by the cells. The microparticles can then be harvested, by separating the microparticles from other components as described above.


A second technique to increase the production of microparticles by the stem cells is to culture the cells under hypoxic conditions. Culturing cells under hypoxic conditions is well-known to the skilled person, and involves culturing the cells in an atmosphere that has less than atmospheric level of O2, i.e. less than 21% O2. This is typically achieved by placing the cells in an incubator that allows oxygen levels to be changed. Hypoxic culture typically involves culturing in an atmosphere containing less than 10% O2, more typically 5% or less O2, for example 4% or less, 3% or less, 2% or less, or 1% or less O2.


The inventors have also realised that co-culturing a stem cell with a different cell type can alter the production of microparticles by the stem cell. The different cell type may be a non-stem cell, i.e. a terminally differentiated cell type. Typically, the different cell type is one with which the stem cell would interact in vivo. In one embodiment, neural stem cells are co-cultured with epithelial cells such as endothelial cells, typically Human Umbilical Vein Endothelial Cells (HUVEC). It has been observed that in vivo, NSCs and the vasculature interact, with proliferating NSCs being localized in close proximity or adjacent to blood vessels. Receptor tyrosine kinase activation and signal protein secretion has also been observed to be upregulated when NSCs are co-cultured with endothelial cells, again indicating that the vasculature modulates the proliferation capacity of NSCs. Without wishing to be bound by theory, the inventors believe that in vivo, there is a pivotal interplay between NSCs and microvessels (i.e. endothelial cells) in the process of tissue regeneration, through amplification of cytokine expression. Microparticles, e.g. exosomes, derived from NSCs (for example CTX0E03 cells) co-cultured with endothelial cells (for example HUVEC) are therefore primed for therapeutic use, because they have been produced in an environment that mimics the in vivo environment in which the stem cells and microparticles are active.


Therefore, culturing a stem cell with a different cell type may improve the amount of microparticles produced and/or may refine the content of the microparticles, typically so that the microparticles produced by the stem cells are biased towards an activated state of tissue repair. Accordingly, microparticles produced by stem cells that have been co-cultured with other cells, e.g. NSCs co-cultured with endothelial cells, are advantageous. These microparticles may be obtained by isolation from the co-cultured stem-cell conditioned media, as described herein.


Surprisingly, the present inventors have realised that the amount of microparticles produced by stem cells can be increased greatly simply by culturing stem cells in a multi-compartment bioreactor. This finding is not limited to neural stem cells and applies generally to the culture of all stem cells. Accordingly, one aspect of the invention provides a method of producing microparticles from stem cells that have been cultured in a multi-compartment bioreactor. The cells from which the microparticles are harvested have typically been cultured for at least one week, typically at least 8, 9, 10, 11, 12, 13 or 14 days, for example 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more, for example at least three weeks, four weeks, five weeks, six weeks or more. It can be seen from FIG. 8 that the increase in microparticle production, week on week, is not merely additive but is exponential. The prolonged culture typically has been observed in the Integra Celline system two-compartment bioreactor (commercially available from Integra Biosciences AG, Zizers, Switzerland) but the findings are not limited to this specific multi-compartment bioreactor; any multi-compartment bioreactor can be used. This culture method can be used to produce microparticles from any stem cell type, including but not limited to neural stem cells and mesenchymal stem cells.


Method of Screening for an Agent that Alters Microparticle Production


The invention provides a method of screening for an agent that alters the production of a microparticle by a conditionally-immortalised cell, typically a conditionally-immortalised stem cell. This method comprises contacting a conditionally-immortalised (e.g. stem) cell with a candidate agent, typically under conditions suitable for microparticle production, and observing whether (i) the rate of production of microparticles by the contacted conditionally-immortalised cell increases or decreases, or (ii) the characteristics (e.g. size, protein, mRNA or miRNA content) of the microparticles changes, compared to a control stem cell that is not contacted with the agent.


Method for Screening Total RNA Composition of Conditioned Medium

Following centrifugation (5 min at 1500 rpm), microparticles are collected from conditioned medium through filtration (0.02-0.2 μm, or 100K MWCO). Total RNA is obtained using trizol based extraction followed by purification using Qiagen RNaesy mini kit. The extract in water has a 260:280 nm absorbance suggesting that it may be RNA. Total RNA is retro-transcribed with either a protocol suitable for mRNA (Superscript II RT, Invitrogen) or miRNA (mScript RT kit, Qiagen). Validation of mRNA and miRNA presence is proven by qRT-PCR using primers for ATP5B and YWHAZ for mRNA, and U6B and 15a for miRNA housekeeping genes respectively. The RNA may be assessed by a generic gene expression analysis assay such as an array (micro array or PCR based array), and sequencing.


Kits

The invention provides a kit for use in a method for producing the microparticle of the invention. The kit comprises a cell culture medium, a conditionally-immortalised cell and instructions for producing the microparticle of claim 22 using the kit. Optionally, the kit comprises one or more components of claim 19 or 20. The kit may also comprise a microparticle according to the invention, for use as a control. The control microparticle is optionally lypohilised. The kit may also contain optionally a detection agent suitable for detection of the produced microparticles, for example an antibody that binds specifically to a marker protein that can be used to identify the microparticle.


The invention is further described with reference to the following non-limiting examples.


EXAMPLES
Example 1
Preparation of Neural Stem Cells and Neural Stem Cell Microparticles for Visualisation by Electron Microscopy
Method
Embedding CTX0E03 Cells for Electron Microscopy





    • 5×70% CTX0E03 cultures

    • Treat with +/−4OHT, IFNγ, TNFα and TGFβ (all at 10 ng for 24 hrs)

    • Detach cells and fix overnight in 2.5% Gluteraldehyde in 0.1M Cacodylate pH7.4

    • Cells spun down 300 g

    • Buffered osmium 2%, 1.5 hrs

    • Spin, wash water, overnight

    • Uranium acetate 2%, 2 hrs

    • Spin, wash water, 30 mins

    • Ethanol gradient 20, 35, 50, 70, 80, 90, 100%, over weekend.

    • 100% propylene oxide (PO), 1 hr

    • Spin, 50% Agar LV resin in PO, 1 hr

    • 75% LV resin/PO 5 hrs

    • 100% resin overnight at 60° C.

    • Cool to RT before cutting (60-80 nm), Imaged TEM at 200 Kv.





Results


FIG. 1A-E shows the electron micrographs of the multivesicular bodies (MVBs) containing exosomes of approximately 30 nm-50 nm in diameter. FIG. 1F shows microvesicles >100 nm in diameter.


Example 2
Production of Neural Stem Cell Microparticles from a Neural Stem Cell Line
Method

5 Sub-confluent flasks containing the same culture of CTX0E03 cells were individually treated with either 10 ng/ml TGF-β, 10 ng/ml IFNγ, or 10 ng/ml TNFα alongside full growth media controls with or without the addition of 4OHT. 72 hours after treatment, the cells were collected using trypzean/EDTA, washed and fixed overnight in 2.5% Gluteraldehyde in 0.1M Cacodylate pH7.4 ready for electron microscopy evaluation.


Results

The frequency of the occurrence of multivesicular bodies (MVBs) was observed to be altered by the presence of TGF-β, IFN-γ or TNF-α. The frequency was highest in the presence of TGF-β, followed by IFN-γ, followed by TNF-α.


Conclusion

The production of microparticles from neural stem cells can be stimulated by the addition of the factors TGF-β, IFN-γ or TNF-α. This has the potential for more efficient production of microparticles.


Example 3
Purification, Quantification and Characterisation of Neural Stem Cell Microparticles
Method

An outline protocol for producing large quantities of microparticles is provided in FIG. 2. The main steps are purification, quantification, characterisation, efficacy testing and manufacture.

    • (1) Purification
      • Microparticles can be purified from stem cell-conditioned medium by ultracentrifugation, e.g. at 100000×g for 1-2 hours. Alternative or additional methods for purification of may be used, such as antibody-based methods, e.g. immunoprecipitation, magnetic bead purification, resin-based purification, using specific antibodies.
    • (2) Quantification
      • Purified microparticles can be quantified by quantification of total nucleic acid or protein levels, e.g. various PCR or colorimetric protein quantification methods such as such as the BCA assay. Other quantification techniques may alternatively be used, including an electron microscopy grid or an immune-assay using antibodies or antibody fragments that specifically bind to microparticle-specific markers (e.g. ELISA, immunoblotting).
    • (3) Characterisation
      • The microparticles can be functionally or structurally characterised. RNA/mRNA/miRNA and protein profiling can be used using methods well known in the art (SDS-PAGE, mass spectrometry, PCR). Constitutively secreted microparticles can be tested and compared to microparticles that have been induced by addition of an inducing agent such as transforming growth factor-beta (TGF-β), interferon-gamma (INF-γ) and/or tumour necrosis factor-alpha (TNF-α).
    • (4) Therapeutic Efficacy
      • The efficacy of the microparticles can be tested by in vitro and in vivo assays. For in vitro evaluation, neural stem cell microparticles can be added to cultures of monocytes, PBMCs, endothelial cells and/or fibroblasts and the effect of the microparticles on these cells evaluated. Administration of neural stem cell microparticles to suitable animal models can be used to evaluate the in vivo efficacy. Clinical trials can be performed to evaluate safety and outcome of neural stem cell microparticles in human subjects.
    • (5) Manufacture/Scale-Up
      • Bioreactors, such as the Integra disposable T1000, can be used for the large-scale manufacture of neural stem cell microparticles. The purified microparticles are then formulated as a therapeutic product.


Example 4
miRNA Characterization in CTX0E03 Microparticles
Methods





    • 3 conditions: CTX0E03 cells in standard culture; microparticles obtained from CTX0E03 cells in standard culture; and purified exosomes derived from CTX0E03 cells in Integra CELLine system (see Examples 7 to 11, below)

    • Investigation of miRNA array using qRT-PCR panel (Qiagen) according to manufacturer's instruction. This assay provides high precision and high sensitivity, with data normalization sensitive to method/choice of reference genes. It does not provide genome wide sequencing.


      Results: A) List of miRNAs with a cp 35 found in (i) standard CTX0E03 cells, (ii) filtered conditioned medium (0.02-0.2 μm filter) i.e. microparticles and (iii) exosomes derived from Integra CELLine system (preliminary miRNA qRT-PCR miscript array (Qiagen) results).





B) Arithmetic and geometric mean of the reference (housekeeping) genes


A



















CTX0E03

CM




std
CM
exosome



Mature miRNA
culture
microparticles
Integra





















hsa-miR-21-5p
19.52
20.9
20.72



hsa-let-7a-5p
22.64
23.11
22.36



hsa-miR-125b-5p
21.64
23.25
21.74



hsa-miR-9-5p
22.58
23.64
22.94



hsa-miR-92a-3p
23.2
23.94
24.01



hsa-miR-24-3p
23.73
24.24
23.83



hsa-miR-20a-5p
23.45
24.43
25.06



hsa-miR-16-5p
23.14
24.72
24.32



hsa-miR-100-5p
23.28
24.74
23.04



hsa-let-7b-5p
24.67
24.75
23.7



hsa-let-7f-5p
23.93
25.09
23.86



hsa-miR-17-5p
24.56
25.24
26.13



hsa-miR-23b-3p
24.3
25.3
24.13



hsa-miR-106b-5p
24.4
25.41
26.16



hsa-miR-222-3p
23.25
25.49
23.17



hsa-let-7e-5p
24.57
25.58
24.16



hsa-miR-26a-5p
23.4
25.63
24.2



hsa-miR-181a-5p
25.16
25.7
24.32



hsa-miR-125a-5p
23.56
25.75
24.88



hsa-miR-103a-3p
24.65
25.8
25.77



hsa-let-7i-5p
24.37
25.98
24.23



hsa-miR-99a-5p
24.44
26.05
23.44



hsa-let-7c
25.76
26.12
24.07



hsa-let-7g
25.2
26.15
25.17



hsa-miR-195-5p
24.72
26.34
25.67



hsa-miR-93-5p
25.15
26.48
26.06



hsa-miR-22-3p
25.03
26.49
25.66



hsa-miR-20b-5p
26.03
26.86
27.42



hsa-miR-18a-5p
26.71
26.87
29.06



hsa-miR-15b-5p
25.1
26.92
26.43



hsa-let-7d-5p
26.84
26.96
26.52



hsa-miR-424-5p
25.56
27.72
26.66



hsa-miR-15a-5p
26.88
27.89
29.3



hsa-miR-130a-3p
27.23
28.26
28.49



hsa-miR-33a-5p
30.34
28.54
34.18



hsa-miR-128-
26.94
28.64
27.66



hsa-miR-218-5p
27.79
28.68
28.03



hsa-miR-301a-3p
29.53
28.69
31.57



hsa-miR-134
28.3
28.76
28.76



hsa-miR-101-3p
28.44
28.82
31.64



hsa-miR-7-5p
29.71
28.82
30.22



hsa-miR-18b-5p
28.83
28.85
35.47



hsa-miR-185-5p
28.34
28.99
28.13



hsa-miR-378-3p
29.76
29.25
28.97



hsa-miR-132-3p
28.65
29.32
27.72



hsa-miR-345-5p
28.49
29.52
29.66



hsa-miR-219-5p
30.58
29.52
32.7



hsa-miR-127-5p
30.05
29.95
31.11



hsa-miR-146b-5p
30.53
30.54
28.07



hsa-miR-10a-5p
27.1
30.69
28.32



hsa-miR-210
29.85
30.83
30.65



hsa-miR-129-5p
32.51
30.98
31.69



hsa-miR-137
31.46
31.13
30.95



hsa-miR-182-5p
28.34
31.64
31.27



hsa-miR-124-3p
33.38
31.71
33.07



hsa-miR-96-5p
29.77
32.27
34.67



hsa-miR-192-5p
31.42
32.42
32.52



hsa-miR-126-3p
31.73
32.44
32.05



hsa-miR-194-5p
31.11
32.49
31.72



hsa-miR-375
33.77
32.94
30.94



hsa-miR-205-5p
35
33.01
32.72



hsa-miR-183-5p
29.88
33.21
31.74



hsa-miR-10b-5p
29.6
33.22
30.79



hsa-miR-302a-3p
29.67
33.6
31.69



hsa-miR-214-3p
34.19
33.76
32.11



hsa-miR-141-3p
35
33.96
34.51



hsa-miR-302c-3p
31.6
34.29
33.93



hsa-miR-196a-5p
35
34.65
35.75



hsa-miR-150-5p
34.59
34.76
34.59



hsa-miR-155-p
32.04
35.75
32.76

























CTX0E03

CM



std
CM
exosome



culture
microparticles
Integra



















Avg. of Arithmetic Mean
23.54
23.82
24.79


Avg. of Geometric Mean
23.48
23.8
24.62









B
Example 5
CTX0E03 Conditioned Medium Analysis Using a Protein Dot Blot
Methods





    • Conditioned 24 hr and 72 hrs conditioned medium (RMM and ITS medium)

    • The collected media has been ‘concentrated’ by dialysis and the proteins biotinylated (typical total protein concentration appears to be 0.5 mg/ml). The media is then incubated with the Raybiotech L507 human protein arrays (total protein concentration 0.1 mg/ml). Following washing and incubation of the array with HRP-conjugated streptavidin, the presence of proteins is detected by chemiluminescence. The array provides qualitative data (i.e. the protein is present, but no indication of its level of expression compared to other proteins).





Results














Cytokine Name
Cytokine Full Name
Function







EDA-A2
ectodysplasin-A2
May be involved in proper




formation of skin appendages


Galectin-3*
Galectin-3
Galactose-specific lectin which




binds IgE. May mediate with the




alpha-3, beta-1 integrin the




stimulation by CSPG4 of




endothelial cells migration.


IGFBP-2
Insulin-like growth factor binding
IGF-binding proteins prolong the



proteins 2
half-life of the IGFs and have




been shown to either inhibit or




stimulate the growth promoting




effects of the IGFs on cell




culture.


IGFBP-rp1/IGFBP-7
Insulin-like Growth Factor
soluble proteins that bind IGFs



Binding Protein Related Protein-
with high affinity.



1 Insulin-like Growth Factor



Binding Protein-7


IL-1a†
Interleukin 1 alpha
potent mediator of inflammation




and immunity


LECT2†
Leukocyte cell-derived
Has a neutrophil chemotactic



chemotaxin-2
activity. Also a positive regulator




of chondrocyte proliferation.


MCP-1†
Monocyte chemoattractant
plays a role in the recruitment of



protein 1
monocytes to sites of injury and




infection.


SPARC*
Secreted Protein, Acidic
matricellular protein that



Cysteine-rich-related modular
modulates cell adhesion and



calcium-binding protein 1
proliferation and is thought to



[Precursor]
function in tissue remodeling




and angiogenesis


TIMP-1*
Tissue inhibitor of
Complexes with



metalloproteinasess-2
metalloproteinases (such as




collagenases) and irreversibly




inactivates them. Also mediates




erythropoiesis in vitro; but,




unlike IL-3, it is species-specific,




stimulating the growth and




differentiation of only human




and murine erythroid




progenitors.


Thrombospondin-1*
Thrombospondin-1
multimodular secreted protein




that associates with the




extracellular matrix and




possesses a variety of biologic




functions, including a potent




angiogenic activity.


VEGF*
Vascular endothelial growth
Growth factor active in



factor
angiogenesis, vasculogenesis




and endothelial cell growth.







These proteins show expression in some instances - though may also be present in media.









EGF R/ErbB1
Epidermal growth factor receptor
Receptor for EGF, but also for




other members of the EGF




family, as TGF-alpha,




amphiregulin, betacellulin,




heparin-binding EGF-like growth




factor


MDC*
A disintegrin and
Probable ligand for integrin in



metalloproteinase domain 11
the brain. This is a non catalytic



Metalloproteinase-like,
metalloprotease-like protein.



disintegrin-like, and cysteine-rich



protein



MDC


Endostatin*
Endostatin
Angiogenesis inhibitor; inhibits




endothelial cell migration but




may not effect proliferation. May




work in balance with VEGF to




maintain level of angiogenesis.


Follistatin
Follistatin
Regulates stem cell renewal




versus differentiation by




inhibiting pro-differentiation




proteins


Csk†
cytoplasmic tyrosine kinase
Activity is required for




interleukin 6 (IL-6) induced




differentiation. May play a role




in the growth and differentiation




of hematopoietic cells. May be




involved in signal transduction




in endocardial and arterial




endothelial cells.





*= angiogenesis


†= inflammation






Example 6
Conditioned Medium Analysis Using Human Angiogenesis ELISA Strips (Signosis)
Method

Human angiogenesis ELISA strips (Signosis) were utilized according to manufacturer's instruction. Fresh RMM medium and 24 hour conditioned CTX0E03 RMM medium were analyzed for 8 angiogenesis cytokines; tumor necrosis factor α (TNFα), insulin-like growth factor 1 (IGF-1), VEGFA, interleukin-6 (IL-6), bFGF, transforming growth factor β 1 (TGFβ1), EGF, and leptin. Individual wells of the strip, coated with each of the primary antibodies directed against the specific angiogenesis cytokines were loaded with test samples. Absorbance was measured by a spectrophotometer at 450 nm. The concentrations of the angiogenesis cytokines were directly proportional to the color intensity of the test sample.


The results are shown in FIG. 3.


Example 7
Integra CELLINE—Disposable Bioreactor for the Production of Micro Particles from CTX0E03 Cells

Efficient micro particle production and harvest from a cell line relies upon maintaining optimal culture conditions for the greatest density of cells. Any restriction in the oxygen or nutrients supplied to the cells or an accumulation of waste metabolic products will limit the life span of the culture, and hence the micro particle production.


The two-compartment CELLine AD 1000 is designed to accommodate adherent cells attached to a matrix inlay within a small cell compartment, separated from a larger media reservoir by means of a 10 kDa semi-permeable membrane. This membrane allows a continuous diffusion of nutrients and removal of waste products, while concentrating any micro particles produced by the cell within the smaller cell compartment. Due to the large volume capacity (1 litre) of the media compartment, the system has the potential to maintain high density cultures for longer periods of time without the need for a media change. The production of exosomes from mesothelioma tumour cell cultures is described in Mitchell et al, 2008.


Method

In order to obtain optimal performance of the CELLine AD1000, place 25 ml of complete growth medium (RMM with growth factors and 4OHT) into the medium compartment of the flask to pre-wet the semi-permeable membrane. Allow the flask to sit for 5 minutes at room temperature before coating the matrix inlay with mouse Laminin by adding 15 ml of laminin solution (20 μg/ml in DMEM/F12) to the cell compartment for a minimum of 1 hour at 37° C. Remove the laminin solution and add 15 ml of warm DMEM/F12 to the cell compartment to remove any excess laminin. Avoiding the matrix inlay drying, slowly introduce approximately 15×106 CTX0E03 cells in a total of 15 ml of complete growth medium. Take care to remove any air bubbles from the cell compartment. Carefully add a further 460 ml of complete growth medium to the cell compartment before incubating the flask overnight in 5% CO2 at 37° C. The next day remove the medium from the cell compartment and replace with 15 ml of pre warmed growth medium.


Every 7 days harvest the microparticles/medium from the cell compartment. Centrifuge the medium at 1500 rpm for 5 minutes to remove any cell debris and store at −80° C. Carefully add another 15 ml of pre-warmed complete growth medium in to the cell compartment and 485 ml of complete growth medium to the medium compartment and incubate for another 7 days. Microparticles were isolated by 100K MWCO filtration. Repeat as necessary.



FIG. 4A shows the amount of protein extracted from 15 ml of media containing microparticles purified using the Integra system compared to normal culture conditions (3 days T175). Milligrams of protein measured by BCA assay. FIG. 5 shows the corresponding quantity of isolated total RNA measured at 260/280 nm.


Marker characterisations indicated that both purified populations (microvesicles and exosomes) express CD63 and CD81 (determined by FACS—FIG. 4B). Only the exosomes express the endosomal marker Alix (determined by Western blot, data not shown).


Example 8
Efficacy Assays

(A) Comparison of the Function of CTX0E03 Conditioned Media with the Function of Purified Exosomes from CTX0E03 Cells in a Wound Healing Assay.


Method—Wound Closure/Scratch Assay





    • Seed 0.25×106 NHDF (normal human dermal fibroblasts) per well of a 12 well plate and allow to become confluent (24 hours)

    • Remove growth factors for 24 hrs

    • Remove cells (scratch) and incubate with exosomes/conditioned media

    • Image effected area over 48 hrs

    • Estimate area using Image J





Results









TABLE 2







Wound closure/scratch assay representing the migration activity of


normal human dermal fibroblasts (NHDF) cultured in CTX0E03


conditioned media or upon the addition of purified exosomes.









Wound closure (%)











0 h
24 h
48 h
















CTX0E03 conditioned media
0%
 100%




2 ug/ml exosomes
0%
95.4%
 100%



Control
0%
48.1%
49.7%










Wound closure was calculated as the area covered by cells in relation to the initial wound area, as determined at 0 h. Wound closure is expressed as the percentage of the initial wound area at time 0 h. These data are also shown, photographically, in FIG. 6A.



FIG. 6B shows that 10 μg CTX0E03 exosomes significantly increase wound closure (as determined in the HDNF scratch/migration assay) after 72 hours, compared to basal conditions (without exosomes).


Further experiments confirmed that exosomes purified (by ultracentrifugation; quantified by BCA protein assay; characterised as >99% positive for CD63 and CD81 and having a greater expression level of Alix compared to the corresponding microparticle fraction) from all time points (weeks 2-6) during continuous culture (using Integra CELLine bioreactors in the presence of growth factors and 4OHT) significantly enhanced fibroblast migration and wound healing, with a peak response between 5-10 μ/ml compared to basal conditions. FIG. 6C shows the % healed areas for basal conditions, 2 μg/ml exosomes, 6 μg/ml exosomes, 20 μg/ml exosomes and an LSGS (low serum growth supplement) positive control. The top panel of FIG. 6C shows exosomes isolated from CTX0E03 cells cultured for 2 weeks in the Integra Celline system and the bottom panel of FIG. 6C shows exosomes isolated from CTX0E03 cells cultured for 6 weeks in the Integra Celline system. These data show that all doses of all tested NSC exosomes provide increased healing compared to basal conditions, with % healing approaching the positive control (LSGS) after 72 hours.


The data in FIG. 6C also show that the exosomes isolated from NSCs cultured for 6 weeks cause faster healing (than 2 week exosomes), with the % healed approaching 100% after only 48 hours, for all doses.



FIG. 6D shows the results of an in vivo injection wound assay in a mouse, confirming that CTX0E03 cells stimulated wound healing to a statistically-significant degree in vivo. This is a simple in vivo bioassay which can be used to confirm the efficacy of microparticles in vivo.


Conclusion Exosomes released from the human neural stem cell line CTX0E03 enhance fibroblast migration in an in vitro model of wound healing, suggesting that exosomes may contribute to the mechanisms by which hNSCs promote repair. Exosomes isolated from cells cultured for 6 weeks show improved wound healing efficacy in vitro, compared to exosomes isolated from cells cultured for 2 weeks.


(B) Stimulation of Angiogenesis

A 24 hour assay to detect angiogenesis on primary HUVECs was carried out using an Ibidi p-slide and automated Wimtube detection and analysis (of tube length and bifurcation points). Microvesicles harvested from Integra flasks at 1, 2, 3, 4 and 6 weeks were added to HUVECs and angiogenesis compared to basal HUVECs (without addition). LSGS (low serum growth supplement) was used as a positive control. The results, depicted in FIG. 12, show that neural stem cell microvesicles increase angiogenesis. Further, these data show that a larger increase in angiogenesis is provided by microvesicles harvested after at least 3 weeks of culture (i.e. after 3 weeks, 4 weeks and 6 weeks culture in an Integra celline bioreactor), than is provided by microvesicles cultured for 1 or 2 weeks. Microvesicles cultured for at least 3 weeks stimulated angiogenesis to a statistically significant level, and a level that approaches that of the positive control. The largest increase in angiogenesis is shown to be provided by microvesicles harvested after 4 weeks; these microvesicles stimulated angiogenesis by the same amount as the positive control.


These data indicate that hNSC microvesicles stimulate angiogenesis.


(C) Stimulation of Neurite Outgrowth

Neurite outgrowth was determined using PC-12 cells though a 1 μm insert. After 72 hours, the PC-12 cell bodies were removed and the neurites stained on the underside of the insert. The stain was then extracted and quantified on a spectrophotometer. Microvesicles harvested from Integra flasks at 2 weeks were added to the cells at 0.03 μg, 0.3 μg and 3 μg, each with 100 ng/ml NGF (nerve growth factor). Neurite outgrowth was compared to basal cells (without addition). 100 ng/ml NGF was used as a control. As shown in FIG. 13, the addition of 3 μg hNSC microvesicles caused a noticeable increase in neurite outgrowth, compared to the addition of NGF alone.


These data indicate that hNSC microvesicles stimulate neurite outgrowth.


Example 9
Production of Exosomes Using the Integra CELLine System

CTX0E03 cells were cultured using the Integra CELLine system and exosomes were purified as described in Example 7. The concentration of exosomes purified from the medium using the CELLine system at the 3 week time point, and as a control a standard T175 system as routinely used in the art, was quantified (using a BCA assay to estimate protein content). FIG. 7 shows that the production of exosomes using the Integra CELLine system is increased several fold, compared to using conventional culture (T175 flasks).


Using the Integra CELLine system, CTX0E03 cells were cultured over a 3-week period and medium was harvested at week 1, 2 and 3 for purification and quantification of exosomes, as described in Example 7. FIG. 8A shows that the production of microparticles increases exponentially over the 3-week culture period, enabling efficient and large-scale production of microparticles. The concentration of exosomes harvested from a single Integra CELLine flask was then monitored over 1-6 weeks of continuous CTX0E03 culture, with the results shown below and depicted in FIG. 8B:















Total quantity of exosomes



Integra time point
(ug)
Exosomes ug/ml

















Week 1
12
0.80


Week 2
112
7.47


Week 3
88
5.87


Week 4
148
9.87


Week 5
240
16.00


Week 6
440
29.33









These results show that exosome production is surprisingly enhanced when stem cells are cultured in a multi-compartment bioreactor for weeks, typically at least three weeks.


Example 10
Characterisation of Phenotype of Cells Obtained from the Integra CELLine and the Standard (T175) Culture System

CTX0E03 cells were cultured using the Integra CELLine bioreactor and standard culture, as described in Example 7. Expression of DCX and GFAP protein markers was confirmed using marker-specific antibodies and fluorescence microscopy.


Expression of DCX, GALC, GFAP, TUBB3, GDNF and IDO markers was detected by qRT-PCR in samples obtained from the cells. Marker expression was compared between microparticles obtained from standard (T175) culture and exosomes obtained from the 3 week cultured Integra CELLine system, assessed against a baseline of the expression level in CTX0E03 cells in standard (T175) culture.


The inventors observed a striking difference in marker expression of cells obtained from the Integra CELLine system as compared to control cells obtained from standard. Markers of partially-differentiated cells were increased several fold in cells cultured in the Integra CELLine system, compared to control cells obtained from standard cultures (FIG. 9). Particularly striking changes are increased expression of the markers DCX1 (doublecortin—a marker for entry into the neural lineage), GFAP (glial fibrillary acidic protein—a marker for entry into the astrocytic lineage), GDNF (glial cell-derived neurotrophic factor) and IDO (indoleamine 2,3-dioxygenase). This indicates that in neural stem cells cultured in a two-compartment bioreactor partially differentiate into cells of neural (DCX+) or astrocytic (GFAP+) lineage. The expression of DCX and GFAP in the Integra-cultured cells was confirmed by fluorescence microscopy, demonstrating that CTX0E03 cells cultured using the Integra CELLine bioreactor have a more differentiated neuronal phenotype than standard CTX0E03 cells.


Example 11
Characterisation of miRNA Expression Profiles of Exosomes Obtained from Integra CELLine Cultures and Microparticles Obtained from Standard (T175) Cultures

CTX0E03 cells were cultured for three weeks using the Integra CELLine culture and in the standard culture in single-compartment 1-175 flasks. Exosomes were purified from the Integra culture and microparticles were purified from the standard T-175 culture as described in Example 7. The relative expression levels of various miRNAs expressed in the exosomes and microparticles obtained from either the standard culture or the Integra CELLine system were determined with an miRNA array using qRT-PCR panel (Qiagen) according to manufacturer's instruction, and converted into fold up and down regulation levels as compared to a standard CTX0E03 cell line control group (see Table 3 and FIG. 10). These data show a differential miRNA expression profile between exosomes obtained from the Integra CELLine culture system for 3 weeks, microparticles, and cells obtained from the standard single-flask culture.









TABLE 3







Fold-regulation of miRNAs in microparticles obtained from standard


culture or exosomes from the Integra CELLine system, relative to


control (CTX0E03 cells).










Standard Culture




(microparticles)
Integra (exosomes)








miRNA
Fold regulation relative to control (CTX0E03 cells)












hsa-miR-146b-5p
−1.0222
10.5805


hsa-let-7c
−1.6954
4.7678


hsa-miR-99a-5p
−3.5349
3.3714


hsa-miR-132-3p
−1.9163
3.088


hsa-miR-378-3p
1.2731
3.0175


hsa-miR-181a-5p
−1.7431
2.9147


hsa-let-7b-5p
−1.4658
2.7574


hsa-miR-100-5p
−3.208
1.977


hsa-let-7e-5p
−2.7101
1.9274


hsa-miR-23b-3p
−2.3322
1.8834


hsa-miR-185-5p
−1.9119
1.8532


hsa-let-7i-5p
−3.5677
1.8404


hsa-let-7a-5p
−1.851
1.7736


hsa-let-7d-5p
−1.5
1.7654


hsa-let-7g-5p
−2.2527
1.7092


hsa-miR-222-3p
−5.8092
1.6779


hsa-let-7f-5p
−2.8712
1.5948


hsa-miR-218-5p
−1.9611
1.5619


hsa-miR-24-3p
−1.6721
1.5511


hsa-miR-9-5p
−2.2475
1.4109


hsa-miR-126-3p
−2.1263
1.203


hsa-miR-134
−1.6567
1.1783


hsa-miR-128
−3.5842
1.0743


hsa-miR-155-5p
−8.8458
1.0425


hsa-miR-22-3p
−3.4782
−1.0023


hsa-miR-26a-5p
−5.3579
−1.0187


hsa-miR-210
−2.3107
−1.0449


hsa-miR-92a-3p
−1.9885
−1.0693


hsa-miR-93-5p
−3.056
−1.1701


hsa-miR-424-5p
−4.9189
−1.2086


hsa-miR-195-5p
−3.8951
−1.2541


hsa-miR-127-5p
−1.1316
−1.2953


hsa-miR-21-5p
−2.8845
−1.3044


hsa-miR-103a-3p
−2.6482
−1.3287


hsa-miR-16-5p
−3.5267
−1.3692


hsa-miR-125a-5p
−5.1159
−1.434


hsa-miR-10a-5p
−14.4701
−1.434


hsa-miR-10b-5p
−15.1194
−1.4373


hsa-miR-345-5p
−2.5521
−1.4406


hsa-miR-130a-3p
−2.6178
−1.5728


hsa-miR-15b-5p
−4.4025
−1.6058


hsa-miR-20b
−2.1312
−1.6096


hsa-miR-20a-5p
−2.3107
−1.8319


hsa-miR-17-5p
−1.9296
−1.8319


hsa-miR-7-5p
−1.5105
−2.042


hsa-miR-106b-5p
−2.4708
−2.1287


hsa-miR-101-3p
1.4794
−2.4453


hsa-miR-302a-3p
−18.0634
−2.4623


hsa-miR-301a-3p
1.4931
−2.5257


hsa-miR-183-5p
−13.9772
−2.5847


hsa-miR-219-5p
1.6994
−2.7321


hsa-miR-18a-5p
−1.4028
−3.2792


hsa-miR-15a-5p
−2.4766
−3.3714


hsa-miR-182-5p
−12.5099
−4.9588


hsa-miR-33a-5p
2.7927
−9.1472


hsa-miR-96-5p
−7.0047
−18.9396


hsa-miR-18b-5p
−1.3519
−49.18









Values were calculated from raw data using the following equations:







Δ





CT






(

sample
/
control

)


=


Average





CT






(
GOI
)


-

Average





CT






(
HKG
)










Fold





expression






(

sample
/
control

)


=

2

-

(

Average





Δ





CT

)










Fold





change

=


Fold





expression






(
sample
)



Fold





expression






(
control
)











If






(

fold





change

)


>

1





then






(

fold





regulation

)



=

(

fold





change

)









If






(

fold





change

)


<

1





then






(

fold





regulation

)



=

-

(

1

fold





change


)






Wherein:


CT=cycle threshold


GOI=gene of interest (investigated miRNA)


HKG=housekeeping genes (reference miRNAs used to normalize the data)


Example 12
Total miRNA Analysis

Cells can shuttle RNA into microparticles determined for release into the extracellular space. This allows the conveyance of genetically encoded messages between cells. We here collectively refer to extracellular RNA as ‘shuttle RNA’. We aimed to analyze comprehensively non coding RNA species released by CTX0E03 neural stem cells (NSCs) using Next Generation Sequencing.


Non coding RNAs are divided in two categories (small and long). Small non coding RNA biotypes include ribosomal RNA (rRNA), small nucleolar (snoRNA), small nuclear RNA (snRNA), microRNA (miRNA), miscellaneous other RNA (misc_RNA, e.g. RMRP, vault RNA, metazoa SRP, and RNY), and long non coding RNA biotypes includes long non-coding RNAs (IncRNAs) and large intergenic non-coding RNAs (lincRNAs).


Here, we characterized shuttle RNAs, including small and long non coding RNAs, released from NSC derived exosomes and microvesicles (MV) and compared with the RNA contents of the producer NSCs.


A) Total RNA Contents in Cells, Exosomes and Microvesicles Identified by Agilent RNA Bioanalyser

The RNA in both exosomes and microvesicles mainly consists of small RNA species as shown in FIG. 14. The majority of the nucleotides (nt) was 200 as shown against the molecular ladder.


B) RNA Composition

Small RNA sequencing libraries were generated to investigate the composition of shuttle and cellular RNA by deep sequencing (Next Generation Sequencing). The results are shown in FIG. 15.


C) Deep Sequencing of CTX0E03 Cell, Microvesicle and Exosome miRNA Expression from Standard (T175) Cultures.


Deep sequencing is based on the preparation of a cDNA library following by sequencing and provides information regarding the total sequence read out of different miRNAs in the microvesicles and exosomes. These deep sequence data complement the qRT-PCR array data shown above and provide a comprehensive analysis of the miRNA profile of the cells and microparticles. Unlike the qRT-PCR array analysis, deep sequencing is not restricted to identification of sequences present in the probe array and so the sequences to be identified do not need to be known in advance. Deep sequencing also provides direct read-out and the ability to sequence very short sequences. However, deep sequencing is not suitable for detection of transcripts with low expression.


Method

The presence of a variety of miRNAs in parental cells and their exosomes (30-100 μm) and microvesicles (100-1000 μm), purified by differential centrifugation, was identified by deep sequencing, following construction of 1 tagged miRNA library for each sample.


Additionally, specific primers for highly shuttled miRNAs (e.g. hsa-miR-1246) were designed and used in real-time reverse transcription PCR (qRT-PCR) to trace exosomes/microvesicles following in vivo implantation.


Deep sequencing was performed by GATC Biotech (Germany) and required the preparation of a tagged miRNA library for each samples followed by sequencing, and miRBase scanning:

    • Construction of tagged miRNA libraries (22 to 30 nt)
      • Sequencing libraries were generated by ligation of specific RNA adapter to both 3′ and 5′ ends for each sample followed by reverse transcription, amplification, and purification of smallRNA libraries (size range of contained smallRNA fraction 22-30 nt).
    • Sequencing on an Illumina HiSeq 2000 (single read)
      • Sequencing was performed using Illumina HiSeq 2000 (single read). Analysis of one pool could include up to 45,000,000 single read, and each read length is up to 50 bases. Sequencing was quality controlled by using FastQ Files (sequences and quality scores).
    • Identification of known miRNAs was performed as followed:
      • RNA adapters were trimmed from resulting sequences and raw data cleaned. Raw data were clustered and for each cluster a number of reads was provided. MiRNAs were identified by miRBase scanning (Ssearch).


Results

Many microvesicle and exosome miRNAs were enriched relative to the cells, indicating that cells specially sort miRNAs for extracellular release. Furthermore, miRNA contents were similar in both exosomes and microvesicles, indicating a common apparatus of selective miRNA uptake in excreted microvesicles. Without wishing to be bound by theory, this may indicate that miRNA content in secreted microvesicles and exosomes can be used as a fingerprint to identify hNSC subtypes.


The deep sequencing analysis therefore identified a unique set of miRNAs in both hNSC exosomes and microvesicles not previously reported. MiRNA content in excreted vesicles is similar, but showed a preferential miRNA uptake compared with hNSC. These findings could support biological effects mediated by shuttle miRNA not previously described for hNSC.


The results are detailed in Tables 4 to 9, below. The data are also depicted in FIG. 11, which clearly shows the significantly different miRNA profiles present in the microvesicles and exosomes, compared to the cells. In summary, these data show a massive increase in the amount (read counts) of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532 in microvesicles and exosomes compared to the cells. Large increases are also seen in hsa-miR-4508, hsa-miR-4516, has-miR-3676-5p and hsa-miR-4485. Massive decreases are seen in the amounts (read counts) of certain miRNAs, including hsa-let-7a-5p, has-miR-92b-3p, has-miR-21-5p. hsa-miR-92a-3p, hsa-miR-10a-5p, hsa-100-5p and hsa-99b-5p.


The presence of each of hsa-miR-1246, hsa-miR-4488, hsa-miR-4492, hsa-miR-4508, hsa-miR-4516 and hsa-miR-4532 in the exosomes was validated by qRT-PCR (data not shown).


Plotting the deep sequencing results in the exosomes and microvesicles as relative fold change compared to the cells confirms that hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532 are significantly upregulated in the exosomes and microvesicles compared to the cells. This comparison also shows that miRNA hsa-miR-3195 is the miRNA that is most upregulated, in both exosomes and microvesicles. Although the absolute reads of hsa-miR-3195 are in the range of ˜40 for exosomes and microvesicles, there is no hsa-miR-3195 present in the cells.


As noted in Example 11 above, miRNA contents in exosomes, microparticles, and parental cells were also tested and validated using PCR array analysis. The following miRNAs were found present by qRT-PCR: hsa-let-7 g-5p, hsa-miR-101-3p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-125b-5p, hsa-miR-128, hsa-miR-130a-3p, hsa-miR-134, hsa-miR-137, hsa-miR-146b-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-181a-5p, hsa-miR-182-5p, hsa-miR-185-5p, hsa-miR-18b-5p, hsa-miR-192-5p, hsa-miR-194-5p, hsa-miR-195-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-210, hsa-miR-21-5p, hsa-miR-218-5p, hsa-miR-219-5p, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23b-3p, hsa-miR-24-3p, hsa-miR-26a-5p, hsa-miR-301a-3p, hsa-miR-302a-3p, hsa-miR-302c-3p, hsa-miR-345-5p, hsa-miR-378a-3p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-9-5p, hsa-miR-96-5p, and hsa-miR-99a-5p.









TABLE 4







Cells EH











Cells: 






CTX0E03 07EH

SEQ ID
MIRNA
READ


MIRNA
MIRNA.SEQUENCE
NO:
LENGTH
COUNTS














hsa-let-7a-5p
UGAGGUAGUAGGUUGUAUAGUU
1
22
75110





hsa-miR-10a-5p
UACCCUGUAGAUCCGAAUUUGUG
2
23
52927





hsa-miR-100-5p
AACCCGUAGAUCCGAACUUGUG
3
22
52451





hsa-miR-99b-5p
CACCCGUAGAACCGACCUUGCG
4
22
39457





hsa-miR-486-5p
UCCUGUACUGAGCUGCCCCGAG
5
22
20310





hsa-miR-27b-3p
UUCACAGUGGCUAAGUUCUGC
6
21
16900





hsa-miR-92a-3p
UAUUGCACUUGUCCCGGCCUGU
7
22
14359





hsa-miR-191-5p
CAACGGAAUCCCAAAAGCAGCUG
8
23
12591





hsa-miR-21-5p
UAGCUUAUCAGACUGAUGUUGA
9
22
11943





hsa-miR-98
UGAGGUAGUAAGUUGUAUUGUU
10
22
11760





hsa-let-7f-5p
UGAGGUAGUAGAUUGUAUAGUU
11
22
10349





hsa-miR-26a-5p
UUCAAGUAAUCCAGGAUAGGCU
12
22
9900





hsa-miR-92b-3p
UAUUGCACUCGUCCCGGCCUCC
13
22
9794





hsa-miR-127-3p
UCGGAUCCGUCUGAGCUUGGCU
14
22
7064





hsa-miR-181a-5p
AACAUUCAACGCUGUCGGUGAGU
15
23
6956





hsa-miR-182-5p
UUUGGCAAUGGUAGAACUCACACU
16
24
5531





hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
17
22
5103





hsa-miR-379-5p
UGGUAGACUAUGGAACGUAGG
18
21
4746





hsa-miR-146b-5p
UGAGAACUGAAUUCCAUAGGCU
19
22
4552





hsa-miR-21-3p
CAACACCAGUCGAUGGGCUGU
20
21
4089





hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
21
19
3973





hsa-let-71-5p
UGAGGUAGUAGUUUGUGCUGUU
22
22
3015





hsa-miR-4532
CCCCGGGGAGCCCGGCG
23
17
2847





hsa-miR-183-5p
UAUGGCACUGGUAGAAUUCACU
24
22
2695





hsa-miR-151a-3p
CUAGACUGAAGCUCCUUGAGG
25
21
2681





hsa-miR-501-3p
AAUGCACCCGGGCAAGGAUUCU
26
22
2649





hsa-let-7e-5p
UGAGGUAGGAGGUUGUAUAGUU
27
22
2449





hsa-let-7b-5p
UGAGGUAGUAGGUUGUGUGGUU
28
22
2435





hsa-miR-16-5p
UAGCAGCACGUAAAUAUUGGCG
29
22
2173





hsa-miR-30a-5p
UGUAAACAUCCUCGACUGGAAG
30
22
2001





hsa-miR-30d-5p
UGUAAACAUCCCCGACUGGAAG
31
22
1977





hsa-miR-409-5p
AGGUUACCCGAGCAACUUUGCAU
32
23
1871





hsa-miR-22-3p
AAGCUGCCAGUUGAAGAACUGU
33
22
1826





hsa-miR-4492
GGGGCUGGGCGCGCGCC
34
17
1754





hsa-miR-125a-5p
UCCCUGAGACCCUUUAACCUGUGA
35
24
1451





hsa-miR-222-3p
AGCUACAUCUGGCUACUGGGU
36
21
1422





hsa-miR-151a-5p
UCGAGGAGCUCACAGUCUAGU
37
21
1386





hsa-miR-181b-5p
AACAUUCAUUGCUGUCGGUGGGU
38
23
1382





hsa-miR-221-5p
ACCUGGCAUACAAUGUAGAUUU
39
22
1363





hsa-miR-186-5p
CAAAGAAUUCUCCUUUUGGGCU
40
22
1225





hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
41
23
1080





hsa-miR-125b-5p
UCCCUGAGACCCUAACUUGUGA
42
22
1002





hsa-let-7g-5p
UGAGGUAGUAGUUUGUACAGUU
43
22
959





hsa-miR-500a-3p
AUGCACCUGGGCAAGGAUUCUG
44
22
923





hsa-miR-30e-5p
UGUAAACAUCCUUGACUGGAAG
45
22
911





hsa-miR-27a-3p
UUCACAGUGGCUAAGUUCCGC
46
21
867





hsa-miR-409-3p
GAAUGUUGCUCGGUGAACCCCU
47
22
865





hsa-miR-148b-3p
UCAGUGCAUCACAGAACUUUGU
48
22
856





hsa-miR-125b-1-3p
ACGGGUUAGGCUCUUGGGAGCU
49
22
851





hsa-miR-410
AAUAUAACACAGAUGGCCUGU
50
21
848





hsa-miR-381
UAUACAAGGGCAAGCUCUCUGU
51
22
842





hsa-miR-99a-5p
AACCCGUAGAUCCGAUCUUGUG
52
22
773





hsa-let-7d-5p
AGAGGUAGUAGGUUGCAUAGUU
53
22
765





hsa-miR-148a-3p
UCAGUGCACUACAGAACUUUGU
54
22
702





hsa-miR-23a-3p
AUCACAUUGCCAGGGAUUUCC
55
21
654





hsa-miR-28-3p
CACUAGAUUGUGAGCUCCUGGA
56
22
593





hsa-miR-423-3p
AGCUCGGUCUGAGGCCCCUCAGU
57
23
557





hsa-miR-9-5p
UCUUUGGUUAUCUAGCUGUAUGA
58
23
518





hsa-miR-23b-3p
AUCACAUUGCCAGGGAUUACC
59
21
508





hsa-miR-941
CACCCGGCUGUGUGCACAUGUGC
60
23
492





hsa-miR-4488
AGGGGGCGGGCUCCGGCG
61
18
485





hsa-miR-103a-3p
AGCAGCAUUGUACAGGGCUAUGA
62
23
459





hsa-miR-25-3p
CAUUGCACUUGUCUCGGUCUGA
63
22
436





hsa-miR-889
UUAAUAUCGGACAACCAUUGU
64
21
411





hsa-miR-378a-3p
ACUGGACUUGGAGUCAGAAGG
65
21
410





hsa-miR-30c-5p
UGUAAACAUCCUACACUCUCAGC
66
23
378





hsa-miR-4485
UAACGGCCGCGGUACCCUAA
67
20
358





hsa-miR-125b-2-3p
UCACAAGUCAGGCUCUUGGGAC
68
22
352





hsa-miR-671-3p
UCCGGUUCUCAGGGCUCCACC
69
21
350





hsa-miR-361-5p
UUAUCAGAAUCUCCAGGGGUAC
70
22
337





hsa-miR-30e-3p
CUUUCAGUCGGAUGUUUACAGC
71
22
294





hsa-miR-1271-5p
CUUGGCACCUAGCAAGCACUCA
72
22
288





hsa-miR-589-5p
UGAGAACCACGUCUGCUCUGAG
73
22
282





hsa-miR-374a-5p
UUAUAAUACAACCUGAUAAGUG
74
22
275





hsa-miR-769-5p
UGAGACCUCUGGGUUCUGAGCU
75
22
263





hsa-miR-345-5p
GCUGACUCCUAGUCCAGGGCUC
76
22
249





hsa-miR-30a-3p
CUUUCAGUCGGAUGUUUGCAGC
77
22
236





hsa-miR-15b-5p
UAGCAGCACAUCAUGGUUUACA
78
22
229





hsa-miR-221-3p
AGCUACAUUGUCUGCUGGGUUUC
79
23
225





hsa-miR-31-5p
AGGCAAGAUGCUGGCAUAGCU
80
21
213





hsa-miR-342-3p
UCUCACACAGAAAUCGCACCCGU
81
23
205





hsa-miR-136-3p
CAUCAUCGUCUCAAAUGAGUCU
82
22
203





hsa-miR-493-3p
UGAAGGUCUACUGUGUGCCAGG
83
22
192





hsa-miR-720
UCUCGCUGGGGCCUCCA
84
17
154





hsa-miR-7-5p
UGGAAGACUAGUGAUUUUGUUGU
85
23
154





hsa-miR-130b-3p
CAGUGCAAUGAUGAAAGGGCAU
86
22
150





hsa-miR-192-5p
CUGACCUAUGAAUUGACAGCC
87
21
138





hsa-miR-493-5p
UUGUACAUGGUAGGCUUUCAUU
88
22
115





hsa-miR-204-5p
UUCCCUUUGUCAUCCUAUGCCU
89
22
113





hsa-miR-26b-5p
UUCAAGUAAUUCAGGAUAGGU
90
21
107





hsa-miR-1307-5p
UCGACCGGACCUCGACCGGCU
91
21
105





hsa-let-7d-3p
CUAUACGACCUGCUGCCUUUCU
92
22
103





hsa-miR-340-5p
UUAUAAAGCAAUGAGACUGAUU
93
22
100





hsa-miR-134
UGUGACUGGUUGACCAGAGGGG
94
22
99





hsa-miR-432-5p
UCUUGGAGUAGGUCAUUGGGUGG
95
23
97





hsa-miR-30b-5p
UGUAAACAUCCUACACUCAGCU
96
22
96





hsa-miR-320a
AAAAGCUGGGUUGAGAGGGCGA
97
22
95





hsa-miR-100-3p
CAAGCUUGUAUCUAUAGGUAUG
98
22
94





hsa-miR-744-5p
UGCGGGGCUAGGGCUAACAGCA
99
22
89





hsa-miR-181a-3p
ACCAUCGACCGUUGAUUGUACC
100
22
86





hsa-miR-34a-5p
UGGCAGUGUCUUAGCUGGUUGU
101
22
85





hsa-miR-181a-2-3p
ACCACUGACCGUUGACUGUACC
102
22
81





hsa-miR-190a
UGAUAUGUUUGAUAUAUUAGGU
103
22
79





hsa-miR-132-3p
UAACAGUCUACAGCCAUGGUCG
104
22
78





hsa-miR-181c-5p
AACAUUCAACCUGUCGGUGAGU
105
22
76





hsa-miR-29a-3p
UAGCACCAUCUGAAAUCGGUUA
106
22
75





hsa-miR-301a-3p
CAGUGCAAUAGUAUUGUCAAAGC
107
23
75





hsa-miR-411-5p
UAGUAGACCGUAUAGCGUACG
108
21
75





hsa-miR-128
UCACAGUGAACCGGUCUCUUU
109
21
74





hsa-miR-4516
GGGAGAAGGGUCGGGGC
110
17
74





hsa-miR-425-5p
AAUGACACGAUCACUCCCGUUGA
111
23
72





hsa-miR-130b-5p
ACUCUUUCCCUGUUGCACUAC
112
21
71





hsa-miR-130a-3p
CAGUGCAAUGUUAAAAGGGCAU
113
22
67





hsa-miR-30d-3p
CUUUCAGUCAGAUGUUUGCUGC
114
22
65





hsa-miR-654-5p
UGGUGGGCCGCAGAACAUGUGC
115
22
65





hsa-miR-93-5p
CAAAGUGCUGUUCGUGCAGGUAG
116
23
65





hsa-miR-487b
AAUCGUACAGGGUCAUCCACUU
117
22
63





hsa-miR-484
UCAGGCUCAGUCCCCUCCCGAU
118
22
62





hsa-miR-24-3p
UGGCUCAGUUCAGCAGGAACAG
119
22
61





hsa-miR-4677-3p
UCUGUGAGACCAAAGAACUACU
120
22
61





hsa-miR-149-5p
UCUGGCUCCGUGUCUUCACUCCC
121
23
56





hsa-miR-197-3p
UUCACCACCUUCUCCACCCAGC
122
22
56





hsa-miR-96-5p
UUUGGCACUAGCACAUUUUUGCU
123
23
56





hsa-miR-1307-3p
ACUCGGCGUGGCGUCGGUCGUG
124
22
55





hsa-miR-34c-5p
AGGCAGUGUAGUUAGCUGAUUGC
125
23
53





hsa-miR-370
GCCUGCUGGGGUGGAACCUGGU
126
22
52





hsa-miR-148b-5p
AAGUUCUGUUAUACACUCAGGC
127
22
51





hsa-miR-335-5p
UCAAGAGCAAUAACGAAAAAUGU
128
23
51





hsa-miR-4461
GAUUGAGACUAGUAGGGCUAGGC
129
23
50





hsa-miR-27a-5p
AGGGCUUAGCUGCUUGUGAGCA
130
22
49





hsa-miR-363-3p
AAUUGCACGGUAUCCAUCUGUA
131
22
47





hsa-miR-431-5p
UGUCUUGCAGGCCGUCAUGCA
132
21
47





hsa-miR-877-5p
GUAGAGGAGAUGGCGCAGGG
133
20
46





hsa-miR-550a-5p
AGUGCCUGAGGGAGUAAGAGCCC
134
23
45





hsa-miR-4508
GCGGGGCUGGGCGCGCG
135
17
44





hsa-miR-541-3p
UGGUGGGCACAGAAUCUGGACU
136
22
42





hsa-miR-135b-5p
UAUGGCUUUUCAUUCCUAUGUGA
137
23
40





hsa-miR-140-3p
UACCACAGGGUAGAACCACGG
138
21
39





hsa-miR-362-5p
AAUCCUUGGAACCUAGGUGUGAGU
139
24
37





hsa-miR-455-3p
GCAGUCCAUGGGCAUAUACAC
140
21
37





hsa-miR-758
UUUGUGACCUGGUCCACUAACC
141
22
37





hsa-miR-101-3p
UACAGUACUGUGAUAACUGAA
142
21
36





hsa-miR-374b-5p
AUAUAAUACAACCUGCUAAGUG
143
22
36





hsa-miR-148a-5p
AAAGUUCUGAGACACUCCGACU
144
22
35





hsa-miR-17-5p
CAAAGUGCUUACAGUGCAGGUAG
145
23
35





hsa-miR-20a-5p
UAAAGUGCUUAUAGUGCAGGUAG
146
23
35





hsa-miR-874
CUGCCCUGGCCCGAGGGACCGA
147
22
35





hsa-miR-193b-3p
AACUGGCCCUCAAAGUCCCGCU
148
22
34





hsa-miR-548ah-3p
CAAAAACUGCAGUUACUUUUGC
149
22
34





hsa-miR-539-3p
AUCAUACAAGGACAAUUUCUUU
150
22
33





hsa-miR-421
AUCAACAGACAUUAAUUGGGCGC
151
23
31





hsa-miR-28-5p
AAGGAGCUCACAGUCUAUUGAG
152
22
30





hsa-miR-485-3p
GUCAUACACGGCUCUCCUCUCU
153
22
29





hsa-miR-2467-5p
UGAGGCUCUGUUAGCCUUGGCUC
154
23
26





hsa-miR-4449
CGUCCCGGGGCUGCGCGAGGCA
155
22
26





hsa-miR-24-2-5p
UGCCUACUGAGCUGAAACACAG
156
22
25





hsa-miR-181d
AACAUUCAUUGUUGUCGGUGGGU
157
23
24





hsa-miR-323a-3p
CACAUUACACGGUCGACCUCU
158
21
24





hsa-miR-106b-3p
CCGCACUGUGGGUACUUGCUGC
159
22
23





hsa-miR-125a-3p
ACAGGUGAGGUUCUUGGGAGCC
160
22
23





hsa-miR-330-5p
UCUCUGGGCCUGUGUCUUAGGC
161
22
23





hsa-miR-1275
GUGGGGGAGAGGCUGUC
162
17
22





hsa-miR-19b-3p
UGUGCAAAUCCAUGCAAAACUGA
163
23
22





hsa-miR-301b
CAGUGCAAUGAUAUUGUCAAAGC
164
23
21





hsa-miR-485-5p
AGAGGCUGGCCGUGAUGAAUUC
165
22
21





hsa-miR-29b-3p
UAGCACCAUUUGAAAUCAGUGUU
166
23
20





hsa-miR-3158-3p
AAGGGCUUCCUCUCUGCAGGAC
167
22
20





hsa-miR-431-3p
CAGGUCGUCUUGCAGGGCUUCU
168
22
20





hsa-miR-454-3p
UAGUGCAAUAUUGCUUAUAGGGU
169
23
20





hsa-miR-106b-5p
UAAAGUGCUGACAGUGCAGAU
170
21
19





hsa-miR-1973
ACCGUGCAAAGGUAGCAUA
171
19
19





hsa-miR-31-3p
UGCUAUGCCAACAUAUUGCCAU
172
22
19





hsa-miR-374a-3p
CUUAUCAGAUUGUAUUGUAAUU
173
22
19





hsa-miR-433
AUCAUGAUGGGCUCCUCGGUGU
174
22
19





hsa-miR-4417
GGUGGGCUUCCCGGAGGG
175
18
19





hsa-miR-143-3p
UGAGAUGAAGCACUGUAGCUC
176
21
18





hsa-miR-19a-3p
UGUGCAAAUCUAUGCAAAACUGA
177
23
18





hsa-miR-532-5p
CAUGCCUUGAGUGUAGGACCGU
178
22
18





hsa-miR-561-5p
AUCAAGGAUCUUAAACUUUGCC
179
22
18





hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
180
22
18





hsa-miR-1301
UUGCAGCUGCCUGGGAGUGACUUC
181
24
17





hsa-miR-299-3p
UAUGUGGGAUGGUAAACCGCUU
182
22
17





hsa-miR-9-3p
AUAAAGCUAGAUAACCGAAAGU
183
22
17





hsa-miR-17-3p
ACUGCAGUGAAGGCACUUGUAG
184
22
15





hsa-miR-376c
AACAUAGAGGAAAUUCCACGU
185
21
15





hsa-miR-424-5p
CAGCAGCAAUUCAUGUUUUGAA
186
22
15





hsa-miR-660-5p
UACCCAUUGCAUAUCGGAGUUG
187
22
15





hsa-miR-153
UUGCAUAGUCACAAAAGUGAUC
188
22
14





hsa-miR-3605-5p
UGAGGAUGGAUAGCAAGGAAGCC
189
23
14





hsa-miR-3687
CCCGGACAGGCGUUCGUGCGACGU
190
24
14





hsa-miR-4284
GGGCUCACAUCACCCCAU
191
18
14





hsa-miR-455-5p
UAUGUGCCUUUGGACUACAUCG
192
22
14





hsa-miR-543
AAACAUUCGCGGUGCACUUCUU
193
22
14





hsa-miR-1276
UAAAGAGCCCUGUGGAGACA
194
20
13





hsa-miR-330-3p
GCAAAGCACACGGCCUGCAGAGA
195
23
13





hsa-miR-369-3p
AAUAAUACAUGGUUGAUCUUU
196
21
13





hsa-miR-4786-5p
UGAGACCAGGACUGGAUGCACC
197
22
13





hsa-miR-548k
AAAAGUACUUGCGGAUUUUGCU
198
22
13





hsa-miR-1226-3p
UCACCAGCCCUGUGUUCCCUAG
199
22
12





hsa-miR-188-3p
CUCCCACAUGCAGGGUUUGCA
200
21
12





hsa-miR-27b-5p
AGAGCUUAGCUGAUUGGUGAAC
201
22
12





hsa-miR-377-5p
AGAGGUUGCCCUUGGUGAAUUC
202
22
12





hsa-miR-487a
AAUCAUACAGGGACAUCCAGUU
203
22
12





hsa-miR-92a-1-5p
AGGUUGGGAUCGGUUGCAAUGCU
204
23
12





hsa-miR-135b-3p
AUGUAGGGCUAAAAGCCAUGGG
205
22
11





hsa-miR-218-5p
UUGUGCUUGAUCUAACCAUGU
206
21
11





hsa-miR-3943
UAGCCCCCAGGCUUCACUUGGCG
207
23
11





hsa-miR-92b-5p
AGGGACGGGACGCGGUGCAGUG
208
22
11





hsa-miR-1185-1-3p
AUAUACAGGGGGAGACUCUUAU
209
22
10





hsa-miR-1273g-3p
ACCACUGCACUCCAGCCUGAG
210
21
10





hsa-miR-2355-5p
AUCCCCAGAUACAAUGGACAA
211
21
10





hsa-miR-23a-5p
GGGGUUCCUGGGGAUGGGAUUU
212
22
10





hsa-miR-30c-1-3p
CUGGGAGAGGGUUGUUUACUCC
213
22
10





hsa-miR-329
AACACACCUGGUUAACCUCUUU
214
22
10





hsa-miR-337-3p
CUCCUAUAUGAUGCCUUUCUUC
215
22
10





hsa-miR-3609
CAAAGUGAUGAGUAAUACUGGCUG
216
24
10





hsa-miR-378a-5p
CUCCUGACUCCAGGUCCUGUGU
217
22
10





hsa-miR-3929
GAGGCUGAUGUGAGUAGACCACU
218
23
10





hsa-miR-4745-5p
UGAGUGGGGCUCCCGGGACGGCG
219
23
10





hsa-miR-5096
GUUUCACCAUGUUGGUCAGGC
220
21
10





hsa-miR-656
AAUAUUAUACAGUCAACCUCU
221
21
10





hsa-let-7a-3p
CUAUACAAUCUACUGUCUUUC
222
21
9





hsa-miR-15a-5p
UAGCAGCACAUAAUGGUUUGUG
223
22
9





hsa-miR-185-5p
UGGAGAGAAAGGCAGUUCCUGA
224
22
9





hsa-miR-25-5p
AGGCGGAGACUUGGGCAAUUG
225
21
9





hsa-miR-3065-5p
UCAACAAAAUCACUGAUGCUGGA
226
23
9





hsa-miR-3176
ACUGGCCUGGGACUACCGG
227
19
9





hsa-miR-339-3p
UGAGCGCCUCGACGACAGAGCCG
228
23
9





hsa-miR-374b-3p
CUUAGCAGGUUGUAUUAUCAUU
229
22
9





hsa-miR-4435
AUGGCCAGAGCUCACACAGAGG
230
22
9





hsa-miR-4448
GGCUCCUUGGUCUAGGGGUA
231
20
9





hsa-miR-4497
CUCCGGGACGGCUGGGC
232
17
9





hsa-miR-4521
GCUAAGGAAGUCCUGUGCUCAG
233
22
9





hsa-miR-539-5p
GGAGAAAUUAUCCUUGGUGUGU
234
22
9





hsa-miR-548ah-5p
AAAAGUGAUUGCAGUGUUUG
235
20
9





hsa-miR-1910
CCAGUCCUGUGCCUGCCGCCU
236
21
8





hsa-miR-376a-3p
AUCAUAGAGGAAAAUCCACGU
237
21
8





hsa-miR-382-5p
GAAGUUGUUCGUGGUGGAUUCG
238
22
8





hsa-miR-3940-3p
CAGCCCGGAUCCCAGCCCACUU
239
22
8





hsa-miR-494
UGAAACAUACACGGGAAACCUC
240
22
8





hsa-miR-495
AAACAAACAUGGUGCACUUCUU
241
22
8





hsa-miR-545-3p
UCAGCAAACAUUUAUUGUGUGC
242
22
8





hsa-miR-99b-3p
CAAGCUCGUGUCUGUGGGUCCG
243
22
8





hsa-miR-1197
UAGGACACAUGGUCUACUUCU
244
21
7





hsa-miR-181b-3p
CUCACUGAACAAUGAAUGCAA
245
21
7





hsa-miR-212-5p
ACCUUGGCUCUAGACUGCUUACU
246
23
7





hsa-miR-3200-3p
CACCUUGCGCUACUCAGGUCUG
247
22
7





hsa-miR-340-3p
UCCGUCUCAGUUACUUUAUAGC
248
22
7





hsa-miR-3607-5p
GCAUGUGAUGAAGCAAAUCAGU
249
22
7





hsa-miR-361-3p
UCCCCCAGGUGUGAUUCUGAUUU
250
23
7





hsa-miR-3656
GGCGGGUGCGGGGGUGG
251
17
7





hsa-miR-532-3p
CCUCCCACACCCAAGGCUUGCA
252
22
7





hsa-miR-574-3p
CACGCUCAUGCACACACCCACA
253
22
7





hsa-miR-107
AGCAGCAUUGUACAGGGCUAUCA
254
23
6





hsa-miR-127-5p
CUGAAGCUCAGAGGGCUCUGAU
255
22
6





hsa-miR-18a-5p
UAAGGUGCAUCUAGUGCAGAUAG
256
23
6





hsa-miR-26a-2-3p
CCUAUUCUUGAUUACUUGUUUC
257
22
6





hsa-miR-296-5p
AGGGCCCCCCCUCAAUCCUGU
258
21
6





hsa-miR-3648
AGCCGCGGGGAUCGCCGAGGG
259
21
6





hsa-miR-382-3p
AAUCAUUCACGGACAACACUU
260
21
6





hsa-miR-3939
UACGCGCAGACCACAGGAUGUC
261
22
6





hsa-miR-432-3p
CUGGAUGGCUCCUCCAUGUCU
262
21
6





hsa-miR-4423-5p
AGUUGCCUUUUUGUUCCCAUGC
263
22
6





hsa-miR-4466
GGGUGCGGGCCGGCGGGG
264
18
6





hsa-miR-454-5p
ACCCUAUCAAUAUUGUCUCUGC
265
22
6





hsa-miR-4746-5p
CCGGUCCCAGGAGAACCUGCAGA
266
23
6





hsa-miR-496
UGAGUAUUACAUGGCCAAUCUC
267
22
6





hsa-miR-548o-3p
CCAAAACUGCAGUUACUUUUGC
268
22
6





hsa-miR-1248
ACCUUCUUGUAUAAGCACUGUGCUAAA
269
27
5





hsa-miR-1254
AGCCUGGAAGCUGGAGCCUGCAGU
270
24
5





hsa-miR-1296
UUAGGGCCCUGGCUCCAUCUCC
271
22
5





hsa-miR-136-5p
ACUCCAUUUGUUUUGAUGAUGGA
272
23
5





hsa-miR-199a-5p
CCCAGUGUUCAGACUACCUGUUC
273
23
5





hsa-miR-296-3p
GAGGGUUGGGUGGAGGCUCUCC
274
22
5





hsa-miR-3177-3p
UGCACGGCACUGGGGACACGU
275
21
5





hsa-miR-324-3p
ACUGCCCCAGGUGCUGCUGG
276
20
5





hsa-miR-337-5p
GAACGGCUUCAUACAGGAGUU
277
21
5





hsa-miR-342-5p
AGGGGUGCUAUCUGUGAUUGA
278
21
5





hsa-miR-365b-3p
UAAUGCCCCUAAAAAUCCUUAU
279
22
5





hsa-miR-3676-5p
AGGAGAUCCUGGGUU
280
15
5





hsa-miR-502-3p
AAUGCACCUGGGCAAGGAUUCA
281
22
5





hsa-miR-505-3p
CGUCAACACUUGCUGGUUUCCU
282
22
5





hsa-miR-550a-3p
UGUCUUACUCCCUCAGGCACAU
283
22
5





hsa-miR-5587-3p
GCCCCGGGCAGUGUGAUCAUC
284
21
5





hsa-miR-641
AAAGACAUAGGAUAGAGUCACCUC
285
24
5





hsa-miR-655
AUAAUACAUGGUUAACCUCUUU
286
22
5





hsa-miR-664-3p
UAUUCAUUUAUCCCCAGCCUACA
287
23
5





hsa-miR-671-5p
AGGAAGCCCUGGAGGGGCUGGAG
288
23
5





hsa-miR-760
CGGCUCUGGGUCUGUGGGGA
289
20
5





hsa-let-7e-3p
CUAUACGGCCUCCUAGCUUUCC
290
22
4





hsa-miR-1268a
CGGGCGUGGUGGUGGGGG
291
18
4





hsa-miR-1273f
GGAGAUGGAGGUUGCAGUG
292
19
4





hsa-miR-1286
UGCAGGACCAAGAUGAGCCCU
293
21
4





hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
294
24
4





hsa-miR-141-3p
UAACACUGUCUGGUAAAGAUGG
295
22
4





hsa-miR-1468
CUCCGUUUGCCUGUUUCGCUG
296
21
4





hsa-miR-328
CUGGCCCUCUCUGCCCUUCCGU
297
22
4





hsa-miR-424-3p
CAAAACGUGAGGCGCUGCUAU
298
21
4





hsa-miR-4454
GGAUCCGAGUCACGGCACCA
299
20
4





hsa-miR-4463
GAGACUGGGGUGGGGCC
300
17
4





hsa-miR-4671-3p
UUAGUGCAUAGUCUUUGGUCU
301
21
4





hsa-miR-4775
UUAAUUUUUUGUUUCGGUCACU
302
22
4





hsa-miR-500a-5p
UAAUCCUUGCUACCUGGGUGAGA
303
23
4





hsa-miR-548b-5p
AAAAGUAAUUGUGGUUUUGGCC
304
22
4





hsa-miR-573
CUGAAGUGAUGUGUAACUGAUCAG
305
24
4





hsa-miR-576-5p
AUUCUAAUUUCUCCACGUCUUU
306
22
4





hsa-miR-625-3p
GACUAUAGAACUUUCCCCCUCA
307
22
4





hsa-miR-652-3p
AAUGGCGCCACUAGGGUUGUG
308
21
4





hsa-miR-665
ACCAGGAGGCUGAGGCCCCU
309
20
4





hsa-miR-766-3p
ACUCCAGCCCCACAGCCUCAGC
310
22
4





hsa-miR-935
CCAGUUACCGCUUCCGCUACCGC
311
23
4





hsa-miR-937
AUCCGCGCUCUGACUCUCUGCC
312
22
4





hsa-miR-1180
UUUCCGGCUCGCGUGGGUGUGU
313
22
3





hsa-miR-1185-2-3p
AUAUACAGGGGGAGACUCUCAU
314
22
3





hsa-miR-132-5p
ACCGUGGCUUUCGAUUGUUACU
315
22
3





hsa-miR-16-2-3p
CCAAUAUUACUGUGCUGCUUUA
316
22
3





hsa-miR-20b-5p
CAAAGUGCUCAUAGUGCAGGUAG
317
23
3





hsa-miR-2116-3p
CCUCCCAUGCCAAGAACUCCC
318
21
3





hsa-miR-299-5p
UGGUUUACCGUCCCACAUACAU
319
22
3





hsa-miR-30b-3p
CUGGGAGGUGGAUGUUUACUUC
320
22
3





hsa-miR-30c-2-3p
CUGGGAGAAGGCUGUUUACUCU
321
22
3





hsa-miR-3187-3p
UUGGCCAUGGGGCUGCGCGG
322
20
3





hsa-miR-3615
UCUCUCGGCUCCUCGCGGCUC
323
21
3





hsa-miR-3620
UCACCCUGCAUCCCGCACCCAG
324
22
3





hsa-miR-3654
GACUGGACAAGCUGAGGAA
325
19
3





hsa-miR-3662
GAAAAUGAUGAGUAGUGACUGAUG
326
24
3





hsa-miR-3681-5p
UAGUGGAUGAUGCACUCUGUGC
327
22
3





hsa-miR-4286
ACCCCACUCCUGGUACC
328
17
3





hsa-miR-4640-3p
CACCCCCUGUUUCCUGGCCCAC
329
22
3





hsa-miR-4717-3p
ACACAUGGGUGGCUGUGGCCU
330
21
3





hsa-miR-542-3p
UGUGACAGAUUGAUAACUGAAA
331
22
3





hsa-miR-5584-5p
CAGGGAAAUGGGAAGAACUAGA
332
22
3





hsa-miR-570-3p
CGAAAACAGCAAUUACCUUUGC
333
22
3





hsa-miR-574-5p
UGAGUGUGUGUGUGUGAGUGUGU
334
23
3





hsa-miR-628-3p
UCUAGUAAGAGUGGCAGUCGA
335
21
3





hsa-miR-654-3p
UAUGUCUGCUGACCAUCACCUU
336
22
3





hsa-miR-769-3p
CUGGGAUCUCCGGGGUCUUGGUU
337
23
3





hsa-miR-943
CUGACUGUUGCCGUCCUCCAG
338
21
3





hsa-let-7b-3p
CUAUACAACCUACUGCCUUCCC
339
22
2





hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
340
26
2





hsa-miR-1255a
AGGAUGAGCAAAGAAAGUAGAUU
341
23
2





hsa-miR-1273e
UUGCUUGAACCCAGGAAGUGGA
342
22
2





hsa-miR-1289
UGGAGUCCAGGAAUCUGCAUUUU
343
23
2





hsa-miR-152
UCAGUGCAUGACAGAACUUGG
344
21
2





hsa-miR-194-5p
UGUAACAGCAACUCCAUGUGGA
345
22
2





hsa-miR-195-5p
UAGCAGCACAGAAAUAUUGGC
346
21
2





hsa-miR-200c-3p
UAAUACUGCCGGGUAAUGAUGGA
347
23
2





hsa-miR-212-3p
UAACAGUCUCCAGUCACGGCC
348
21
2





hsa-miR-222-5p
CUCAGUAGCCAGUGUAGAUCCU
349
22
2





hsa-miR-3065-3p
UCAGCACCAGGAUAUUGUUGGAG
350
23
2





hsa-miR-3115
AUAUGGGUUUACUAGUUGGU
351
20
2





hsa-miR-3126-5p
UGAGGGACAGAUGCCAGAAGCA
352
22
2





hsa-miR-3174
UAGUGAGUUAGAGAUGCAGAGCC
353
23
2





hsa-miR-324-5p
CGCAUCCCCUAGGGCAUUGGUGU
354
23
2





hsa-miR-33a-5p
GUGCAUUGUAGUUGCAUUGCA
355
21
2





hsa-miR-3677-3p
CUCGUGGGCUCUGGCCACGGCC
356
22
2





hsa-miR-369-5p
AGAUCGACCGUGUUAUAUUCGC
357
22
2





hsa-miR-425-3p
AUCGGGAAUGUCGUGUCCGCCC
358
22
2





hsa-miR-4426
GAAGAUGGACGUACUUU
359
17
2





hsa-miR-4467
UGGCGGCGGUAGUUAUGGGCUU
360
22
2





hsa-miR-4482-3p
UUUCUAUUUCUCAGUGGGGCUC
361
22
2





hsa-miR-4515
AGGACUGGACUCCCGGCAGCCC
362
22
2





hsa-miR-4792
CGGUGAGCGCUCGCUGGC
363
18
2





hsa-miR-659-5p
AGGACCUUCCCUGAACCAAGGA
364
22
2





hsa-miR-663a
AGGCGGGGCGCCGCGGGACCGC
365
22
2





hsa-miR-940
AAGGCAGGGCCCCCGCUCCCC
366
21
2





hsa-miR-99a-3p
CAAGCUCGCUUCUAUGGGUCUG
367
22
2





hsa-miR-1185-5p
AGAGGAUACCCUUUGUAUGUU
368
21
1





hsa-miR-1225-3p
UGAGCCCCUGUGCCGCCCCCAG
369
22
1





hsa-miR-1237
UCCUUCUGCUCCGUCCCCCAG
370
21
1





hsa-miR-1252
AGAAGGAAAUUGAAUUCAUUUA
371
22
1





hsa-miR-1257
AGUGAAUGAUGGGUUCUGACC
372
21
1





hsa-miR-1260b
AUCCCACCACUGCCACCAU
373
19
1





hsa-miR-1273d
GAACCCAUGAGGUUGAGGCUGCAGU
374
25
1





hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
375
19
1





hsa-miR-1306-3p
ACGUUGGCUCUGGUGGUG
376
18
1





hsa-miR-1321
CAGGGAGGUGAAUGUGAU
377
18
1





hsa-miR-1343
CUCCUGGGGCCCGCACUCUCGC
378
22
1





hsa-miR-138-5p
AGCUGGUGUUGUGAAUCAGGCCG
379
23
1





hsa-miR-140-5p
CAGUGGUUUUACCCUAUGGUAG
380
22
1





hsa-miR-146b-3p
UGCCCUGUGGACUCAGUUCUGG
381
22
1





hsa-miR-186-3p
GCCCAAAGGUGAAUUUUUUGGG
382
22
1





hsa-miR-1908
CGGCGGGGACGGCGAUUGGUC
383
21
1





hsa-miR-1915-3p
CCCCAGGGCGACGCGGCGGG
384
20
1





hsa-miR-1915-5p
ACCUUGCCUUGCUGCCCGGGCC
385
22
1





hsa-miR-193a-3p
AACUGGCCUACAAAGUCCCAGU
386
22
1





hsa-miR-19b-1-5p
AGUUUUGCAGGUUUGCAUCCAGC
387
23
1





hsa-miR-208b
AUAAGACGAACAAAAGGUUUGU
388
22
1





hsa-miR-2110
UUGGGGAAACGGCCGCUGAGUG
389
22
1





hsa-miR-219-1-3p
AGAGUUGAGUCUGGACGUCCCG
390
22
1





hsa-miR-26b-3p
CCUGUUCUCCAUUACUUGGCUC
391
22
1





hsa-miR-2964a-3p
AGAAUUGCGUUUGGACAAUCAGU
392
23
1





hsa-miR-29a-5p
ACUGAUUUCUUUUGGUGUUCAG
393
22
1





hsa-miR-3126-3p
CAUCUGGCAUCCGUCACACAGA
394
22
1





hsa-miR-3130-3p
GCUGCACCGGAGACUGGGUAA
395
21
1





hsa-miR-3130-5p
UACCCAGUCUCCGGUGCAGCC
396
21
1





hsa-miR-3140-5p
ACCUGAAUUACCAAAAGCUUU
397
21
1





hsa-miR-3155a
CCAGGCUCUGCAGUGGGAACU
398
21
1





hsa-miR-3157-3p
CUGCCCUAGUCUAGCUGAAGCU
399
22
1





hsa-miR-3180-3p
UGGGGCGGAGCUUCCGGAGGCC
400
22
1





hsa-miR-323b-5p
AGGUUGUCCGUGGUGAGUUCGCA
401
23
1





hsa-miR-339-5p
UCCCUGUCCUCCAGGAGCUCACG
402
23
1





hsa-miR-34a-3p
CAAUCAGCAAGUAUACUGCCCU
403
22
1





hsa-miR-34b-3p
CAAUCACUAACUCCACUGCCAU
404
22
1





hsa-miR-34c-3p
AAUCACUAACCACACGGCCAGG
405
22
1





hsa-miR-3658
UUUAAGAAAACACCAUGGAGAU
406
22
1





hsa-miR-365a-5p
AGGGACUUUUGGGGGCAGAUGUG
407
23
1





hsa-miR-3676-3p
CCGUGUUUCCCCCACGCUUU
408
20
1





hsa-miR-3691-5p
AGUGGAUGAUGGAGACUCGGUAC
409
23
1





hsa-miR-376a-5p
GUAGAUUCUCCUUCUAUGAGUA
410
22
1





hsa-miR-378g
ACUGGGCUUGGAGUCAGAAG
411
20
1





hsa-miR-3909
UGUCCUCUAGGGCCUGCAGUCU
412
22
1





hsa-miR-3928
GGAGGAACCUUGGAGCUUCGGC
413
22
1





hsa-miR-3942-3p
UUUCAGAUAACAGUAUUACAU
414
21
1





hsa-miR-3944-5p
UGUGCAGCAGGCCAACCGAGA
415
21
1





hsa-miR-3960
GGCGGCGGCGGAGGCGGGGG
416
20
1





hsa-miR-4326
UGUUCCUCUGUCUCCCAGAC
417
20
1





hsa-miR-4444
CUCGAGUUGGAAGAGGCG
418
18
1





hsa-miR-4450
UGGGGAUUUGGAGAAGUGGUGA
419
22
1





hsa-miR-4642
AUGGCAUCGUCCCCUGGUGGCU
420
22
1





hsa-miR-4668-5p
AGGGAAAAAAAAAAGGAUUUGUC
421
23
1





hsa-miR-4673
UCCAGGCAGGAGCCGGACUGGA
422
22
1





hsa-miR-4688
UAGGGGCAGCAGAGGACCUGGG
423
22
1





hsa-miR-4700-3p
CACAGGACUGACUCCUCACCCCAGUG
424
26
1





hsa-miR-4731-3p
CACACAAGUGGCCCCCAACACU
425
22
1





hsa-miR-4749-3p
CGCCCCUCCUGCCCCCACAG
426
20
1





hsa-miR-4769-5p
GGUGGGAUGGAGAGAAGGUAUGAG
427
24
1





hsa-miR-4800-5p
AGUGGACCGAGGAAGGAAGGA
428
21
1





hsa-miR-491-5p
AGUGGGGAACCCUUCCAUGAGG
429
22
1





hsa-miR-501-5p
AAUCCUUUGUCCCUGGGUGAGA
430
22
1





hsa-miR-5092
AAUCCACGCUGAGCUUGGCAUC
431
22
1





hsa-miR-541-5p
AAAGGAUUCUGCUGUCGGUCCCACU
432
25
1





hsa-miR-542-5p
UCGGGGAUCAUCAUGUCACGAGA
433
23
1





hsa-miR-551b-3p
GCGACCCAUACUUGGUUUCAG
434
21
1





hsa-miR-5690
UCAGCUACUACCUCUAUUAGG
435
21
1





hsa-miR-577
UAGAUAAAAUAUUGGUACCUG
436
21
1





hsa-miR-584-3p
UCAGUUCCAGGCCAACCAGGCU
437
22
1





hsa-miR-589-3p
UCAGAACAAAUGCCGGUUCCCAGA
438
24
1





hsa-miR-616-5p
ACUCAAAACCCUUCAGUGACUU
439
22
1





hsa-miR-628-5p
AUGCUGACAUAUUUACUAGAGG
440
22
1





hsa-miR-629-5p
UGGGUUUACGUUGGGAGAACU
441
21
1





hsa-miR-644b-3p
UUCAUUUGCCUCCCAGCCUACA
442
22
1





hsa-miR-664-5p
ACUGGCUAGGGAAAAUGAUUGGAU
443
24
1





hsa-miR-922
GCAGCAGAGAAUAGGACUACGUC
444
23
1
















TABLE 5







Cells EI











CELLS-






CTX0E03 07E1

SEQ ID
MIRNA
READ


MIRNA
MIRNA.SEQUENCE
NO:
LENGTH
COUNTS














hsa-let-7a-5p
UGAGGUAGUAGGUUGUAUAGUU
1
22
305060





hsa-miR-92b-3p
UAUUGCACUCGUCCCGGCCUCC
13
22
242715





hsa-miR-21-5p
UAGCUUAUCAGACUGAUGUUGA
9
22
154626





hsa-miR-92a-3p
UAUUGCACUUGUCCCGGCCUGU
7
22
137412





hsa-miR-127-3p
UCGGAUCCGUCUGAGCUUGGCU
14
22
110806





hsa-miR-100-5p
AACCCGUAGAUCCGAACUUGUG
3
22
109290





hsa-miR-27b-3p
UUCACAGUGGCUAAGUUCUGC
6
21
91902





hsa-miR-191-5p
CAACGGAAUCCCAAAAGCAGCUG
8
23
89150





hsa-miR-26a-5p
UUCAAGUAAUCCAGGAUAGGCU
12
22
88724





hsa-miR-99b-5p
CACCCGUAGAACCGACCUUGCG
4
22
87399





hsa-let-7f-5p
UGAGGUAGUAGAUUGUAUAGUU
11
22
78395





hsa-miR-181a-5p
AACAUUCAACGCUGUCGGUGAGU
15
23
47686





hsa-miR-486-5p
UCCUGUACUGAGCUGCCCCGAG
5
22
41639





hsa-miR-30a-5p
UGUAAACAUCCUCGACUGGAAG
30
22
35465





hsa-miR-98
UGAGGUAGUAAGUUGUAUUGUU
10
22
30440





hsa-miR-151a-3p
CUAGACUGAAGCUCCUUGAGG
25
21
29047





hsa-miR-21-3p
CAACACCAGUCGAUGGGCUGU
20
21
27733





hsa-miR-30d-5p
UGUAAACAUCCCCGACUGGAAG
31
22
27307





hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
17
22
27224





hsa-miR-10a-5p
UACCCUGUAGAUCCGAAUUUGUG
2
23
26908





hsa-miR-22-3p
AAGCUGCCAGUUGAAGAACUGU
33
22
26456





hsa-miR-182-5p
UUUGGCAAUGGUAGAACUCACACU
16
24
25885





hsa-miR-222-3p
AGCUACAUCUGGCUACUGGGU
36
21
22187





hsa-miR-125a-5p
UCCCUGAGACCCUUUAACCUGUGA
35
24
20960





hsa-miR-16-5p
UAGCAGCACGUAAAUAUUGGCG
29
22
19856





hsa-let-7b-5p
UGAGGUAGUAGGUUGUGUGGUU
28
22
19774





hsa-miR-151a-5p
UCGAGGAGCUCACAGUCUAGU
37
21
19773





hsa-let-7e-5p
UGAGGUAGGAGGUUGUAUAGUU
27
22
19035





hsa-miR-125b-5p
UCCCUGAGACCCUAACUUGUGA
42
22
17965





hsa-let-7i-5p
UGAGGUAGUAGUUUGUGCUGUU
22
22
17802





hsa-let-7g-5p
UGAGGUAGUAGUUUGUACAGUU
43
22
15467





hsa-miR-409-3p
GAAUGUUGCUCGGUGAACCCCU
47
22
14133





hsa-miR-30e-5p
UGUAAACAUCCUUGACUGGAAG
45
22
13889





hsa-miR-181b-5p
AACAUUCAUUGCUGUCGGUGGGU
38
23
12606





hsa-miR-186-5p
CAAAGAAUUCUCCUUUUGGGCU
40
22
12441





hsa-miR-381
UAUACAAGGGCAAGCUCUCUGU
51
22
9851





hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
41
23
8893





hsa-miR-30c-5p
UGUAAACAUCCUACACUCUCAGC
66
23
8737





hsa-miR-410
AAUAUAACACAGAUGGCCUGU
50
21
8509





hsa-miR-146b-5p
UGAGAACUGAAUUCCAUAGGCU
19
22
8434





hsa-miR-654-3p
UAUGUCUGCUGACCAUCACCUU
336
22
8392





hsa-miR-9-5p
UCUUUGGUUAUCUAGCUGUAUGA
58
23
7957





hsa-miR-28-3p
CACUAGAUUGUGAGCUCCUGGA
56
22
7767





hsa-miR-148a-3p
UCAGUGCACUACAGAACUUUGU
54
22
6599





hsa-miR-379-5p
UGGUAGACUAUGGAACGUAGG
18
21
6135





hsa-let-7d-5p
AGAGGUAGUAGGUUGCAUAGUU
53
22
5972





hsa-miR-183-5p
UAUGGCACUGGUAGAAUUCACU
24
22
5477





hsa-miR-25-3p
CAUUGCACUUGUCUCGGUCUGA
63
22
5303





hsa-miR-423-3p
AGCUCGGUCUGAGGCCCCUCAGU
57
23
5225





hsa-miR-889
UUAAUAUCGGACAACCAUUGU
64
21
4597





hsa-miR-221-5p
ACCUGGCAUACAAUGUAGAUUU
39
22
4379





hsa-miR-125b-1-3p
ACGGGUUAGGCUCUUGGGAGCU
49
22
4192





hsa-miR-409-5p
AGGUUACCCGAGCAACUUUGCAU
32
23
3970





hsa-miR-4492
GGGGCUGGGCGCGCGCC
34
17
3864





hsa-miR-148b-3p
UCAGUGCAUCACAGAACUUUGU
48
22
3593





hsa-miR-103a-3p
AGCAGCAUUGUACAGGGCUAUGA
62
23
3518





hsa-miR-1271-5p
CUUGGCACCUAGCAAGCACUCA
72
22
3477





hsa-miR-136-3p
CAUCAUCGUCUCAAAUGAGUCU
82
22
3373





hsa-miR-769-5p
UGAGACCUCUGGGUUCUGAGCU
75
22
2957





hsa-miR-4532
CCCCGGGGAGCCCGGCG
23
17
2915





hsa-miR-378a-3p
ACUGGACUUGGAGUCAGAAGG
65
21
2895





hsa-miR-99a-5p
AACCCGUAGAUCCGAUCUUGUG
52
22
2767





hsa-miR-221-3p
AGCUACAUUGUCUGCUGGGUUUC
79
23
2764





hsa-miR-30e-3p
CUUUCAGUCGGAUGUUUACAGC
71
22
2441





hsa-miR-26b-5p
UUCAAGUAAUUCAGGAUAGGU
90
21
2432





hsa-miR-4488
AGGGGGCGGGCUCCGGCG
61
18
2391





hsa-miR-27a-3p
UUCACAGUGGCUAAGUUCCGC
46
21
2385





hsa-miR-23b-3p
AUCACAUUGCCAGGGAUUACC
59
21
2316





hsa-miR-500a-3p
AUGCACCUGGGCAAGGAUUCUG
44
22
2144





hsa-miR-941
CACCCGGCUGUGUGCACAUGUGC
60
23
2114





hsa-miR-23a-3p
AUCACAUUGCCAGGGAUUUCC
55
21
2086





hsa-miR-30a-3p
CUUUCAGUCGGAUGUUUGCAGC
77
22
2045





hsa-miR-30b-5p
UGUAAACAUCCUACACUCAGCU
96
22
1936





hsa-miR-501-3p
AAUGCACCCGGGCAAGGAUUCU
26
22
1895





hsa-miR-130b-3p
CAGUGCAAUGAUGAAAGGGCAU
86
22
1862





hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
21
19
1783





hsa-miR-140-3p
UACCACAGGGUAGAACCACGG
138
21
1735





hsa-miR-31-5p
AGGCAAGAUGCUGGCAUAGCU
80
21
1705





hsa-miR-493-3p
UGAAGGUCUACUGUGUGCCAGG
83
22
1698





hsa-miR-181c-5p
AACAUUCAACCUGUCGGUGAGU
105
22
1554





hsa-miR-93-5p
CAAAGUGCUGUUCGUGCAGGUAG
116
23
1492





hsa-miR-181a-2-3p
ACCACUGACCGUUGACUGUACC
102
22
1491





hsa-miR-15b-5p
UAGCAGCACAUCAUGGUUUACA
78
22
1465





hsa-miR-7-5p
UGGAAGACUAGUGAUUUUGUUGU
85
23
1460





hsa-miR-192-5p
CUGACCUAUGAAUUGACAGCC
87
21
1453





hsa-miR-425-5p
AAUGACACGAUCACUCCCGUUGA
111
23
1432





hsa-miR-204-5p
UUCCCUUUGUCAUCCUAUGCCU
89
22
1378





hsa-miR-340-5p
UUAUAAAGCAAUGAGACUGAUU
93
22
1360





hsa-miR-190a
UGAUAUGUUUGAUAUAUUAGGU
103
22
1305





hsa-miR-34a-5p
UGGCAGUGUCUUAGCUGGUUGU
101
22
1283





hsa-miR-20a-5p
UAAAGUGCUUAUAGUGCAGGUAG
146
23
1257





hsa-miR-29a-3p
UAGCACCAUCUGAAAUCGGUUA
106
22
1206





hsa-miR-361-5p
UUAUCAGAAUCUCCAGGGGUAC
70
22
1173





hsa-miR-671-3p
UCCGGUUCUCAGGGCUCCACC
69
21
1166





hsa-miR-411-5p
UAGUAGACCGUAUAGCGUACG
108
21
1130





hsa-miR-589-5p
UGAGAACCACGUCUGCUCUGAG
73
22
1067





hsa-miR-130a-3p
CAGUGCAAUGUUAAAAGGGCAU
113
22
1020





hsa-miR-320a
AAAAGCUGGGUUGAGAGGGCGA
97
22
994





hsa-miR-149-5p
UCUGGCUCCGUGUCUUCACUCCC
121
23
948





hsa-miR-335-5p
UCAAGAGCAAUAACGAAAAAUGU
128
23
945





hsa-miR-134
UGUGACUGGUUGACCAGAGGGG
94
22
941





hsa-miR-17-5p
CAAAGUGCUUACAGUGCAGGUAG
145
23
939





hsa-miR-493-5p
UUGUACAUGGUAGGCUUUCAUU
88
22
876





hsa-miR-34c-5p
AGGCAGUGUAGUUAGCUGAUUGC
125
23
846





hsa-miR-484
UCAGGCUCAGUCCCCUCCCGAU
118
22
835





hsa-miR-181a-3p
ACCAUCGACCGUUGAUUGUACC
100
22
803





hsa-miR-24-3p
UGGCUCAGUUCAGCAGGAACAG
119
22
740





hsa-miR-128
UCACAGUGAACCGGUCUCUUU
109
21
707





hsa-miR-342-3p
UCUCACACAGAAAUCGCACCCGU
81
23
698





hsa-miR-454-3p
UAGUGCAAUAUUGCUUAUAGGGU
169
23
690





hsa-miR-1307-5p
UCGACCGGACCUCGACCGGCU
91
21
616





hsa-miR-487b
AAUCGUACAGGGUCAUCCACUU
117
22
590





hsa-miR-130b-5p
ACUCUUUCCCUGUUGCACUAC
112
21
568





hsa-miR-197-3p
UUCACCACCUUCUCCACCCAGC
122
22
544





hsa-miR-432-5p
UCUUGGAGUAGGUCAUUGGGUGG
95
23
542





hsa-miR-374a-5p
UUAUAAUACAACCUGAUAAGUG
74
22
537





hsa-miR-345-5p
GCUGACUCCUAGUCCAGGGCUC
76
22
527





hsa-miR-744-5p
UGCGGGGCUAGGGCUAACAGCA
99
22
515





hsa-miR-376c
AACAUAGAGGAAAUUCCACGU
185
21
506





hsa-miR-181d
AACAUUCAUUGUUGUCGGUGGGU
157
23
497





hsa-miR-363-3p
AAUUGCACGGUAUCCAUCUGUA
131
22
493





hsa-miR-539-3p
AUCAUACAAGGACAAUUUCUUU
150
22
493





hsa-miR-758
UUUGUGACCUGGUCCACUAACC
141
22
477





hsa-miR-323a-3p
CACAUUACACGGUCGACCUCU
158
21
443





hsa-miR-107
AGCAGCAUUGUACAGGGCUAUCA
254
23
431





hsa-miR-720
UCUCGCUGGGGCCUCCA
84
17
427





hsa-miR-654-5p
UGGUGGGCCGCAGAACAUGUGC
115
22
409





hsa-miR-370
GCCUGCUGGGGUGGAACCUGGU
126
22
406





hsa-miR-421
AUCAACAGACAUUAAUUGGGCGC
151
23
399





hsa-miR-30d-3p
CUUUCAGUCAGAUGUUUGCUGC
114
22
358





hsa-miR-148b-5p
AAGUUCUGUUAUACACUCAGGC
127
22
354





hsa-miR-1301
UUGCAGCUGCCUGGGAGUGACUUC
181
24
346





hsa-miR-374b-5p
AUAUAAUACAACCUGCUAAGUG
143
22
339





hsa-miR-125b-2-3p
UCACAAGUCAGGCUCUUGGGAC
68
22
333





hsa-miR-28-5p
AAGGAGCUCACAGUCUAUUGAG
152
22
332





hsa-miR-495
AAACAAACAUGGUGCACUUCUU
241
22
321





hsa-miR-15a-5p
UAGCAGCACAUAAUGGUUUGUG
223
22
320





hsa-miR-100-3p
CAAGCUUGUAUCUAUAGGUAUG
98
22
314





hsa-miR-193b-3p
AACUGGCCCUCAAAGUCCCGCU
148
22
305





hsa-miR-330-5p
UCUCUGGGCCUGUGUCUUAGGC
161
22
303





hsa-miR-376a-3p
AUCAUAGAGGAAAAUCCACGU
237
21
298





hsa-miR-135b-5p
UAUGGCUUUUCAUUCCUAUGUGA
137
23
289





hsa-miR-301a-3p
CAGUGCAAUAGUAUUGUCAAAGC
107
23
280





hsa-miR-218-5p
UUGUGCUUGAUCUAACCAUGU
206
21
276





hsa-miR-143-3p
UGAGAUGAAGCACUGUAGCUC
176
21
256





hsa-miR-27b-5p
AGAGCUUAGCUGAUUGGUGAAC
201
22
255





hsa-miR-369-3p
AAUAAUACAUGGUUGAUCUUU
196
21
255





hsa-miR-877-5p
GUAGAGGAGAUGGCGCAGGG
133
20
249





hsa-miR-19b-3p
UGUGCAAAUCCAUGCAAAACUGA
163
23
246





hsa-miR-424-5p
CAGCAGCAAUUCAUGUUUUGAA
186
22
245





hsa-miR-660-5p
UACCCAUUGCAUAUCGGAGUUG
187
22
244





hsa-miR-532-5p
CAUGCCUUGAGUGUAGGACCGU
178
22
238





hsa-miR-299-3p
UAUGUGGGAUGGUAAACCGCUU
182
22
235





hsa-miR-431-3p
CAGGUCGUCUUGCAGGGCUUCU
168
22
231





hsa-miR-374a-3p
CUUAUCAGAUUGUAUUGUAAUU
173
22
220





hsa-miR-148a-5p
AAAGUUCUGAGACACUCCGACU
144
22
214





hsa-miR-4516
GGGAGAAGGGUCGGGGC
110
17
207





hsa-miR-92b-5p
AGGGACGGGACGCGGUGCAGUG
208
22
206





hsa-miR-16-2-3p
CCAAUAUUACUGUGCUGCUUUA
316
22
202





hsa-miR-101-3p
UACAGUACUGUGAUAACUGAA
142
21
201





hsa-let-7a-3p
CUAUACAAUCUACUGUCUUUC
222
21
199





hsa-miR-4485
UAACGGCCGCGGUACCCUAA
67
20
195





hsa-miR-455-3p
GCAGUCCAUGGGCAUAUACAC
140
21
192





hsa-miR-185-5p
UGGAGAGAAAGGCAGUUCCUGA
224
22
188





hsa-miR-1185-1-3p
AUAUACAGGGGGAGACUCUUAU
209
22
187





hsa-miR-1197
UAGGACACAUGGUCUACUUCU
244
21
185





hsa-miR-106b-3p
CCGCACUGUGGGUACUUGCUGC
159
22
178





hsa-miR-24-2-5p
UGCCUACUGAGCUGAAACACAG
156
22
178





hsa-miR-4677-3p
UCUGUGAGACCAAAGAACUACU
120
22
177





hsa-miR-380-3p
UAUGUAAUAUGGUCCACAUCUU
445
22
174





hsa-miR-548k
AAAAGUACUUGCGGAUUUUGCU
198
22
171





hsa-miR-1307-3p
ACUCGGCGUGGCGUCGGUCGUG
124
22
169





hsa-miR-485-3p
GUCAUACACGGCUCUCCUCUCU
153
22
168





hsa-miR-494
UGAAACAUACACGGGAAACCUC
240
22
165





hsa-miR-17-3p
ACUGCAGUGAAGGCACUUGUAG
184
22
163





hsa-miR-561-5p
AUCAAGGAUCUUAAACUUUGCC
179
22
160





hsa-miR-27a-5p
AGGGCUUAGCUGCUUGUGAGCA
130
22
158





hsa-miR-874
CUGCCCUGGCCCGAGGGACCGA
147
22
151





hsa-miR-9-3p
AUAAAGCUAGAUAACCGAAAGU
183
22
151





hsa-miR-96-5p
UUUGGCACUAGCACAUUUUUGCU
123
23
151





hsa-miR-656
AAUAUUAUACAGUCAACCUCU
221
21
147





hsa-miR-379-3p
UAUGUAACAUGGUCCACUAACU
446
22
145





hsa-miR-382-5p
GAAGUUGUUCGUGGUGGAUUCG
238
22
144





hsa-miR-541-3p
UGGUGGGCACAGAAUCUGGACU
136
22
141





hsa-miR-337-3p
CUCCUAUAUGAUGCCUUUCUUC
215
22
139





hsa-miR-15b-3p
CGAAUCAUUAUUUGCUGCUCUA
447
22
137





hsa-miR-20b-5p
CAAAGUGCUCAUAGUGCAGGUAG
317
23
136





hsa-miR-329
AACACACCUGGUUAACCUCUUU
214
22
136





hsa-miR-3676-5p
AGGAGAUCCUGGGUU
280
15
134





hsa-miR-543
AAACAUUCGCGGUGCACUUCUU
193
22
134





hsa-miR-365b-3p
UAAUGCCCCUAAAAAUCCUUAU
279
22
133





hsa-miR-125a-3p
ACAGGUGAGGUUCUUGGGAGCC
160
22
131





hsa-miR-3065-5p
UCAACAAAAUCACUGAUGCUGGA
226
23
130





hsa-miR-1296
UUAGGGCCCUGGCUCCAUCUCC
271
22
126





hsa-miR-935
CCAGUUACCGCUUCCGCUACCGC
311
23
118





hsa-miR-132-3p
UAACAGUCUACAGCCAUGGUCG
104
22
116





hsa-miR-4284
GGGCUCACAUCACCCCAU
191
18
116





hsa-miR-487a
AAUCAUACAGGGACAUCCAGUU
203
22
113





hsa-miR-574-5p
UGAGUGUGUGUGUGUGAGUGUGU
334
23
113





hsa-miR-301b
CAGUGCAAUGAUAUUGUCAAAGC
164
23
111





hsa-miR-548o-3p
CCAAAACUGCAGUUACUUUUGC
268
22
105





hsa-miR-18a-5p
UAAGGUGCAUCUAGUGCAGAUAG
256
23
104





hsa-miR-485-5p
AGAGGCUGGCCGUGAUGAAUUC
165
22
104





hsa-miR-548ah-5p
AAAAGUGAUUGCAGUGUUUG
235
20
103





hsa-miR-361-3p
UCCCCCAGGUGUGAUUCUGAUUU
250
23
101





hsa-miR-433
AUCAUGAUGGGCUCCUCGGUGU
174
22
101





hsa-miR-337-5p
GAACGGCUUCAUACAGGAGUU
277
21
100





hsa-miR-1276
UAAAGAGCCCUGUGGAGACA
194
20
99





hsa-miR-30c-1-3p
CUGGGAGAGGGUUGUUUACUCC
213
22
99





hsa-miR-31-3p
UGCUAUGCCAACAUAUUGCCAU
172
22
96





hsa-miR-424-3p
CAAAACGUGAGGCGCUGCUAU
298
21
96





hsa-miR-550a-5p
AGUGCCUGAGGGAGUAAGAGCCC
134
23
95





hsa-miR-4454
GGAUCCGAGUCACGGCACCA
299
20
94





hsa-miR-541-5p
AAAGGAUUCUGCUGUCGGUCCCACU
432
25
92





hsa-miR-106b-5p
UAAAGUGCUGACAGUGCAGAU
170
21
89





hsa-miR-153
UUGCAUAGUCACAAAAGUGAUC
188
22
88





hsa-miR-135b-3p
AUGUAGGGCUAAAAGCCAUGGG
205
22
87





hsa-miR-574-3p
CACGCUCAUGCACACACCCACA
253
22
87





hsa-miR-1226-3p
UCACCAGCCCUGUGUUCCCUAG
199
22
85





hsa-miR-576-5p
AUUCUAAUUUCUCCACGUCUUU
306
22
84





hsa-miR-127-5p
CUGAAGCUCAGAGGGCUCUGAU
255
22
83





hsa-miR-155-5p
UUAAUGCUAAUCGUGAUAGGGGU
448
23
83





hsa-miR-3176
ACUGGCCUGGGACUACCGG
227
19
83





hsa-miR-382-3p
AAUCAUUCACGGACAACACUU
260
21
83





hsa-miR-1275
GUGGGGGAGAGGCUGUC
162
17
82





hsa-miR-671-5p
AGGAAGCCCUGGAGGGGCUGGAG
288
23
82





hsa-miR-23a-5p
GGGGUUCCUGGGGAUGGGAUUU
212
22
81





hsa-miR-25-5p
AGGCGGAGACUUGGGCAAUUG
225
21
80





hsa-miR-641
AAAGACAUAGGAUAGAGUCACCUC
285
24
80





hsa-miR-19a-3p
UGUGCAAAUCUAUGCAAAACUGA
177
23
79





hsa-miR-377-3p
AUCACACAAAGGCAACUUUUGU
449
22
78





hsa-miR-454-5p
ACCCUAUCAAUAUUGUCUCUGC
265
22
78





hsa-miR-496
UGAGUAUUACAUGGCCAAUCUC
267
22
78





hsa-miR-29b-3p
UAGCACCAUUUGAAAUCAGUGUU
166
23
77





hsa-miR-26a-2-3p
CCUAUUCUUGAUUACUUGUUUC
257
22
76





hsa-miR-1260b
AUCCCACCACUGCCACCAU
373
19
74





hsa-miR-2467-5p
UGAGGCUCUGUUAGCCUUGGCUC
154
23
74





hsa-miR-377-5p
AGAGGUUGCCCUUGGUGAAUUC
202
22
74





hsa-miR-330-3p
GCAAAGCACACGGCCUGCAGAGA
195
23
73





hsa-miR-1180
UUUCCGGCUCGCGUGGGUGUGU
313
22
71





hsa-miR-99b-3p
CAAGCUCGUGUCUGUGGGUCCG
243
22
71





hsa-miR-299-5p
UGGUUUACCGUCCCACAUACAU
319
22
69





hsa-miR-374b-3p
CUUAGCAGGUUGUAUUAUCAUU
229
22
69





hsa-miR-4746-5p
CCGGUCCCAGGAGAACCUGCAGA
266
23
69





hsa-miR-331-3p
GCCCCUGGGCCUAUCCUAGAA
450
21
68





hsa-miR-340-3p
UCCGUCUCAGUUACUUUAUAGC
248
22
68





hsa-miR-92a-1-5p
AGGUUGGGAUCGGUUGCAAUGCU
204
23
68





hsa-miR-542-3p
UGUGACAGAUUGAUAACUGAAA
331
22
66





hsa-miR-431-5p
UGUCUUGCAGGCCGUCAUGCA
132
21
65





hsa-miR-1254
AGCCUGGAAGCUGGAGCCUGCAGU
270
24
61





hsa-miR-3158-3p
AAGGGCUUCCUCUCUGCAGGAC
167
22
61





hsa-miR-362-5p
AAUCCUUGGAACCUAGGUGUGAGU
139
24
61





hsa-miR-30c-2-3p
CUGGGAGAAGGCUGUUUACUCU
321
22
59





hsa-miR-4461
GAUUGAGACUAGUAGGGCUAGGC
129
23
59





hsa-miR-3200-3p
CACCUUGCGCUACUCAGGUCUG
247
22
57





hsa-miR-215
AUGACCUAUGAAUUGACAGAC
451
21
56





hsa-miR-1185-5p
AGAGGAUACCCUUUGUAUGUU
368
21
55





hsa-miR-328
CUGGCCCUCUCUGCCCUUCCGU
297
22
55





hsa-miR-655
AUAAUACAUGGUUAACCUCUUU
286
22
55





hsa-miR-181b-3p
CUCACUGAACAAUGAAUGCAA
245
21
54





hsa-miR-376b
AUCAUAGAGGAAAAUCCAUGUU
452
22
54





hsa-miR-486-3p
CGGGGCAGCUCAGUACAGGAU
453
21
54





hsa-miR-760
CGGCUCUGGGUCUGUGGGGA
289
20
54





hsa-miR-3909
UGUCCUCUAGGGCCUGCAGUCU
412
22
53





hsa-miR-4508
GCGGGGCUGGGCGCGCG
135
17
53





hsa-miR-4521
GCUAAGGAAGUCCUGUGCUCAG
233
22
53





hsa-let-7e-3p
CUAUACGGCCUCCUAGCUUUCC
290
22
52





hsa-miR-455-5p
UAUGUGCCUUUGGACUACAUCG
192
22
52





hsa-miR-93-3p
ACUGCUGAGCUAGCACUUCCCG
454
22
51





hsa-miR-151b
UCGAGGAGCUCACAGUCU
455
18
49





hsa-miR-887
GUGAACGGGCGCCAUCCCGAGG
456
22
49





hsa-miR-152
UCAGUGCAUGACAGAACUUGG
344
21
48





hsa-miR-324-3p
ACUGCCCCAGGUGCUGCUGG
276
20
48





hsa-miR-1266
CCUCAGGGCUGUAGAACAGGGCU
457
23
47





hsa-miR-302b-3p
UAAGUGCUUCCAUGUUUUAGUAG
458
23
47





hsa-miR-548e
AAAAACUGAGACUACUUUUGCA
459
22
47





hsa-miR-502-3p
AAUGCACCUGGGCAAGGAUUCA
281
22
46





hsa-miR-302d-3p
UAAGUGCUUCCAUGUUUGAGUGU
460
23
45





hsa-miR-3943
UAGCCCCCAGGCUUCACUUGGCG
207
23
45





hsa-miR-1286
UGCAGGACCAAGAUGAGCCCU
293
21
44





hsa-miR-3605-5p
UGAGGAUGGAUAGCAAGGAAGCC
189
23
44





hsa-miR-505-3p
CGUCAACACUUGCUGGUUUCCU
282
22
44





hsa-miR-3615
UCUCUCGGCUCCUCGCGGCUC
323
21
43





hsa-miR-4435
AUGGCCAGAGCUCACACAGAGG
230
22
43





hsa-miR-598
UACGUCAUCGUUGUCAUCGUCA
461
22
43





hsa-miR-126-5p
CAUUAUUACUUUUGGUACGCG
462
21
42





hsa-miR-4671-3p
UUAGUGCAUAGUCUUUGGUCU
301
21
41





hsa-miR-652-3p
AAUGGCGCCACUAGGGUUGUG
442
21
41





hsa-miR-3687
CCCGGACAGGCGUUCGUGCGACGU
190
24
40





hsa-miR-4286
ACCCCACUCCUGGUACC
328
17
40





hsa-miR-590-3p
UAAUUUUAUGUAUAAGCUAGU
463
21
40





hsa-miR-1285-3p
UCUGGGCAACAAAGUGAGACCU
464
22
39





hsa-miR-2355-5p
AUCCCCAGAUACAAUGGACAA
593
21
38





hsa-miR-550a-3p
UGUCUUACUCCCUCAGGCACAU
283
22
38





hsa-let-7d-3p
CUAUACGACCUGCUGCCUUUCU
92
22
37





hsa-miR-136-5p
ACUCCAUUUGUUUUGAUGAUGGA
272
23
37





hsa-miR-1468
CUCCGUUUGCCUGUUUCGCUG
296
21
37





hsa-miR-3609
CAAAGUGAUGAGUAAUACUGGCUG
216
24
37





hsa-miR-548b-5p
AAAAGUAAUUGUGGUUUUGGCC
304
22
37





hsa-miR-664-3p
UAUUCAUUUAUCCCCAGCCUACA
287
23
37





hsa-miR-99a-3p
CAAGCUCGCUUCUAUGGGUCUG
367
22
37





hsa-miR-532-3p
CCUCCCACACCCAAGGCUUGCA
252
22
36





hsa-miR-10b-5p
UACCCUGUAGAACCGAAUUUGUG
465
23
33





hsa-miR-369-5p
AGAUCGACCGUGUUAUAUUCGC
357
22
33





hsa-miR-3161
CUGAUAAGAACAGAGGCCCAGAU
466
23
32





hsa-miR-3940-3p
CAGCCCGGAUCCCAGCCCACUU
239
22
32





hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
180
22
32





hsa-miR-219-2-3p
AGAAUUGUGGCUGGACAUCUGU
467
22
31





hsa-miR-2277-5p
AGCGCGGGCUGAGCGCUGCCAGUC
735
24
31





hsa-miR-4448
GGCUCCUUGGUCUAGGGGUA
231
20
31





hsa-miR-339-5p
UCCCUGUCCUCCAGGAGCUCACG
402
23
30





hsa-miR-3613-5p
UGUUGUACUUUUUUUUUUGUUC
469
22
30





hsa-miR-4775
UUAAUUUUUUGUUUCGGUCACU
302
22
30





hsa-miR-212-5p
ACCUUGGCUCUAGACUGCUUACU
246
23
29





hsa-miR-324-5p
CGCAUCCCCUAGGGCAUUGGUGU
354
23
27





hsa-miR-4326
UGUUCCUCUGUCUCCCAGAC
417
20
27





hsa-miR-582-3p
UAACUGGUUGAACAACUGAACC
470
22
27





hsa-miR-34a-3p
CAAUCAGCAAGUAUACUGCCCU
403
22
26





hsa-miR-106a-5p
AAAAGUGCUUACAGUGCAGGUAG
471
23
25





hsa-miR-4745-5p
UGAGUGGGGCUCCCGGGACGGCG
219
23
25





hsa-miR-769-3p
CUGGGAUCUCCGGGGUCUUGGUU
337
23
25





hsa-miR-1268a
CGGGCGUGGUGGUGGGGG
291
18
24





hsa-miR-154-3p
AAUCAUACACGGUUGACCUAUU
472
22
24





hsa-miR-188-3p
CUCCCACAUGCAGGGUUUGCA
200
21
24





hsa-miR-29c-3p
UAGCACCAUUUGAAAUCGGUUA
473
22
24





hsa-miR-539-5p
GGAGAAAUUAUCCUUGGUGUGU
234
22
24





hsa-miR-766-3p
ACUCCAGCCCCACAGCCUCAGC
310
22
24





hsa-miR-30b-3p
CUGGGAGGUGGAUGUUUACUUC
320
22
23





hsa-miR-3177-3p
UGCACGGCACUGGGGACACGU
275
21
23





hsa-miR-191-3p
GCUGCGCUUGGAUUUCGUCCCC
474
22
22





hsa-miR-296-3p
GAGGGUUGGGUGGAGGCUCUCC
274
22
22





hsa-miR-296-5p
AGGGCCCCCCCUCAAUCCUGU
258
21
22





hsa-miR-339-3p
UGAGCGCCUCGACGACAGAGCCG
228
23
22





hsa-miR-501-5p
AAUCCUUUGUCCCUGGGUGAGA
430
22
22





hsa-miR-200b-3p
UAAUACUGCCUGGUAAUGAUGA
475
22
21





hsa-miR-212-3p
UAACAGUCUCCAGUCACGGCC
348
21
21





hsa-miR-26b-3p
CCUGUUCUCCAUUACUUGGCUC
391
22
21





hsa-miR-665
ACCAGGAGGCUGAGGCCCCU
309
20
21





hsa-miR-668
UGUCACUCGGCUCGGCCCACUAC
476
23
21





hsa-miR-146a-5p
UGAGAACUGAAUUCCAUGGGUU
477
22
20





hsa-miR-1973
ACCGUGCAAAGGUAGCAUA
171
19
20





hsa-miR-210
CUGUGCGUGUGACAGCGGCUGA
478
22
20





hsa-miR-3607-5p
GCAUGUGAUGAAGCAAAUCAGU
249
22
20





hsa-miR-378a-5p
CUCCUGACUCCAGGUCCUGUGU
217
22
20





hsa-miR-4449
CGUCCCGGGGCUGCGCGAGGCA
155
22
20





hsa-miR-138-5p
AGCUGGUGUUGUGAAUCAGGCCG
379
23
19





hsa-miR-146b-3p
UGCCCUGUGGACUCAGUUCUGG
381
22
18





hsa-miR-3065-3p
UCAGCACCAGGAUAUUGUUGGAG
350
23
18





hsa-miR-4417
GGUGGGCUUCCCGGAGGG
175
18
18





hsa-miR-497-5p
CAGCAGCACACUGUGGUUUGU
479
21
18





hsa-miR-500a-5p
UAAUCCUUGCUACCUGGGUGAGA
303
23
18





hsa-miR-625-3p
GACUAUAGAACUUUCCCCCUCA
307
22
18





hsa-miR-628-3p
UCUAGUAAGAGUGGCAGUCGA
335
21
18





hsa-miR-1343
CUCCUGGGGCCCGCACUCUCGC
378
22
17





hsa-miR-3648
AGCCGCGGGGAUCGCCGAGGG
259
21
17





hsa-miR-432-3p
CUGGAUGGCUCCUCCAUGUCU
262
21
17





hsa-miR-4482-3p
UUUCUAUUUCUCAGUGGGGCUC
361
22
17





hsa-miR-542-5p
UCGGGGAUCAUCAUGUCACGAGA
433
23
17





hsa-miR-551b-3p
GCGACCCAUACUUGGUUUCAG
434
21
17





hsa-miR-7-1-3p
CAACAAAUCACAGUCUGCCAUA
480
22
17





hsa-miR-219-1-3p
AGAGUUGAGUCUGGACGUCCCG
390
22
16





hsa-miR-3656
GGCGGGUGCGGGGGUGG
251
17
16





hsa-miR-3661
UGACCUGGGACUCGGACAGCUG
481
22
16





hsa-miR-411-3p
UAUGUAACACGGUCCACUAACC
482
22
16





hsa-miR-5096
GUUUCACCAUGUUGGUCAGGC
220
21
16





hsa-miR-577
UAGAUAAAAUAUUGGUACCUG
436
21
16





hsa-let-71-3p
CUGCGCAAGCUACUGCCUUGCU
483
22
15





hsa-miR-132-5p
ACCGUGGCUUUCGAUUGUUACU
315
22
15





hsa-miR-140-5p
CAGUGGUUUUACCCUAUGGUAG
380
22
15





hsa-miR-195-5p
UAGCAGCACAGAAAUAUUGGC
346
21
15





hsa-miR-3187-3p
UUGGCCAUGGGGCUGCGCGG
322
20
15





hsa-miR-342-5p
AGGGGUGCUAUCUGUGAUUGA
278
21
15





hsa-miR-34b-3p
CAAUCACUAACUCCACUGCCAU
404
22
15





hsa-miR-4661-5p
AACUAGCUCUGUGGAUCCUGAC
484
22
15





hsa-miR-584-5p
UUAUGGUUUGCCUGGGACUGAG
485
22
15





hsa-miR-744-3p
CUGUUGCCACUAACCUCAACCU
486
22
15





hsa-miR-770-5p
UCCAGUACCACGUGUCAGGGCCA
487
23
15





hsa-miR-3677-3p
CUCGUGGGCUCUGGCCACGGCC
356
22
14





hsa-miR-425-3p
AUCGGGAAUGUCGUGUCCGCCC
358
22
14





hsa-miR-548ah-3p
CAAAAACUGCAGUUACUUUUGC
149
22
14





hsa-miR-5699
UCCUGUCUUUCCUUGUUGGAGC
488
22
14





hsa-miR-582-5p
UUACAGUUGUUCAACCAGUUACU
489
23
14





hsa-miR-1185-2-3p
AUAUACAGGGGGAGACUCUCAU
314
22
13





hsa-miR-1249
ACGCCCUUCCCCCCCUUCUUCA
490
22
13





hsa-miR-1255a
AGGAUGAGCAAAGAAAGUAGAUU
341
23
13





hsa-miR-1910
CCAGUCCUGUGCCUGCCGCCU
236
21
13





hsa-miR-301a-5p
GCUCUGACUUUAUUGCACUACU
491
22
13





hsa-miR-5001-3p
UUCUGCCUCUGUCCAGGUCCUU
492
22
13





hsa-miR-5094
AAUCAGUGAAUGCCUUGAACCU
493
22
13





hsa-miR-628-5p
AUGCUGACAUAUUUACUAGAGG
440
22
13





hsa-miR-629-5p
UGGGUUUACGUUGGGAGAACU
441
21
13





hsa-miR-937
AUCCGCGCUCUGACUCUCUGCC
312
22
13





hsa-miR-940
AAGGCAGGGCCCCCGCUCCCC
366
21
13





hsa-miR-1248
ACCUUCUUGUAUAAGCACUGUGCUAAA
269
27
12





hsa-miR-194-5p
UGUAACAGCAACUCCAUGUGGA
345
22
12





hsa-miR-199b-3p
ACAGUAGUCUGCACAUUGGUUA
494
22
12





hsa-miR-22-5p
AGUUCUUCAGUGGCAAGCUUUA
495
22
12





hsa-miR-3605-3p
CCUCCGUGUUACCUGUCCUCUAG
496
23
12





hsa-miR-3654
GACUGGACAAGCUGAGGAA
325
19
12





hsa-miR-504
AGACCCUGGUCUGCACUCUAUC
497
22
12





hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
294
24
11





hsa-miR-1299
UUCUGGAAUUCUGUGUGAGGGA
498
22
11





hsa-miR-188-5p
CAUCCCUUGCAUGGUGGAGGG
499
21
11





hsa-miR-222-5p
CUCAGUAGCCAGUGUAGAUCCU
349
22
11





hsa-miR-331-5p
CUAGGUAUGGUCCCAGGGAUCC
500
22
11





hsa-miR-3939
UACGCGCAGACCACAGGAUGUC
261
22
11





hsa-miR-154-5p
UAGGUUAUCCGUGUUGCCUUCG
501
22
10





hsa-miR-18a-3p
ACUGCCCUAAGUGCUCCUUCUGG
502
23
10





hsa-miR-1908
CGGCGGGGACGGCGAUUGGUC
383
21
10





hsa-miR-200c-3p
UAAUACUGCCGGGUAAUGAUGGA
347
23
10





hsa-miR-2116-3p
CCUCCCAUGCCAAGAACUCCC
318
21
10





hsa-miR-302a-3p
UAAGUGCUUCCAUGUUUUGGUGA
503
23
10





hsa-miR-3174
UAGUGAGUUAGAGAUGCAGAGCC
353
23
10





hsa-miR-326
CCUCUGGGCCCUUCCUCCAG
504
20
10





hsa-let-7g-3p
CUGUACAGGCCACUGCCUUGC
505
21
9





hsa-miR-141-3p
UAACACUGUCUGGUAAAGAUGG
295
22
9





hsa-miR-24-1-5p
UGCCUACUGAGCUGAUAUCAGU
506
22
9





hsa-miR-3115
AUAUGGGUUUACUAGUUGGU
351
20
9





hsa-miR-3180-3p
UGGGGCGGAGCUUCCGGAGGCC
400
22
9





hsa-miR-33a-5p
GUGCAUUGUAGUUGCAUUGCA
355
21
9





hsa-miR-34c-3p
AAUCACUAACCACACGGCCAGG
405
22
9





hsa-miR-3929
GAGGCUGAUGUGAGUAGACCACU
218
23
9





hsa-miR-4517
AAAUAUGAUGAAACUCACAGCUGAG
507
25
9





hsa-miR-576-3p
AAGAUGUGGAAAAAUUGGAAUC
508
22
9





hsa-miR-1229
CUCUCACCACUGCCCUCCCACAG
509
23
8





hsa-miR-1289
UGGAGUCCAGGAAUCUGCAUUUU
343
23
8





hsa-miR-1915-5p
ACCUUGCCUUGCUGCCCGGGCC
385
22
8





hsa-miR-23b-5p
UGGGUUCCUGGCAUGCUGAUUU
510
22
8





hsa-miR-302a-5p
ACUUAAACGUGGAUGUACUUGCU
511
23
8





hsa-miR-3938
AAUUCCCUUGUAGAUAACCCGG
512
22
8





hsa-miR-4466
GGGUGCGGGCCGGCGGGG
264
18
8





hsa-miR-4786-5p
UGAGACCAGGACUGGAUGCACC
197
22
8





hsa-miR-589-3p
UCAGAACAAAUGCCGGUUCCCAGA
438
24
8





hsa-miR-616-5p
ACUCAAAACCCUUCAGUGACUU
439
22
8





hsa-miR-943
CUGACUGUUGCCGUCCUCCAG
338
21
8





hsa-miR-1237
UCCUUCUGCUCCGUCCCCCAG
370
21
7





hsa-miR-1915-3p
CCCCAGGGCGACGCGGCGGG
384
20
7





hsa-miR-3620
UCACCCUGCAUCCCGCACCCAG
324
22
7





hsa-miR-3691-5p
AGUGGAUGAUGGAGACUCGGUAC
409
23
7





hsa-miR-4426
GAAGAUGGACGUACUUU
359
17
7





hsa-let-7a-2-3p
CUGUACAGCCUCCUAGCUUUCC
513
22
6





hsa-miR-10a-3p
CAAAUUCGUAUCUAGGGGAAUA
514
22
6





hsa-miR-1287
UGCUGGAUCAGUGGUUCGAGUC
515
22
6





hsa-miR-145-5p
GUCCAGUUUUCCCAGGAAUCCCU
516
23
6





hsa-miR-29b-1-5p
GCUGGUUUCAUAUGGUGGUUUAGA
517
24
6





hsa-miR-3128
UCUGGCAAGUAAAAAACUCUCAU
518
23
6





hsa-miR-33b-5p
GUGCAUUGCUGUUGCAUUGC
519
20
6





hsa-miR-3681-5p
UAGUGGAUGAUGCACUCUGUGC
327
22
6





hsa-miR-3685
UUUCCUACCCUACCUGAAGACU
520
22
6





hsa-miR-3918
ACAGGGCCGCAGAUGGAGACU
521
21
6





hsa-miR-551b-5p
GAAAUCAAGCGUGGGUGAGACC
522
22
6





hsa-miR-1273f
GGAGAUGGAGGUUGCAGUG
292
19
5





hsa-miR-1273g-3p
ACCACUGCACUCCAGCCUGAG
210
21
5





hsa-miR-1304-5p
UUUGAGGCUACAGUGAGAUGUG
523
22
5





hsa-miR-1538
CGGCCCGGGCUGCUGCUGUUCCU
524
23
5





hsa-miR-181c-3p
AACCAUCGACCGUUGAGUGGAC
525
22
5





hsa-miR-193a-5p
UGGGUCUUUGCGGGCGAGAUGA
526
22
5





hsa-miR-208b
AUAAGACGAACAAAAGGUUUGU
388
22
5





hsa-miR-219-5p
UGAUUGUCCAAACGCAAUUCU
527
21
5





hsa-miR-3159
UAGGAUUACAAGUGUCGGCCAC
528
22
5





hsa-miR-3173-5p
UGCCCUGCCUGUUUUCUCCUUU
529
22
5





hsa-miR-3175
CGGGGAGAGAACGCAGUGACGU
530
22
5





hsa-miR-3200-5p
AAUCUGAGAAGGCGCACAAGGU
531
22
5





hsa-miR-3662
GAAAAUGAUGAGUAGUGACUGAUG
326
24
5





hsa-miR-3928
GGAGGAACCUUGGAGCUUCGGC
413
22
5





hsa-miR-4709-3p
UUGAAGAGGAGGUGCUCUGUAGC
532
23
5





hsa-miR-4787-3p
GAUGCGCCGCCCACUGCCCCGCGC
533
24
5





hsa-miR-499a-5p
UUAAGACUUGCAGUGAUGUUU
534
21
5





hsa-miR-545-3p
UCAGCAAACAUUUAUUGUGUGC
242
22
5





hsa-miR-548u
CAAAGACUGCAAUUACUUUUGCG
535
23
5





hsa-miR-659-5p
AGGACCUUCCCUGAACCAAGGA
364
22
5





hsa-miR-1257
AGUGAAUGAUGGGUUCUGACC
372
21
4





hsa-miR-1292
UGGGAACGGGUUCCGGCAGACGCUG
536
25
4





hsa-miR-1914-5p
CCCUGUGCCCGGCCCACUUCUG
537
22
4





hsa-miR-195-3p
CCAAUAUUGGCUGUGCUGCUCC
538
22
4





hsa-miR-2110
UUGGGGAAACGGCCGCUGAGUG
389
22
4





hsa-miR-302c-5p
UUUAACAUGGGGGUACCUGCUG
539
22
4





hsa-miR-3126-3p
CAUCUGGCAUCCGUCACACAGA
394
22
4





hsa-miR-3126-5p
UGAGGGACAGAUGCCAGAAGCA
352
22
4





hsa-miR-3150a-5p
CAACCUCGACGAUCUCCUCAGC
540
22
4





hsa-miR-3157-3p
CUGCCCUAGUCUAGCUGAAGCU
399
22
4





hsa-miR-323b-3p
CCCAAUACACGGUCGACCUCUU
541
22
4





hsa-miR-335-3p
UUUUUCAUUAUUGCUCCUGACC
542
22
4





hsa-miR-3607-3p
ACUGUAAACGCUUUCUGAUG
543
20
4





hsa-miR-3653
CUAAGAAGUUGACUGAAG
544
18
4





hsa-miR-3663-3p
UGAGCACCACACAGGCCGGGCGC
545
23
4





hsa-miR-376a-5p
GUAGAUUCUCCUUCUAUGAGUA
410
22
4





hsa-miR-4423-3p
AUAGGCACCAAAAAGCAACAA
662
21
4





hsa-miR-4423-5p
AGUUGCCUUUUUGUUCCCAUGC
263
22
4





hsa-miR-4463
GAGACUGGGGUGGGGCC
300
17
4





hsa-miR-449a
UGGCAGUGUAUUGUUAGCUGGU
547
22
4





hsa-miR-4511
GAAGAACUGUUGCAUUUGCCCU
548
22
4





hsa-miR-4640-3p
CACCCCCUGUUUCCUGGCCCAC
329
22
4





hsa-miR-4800-3p
CAUCCGUCCGUCUGUCCAC
549
19
4





hsa-miR-505-5p
GGGAGCCAGGAAGUAUUGAUGU
550
22
4





hsa-miR-548a-3p
CAAAACUGGCAAUUACUUUUGC
551
22
4





hsa-miR-570-3p
CGAAAACAGCAAUUACCUUUGC
333
22
4





hsa-miR-663a
AGGCGGGGCGCCGCGGGACCGC
365
22
4





hsa-miR-877-3p
UCCUCUUCUCCCUCCUCCCAG
552
21
4





hsa-miR-103a-2-5p
AGCUUCUUUACAGUGCUGCCUUG
553
23
3





hsa-miR-1268b
CGGGCGUGGUGGUGGGGGUG
554
20
3





hsa-miR-1270
CUGGAGAUAUGGAAGAGCUGUGU
555
23
3





hsa-miR-1293
UGGGUGGUCUGGAGAUUUGUGC
556
22
3





hsa-miR-1322
GAUGAUGCUGCUGAUGCUG
557
19
3





hsa-miR-150-5p
UCUCCCAACCCUUGUACCAGUG
558
22
3





hsa-miR-190b
UGAUAUGUUUGAUAUUGGGUU
559
21
3





hsa-miR-193a-3p
AACUGGCCUACAAAGUCCCAGU
386
22
3





hsa-miR-193b-5p
CGGGGUUUUGAGGGCGAGAUGA
560
22
3





hsa-miR-199a-5p
CCCAGUGUUCAGACUACCUGUUC
273
23
3





hsa-miR-20a-3p
ACUGCAUUAUGAGCACUUAAAG
561
22
3





hsa-miR-216a
UAAUCUCAGCUGGCAACUGUGA
562
22
3





hsa-miR-2682-5p
CAGGCAGUGACUGUUCAGACGUC
563
23
3





hsa-miR-2964a-5p
AGAUGUCCAGCCACAAUUCUCG
564
22
3





hsa-miR-3177-5p
UGUGUACACACGUGCCAGGCGCU
565
23
3





hsa-miR-320c
AAAAGCUGGGUUGAGAGGGU
566
20
3





hsa-miR-323a-5p
AGGUGGUCCGUGGCGCGUUCGC
567
22
3





hsa-miR-3622a-5p
CAGGCACGGGAGCUCAGGUGAG
568
22
3





hsa-miR-3912
UAACGCAUAAUAUGGACAUGU
569
21
3





hsa-miR-3934
UCAGGUGUGGAAACUGAGGCAG
570
22
3





hsa-miR-3942-3p
UUUCAGAUAACAGUAUUACAU
414
21
3





hsa-miR-3942-5p
AAGCAAUACUGUUACCUGAAAU
571
22
3





hsa-miR-4523
GACCGAGAGGGCCUCGGCUGU
572
21
3





hsa-miR-4640-5p
UGGGCCAGGGAGCAGCUGGUGGG
573
23
3





hsa-miR-4671-5p
ACCGAAGACUGUGCGCUAAUCU
574
22
3





hsa-miR-4709-5p
ACAACAGUGACUUGCUCUCCAA
575
22
3





hsa-miR-4731-3p
CACACAAGUGGCCCCCAACACU
425
22
3





hsa-miR-4731-5p
UGCUGGGGGCCACAUGAGUGUG
576
22
3





hsa-miR-4762-5p
CCAAAUCUUGAUCAGAAGCCU
577
21
3





hsa-miR-5010-5p
AGGGGGAUGGCAGAGCAAAAUU
578
22
3





hsa-miR-502-5p
AUCCUUGCUAUCUGGGUGCUA
579
21
3





hsa-miR-548d-5p
AAAAGUAAUUGUGGUUUUUGCC
580
22
3





hsa-miR-548i
AAAAGUAAUUGCGGAUUUUGCC
581
22
3





hsa-miR-548j
AAAAGUAAUUGCGGUCUUUGGU
582
22
3





hsa-miR-5587-3p
GCCCCGGGCAGUGUGAUCAUC
284
21
3





hsa-miR-1225-3p
UGAGCCCCUGUGCCGCCCCCAG
369
22
2





hsa-miR-1227
CGUGCCACCCUUUUCCCCAG
583
20
2





hsa-miR-1252
AGAAGGAAAUUGAAUUCAUUUA
371
22
2





hsa-miR-1280
UCCCACCGCUGCCACCC
584
17
2





hsa-miR-1288
UGGACUGCCCUGAUCUGGAGA
585
21
2





hsa-miR-1303
UUUAGAGACGGGGUCUUGCUCU
586
22
2





hsa-miR-1306-3p
ACGUUGGCUCUGGUGGUG
376
18
2





hsa-miR-139-5p
UCUACAGUGCACGUGUCUCCAG
587
22
2





hsa-miR-149-3p
AGGGAGGGACGGGGGCUGUGC
588
21
2





hsa-miR-16-1-3p
CCAGUAUUAACUGUGCUGCUGA
589
22
2





hsa-miR-1909-5p
UGAGUGCCGGUGCCUGCCCUG
590
21
2





hsa-miR-224-5p
CAAGUCACUAGUGGUUCCGUU
591
21
2





hsa-miR-2276
UCUGCAAGUGUCAGAGGCGAGG
592
22
2





hsa-miR-2355-3p
AUUGUCCUUGCUGUUUGGAGAU
468
22
2





hsa-miR-2964a-3p
AGAAUUGCGUUUGGACAAUCAGU
392
23
2





hsa-miR-29c-5p
UGACCGAUUUCUCCUGGUGUUC
594
22
2





hsa-miR-3074-3p
GAUAUCAGCUCAGUAGGCACCG
595
22
2





hsa-miR-3120-3p
CACAGCAAGUGUAGACAGGCA
596
21
2





hsa-miR-3130-5p
UACCCAGUCUCCGGUGCAGCC
396
21
2





hsa-miR-3140-3p
AGCUUUUGGGAAUUCAGGUAGU
597
22
2





hsa-miR-3155a
CCAGGCUCUGCAGUGGGAACU
398
21
2





hsa-miR-3163
UAUAAAAUGAGGGCAGUAAGAC
598
22
2





hsa-miR-3167
AGGAUUUCAGAAAUACUGGUGU
599
22
2





hsa-miR-363-5p
CGGGUGGAUCACGAUGCAAUUU
600
22
2





hsa-miR-3676-3p
CCGUGUUUCCCCCACGCUUU
408
20
2





hsa-miR-378g
ACUGGGCUUGGAGUCAGAAG
411
20
2





hsa-miR-4467
UGGCGGCGGUAGUUAUGGGCUU
360
22
2





hsa-miR-4498
UGGGCUGGCAGGGCAAGUGCUG
601
22
2





hsa-miR-4654
UGUGGGAUCUGGAGGCAUCUGG
420
22
2





hsa-miR-4659a-3p
UUUCUUCUUAGACAUGGCAACG
603
22
2





hsa-miR-4662a-5p
UUAGCCAAUUGUCCAUCUUUAG
604
22
2





hsa-miR-4683
UGGAGAUCCAGUGCUCGCCCGAU
605
23
2





hsa-miR-4738-3p
UGAAACUGGAGCGCCUGGAGGA
606
22
2





hsa-miR-4746-3p
AGCGGUGCUCCUGCGGGCCGA
607
21
2





hsa-miR-4748
GAGGUUUGGGGAGGAUUUGCU
608
21
2





hsa-miR-4792
CGGUGAGCGCUCGCUGGC
363
18
2





hsa-miR-491-5p
AGUGGGGAACCCUUCCAUGAGG
429
22
2





hsa-miR-5000-3p
UCAGGACACUUCUGAACUUGGA
609
22
2





hsa-miR-503
UAGCAGCGGGAACAGUUCUGCAG
610
23
2





hsa-miR-5189
UCUGGGCACAGGCGGAUGGACAGG
611
24
2





hsa-miR-548aq-3p
CAAAAACUGCAAUUACUUUUGC
612
22
2





hsa-miR-548av-3p
AAAACUGCAGUUACUUUUGC
613
20
2





hsa-miR-5584-5p
CAGGGAAAUGGGAAGAACUAGA
332
22
2





hsa-miR-5690
UCAGCUACUACCUCUAUUAGG
435
21
2





hsa-miR-573
CUGAAGUGAUGUGUAACUGAUCAG
305
24
2





hsa-miR-597
UGUGUCACUCGAUGACCACUGU
614
22
2





hsa-miR-622
ACAGUCUGCUGAGGUUGGAGC
615
21
2





hsa-miR-636
UGUGCUUGCUCGUCCCGCCCGCA
616
23
2





hsa-miR-1193
GGGAUGGUAGACCGGUGACGUGC
617
23
1





hsa-miR-1224-3p
CCCCACCUCCUCUCUCCUCAG
618
21
1





hsa-miR-122-5p
UGGAGUGUGACAAUGGUGUUUG
720
22
1





hsa-miR-1228-5p
GUGGGCGGGGGCAGGUGUGUG
620
21
1





hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
340
26
1





hsa-miR-1247-5p
ACCCGUCCCGUUCGUCCCCGGA
621
22
1





hsa-miR-1255b-5p
CGGAUGAGCAAAGAAAGUGGUU
622
22
1





hsa-miR-1269b
CUGGACUGAGCCAUGCUACUGG
623
22
1





hsa-miR-1272
GAUGAUGAUGGCAGCAAAUUCUGAAA
624
26
1





hsa-miR-1273c
GGCGACAAAACGAGACCCUGUC
625
22
1





hsa-miR-1273e
UUGCUUGAACCCAGGAAGUGGA
342
22
1





hsa-miR-1282
UCGUUUGCCUUUUUCUGCUU
626
20
1





hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
375
19
1





hsa-miR-1294
UGUGAGGUUGGCAUUGUUGUCU
627
22
1





hsa-miR-1306-5p
CCACCUCCCCUGCAAACGUCCA
628
22
1





hsa-miR-1321
CAGGGAGGUGAAUGUGAU
377
18
1





hsa-miR-135a-5p
UAUGGCUUUUUAUUCCUAUGUGA
629
23
1





hsa-miR-137
UUAUUGCUUAAGAAUACGCGUAG
630
23
1





hsa-miR-142-5p
CAUAAAGUAGAAAGCACUACU
631
21
1





hsa-miR-143-5p
GGUGCAGUGCUGCAUCUCUGGU
632
22
1





hsa-miR-15a-3p
CAGGCCAUAUUGUGCUGCCUCA
633
22
1





hsa-miR-186-3p
GCCCAAAGGUGAAUUUUUUGGG
382
22
1





hsa-miR-192-3p
CUGCCAAUUCCAUAGGUCACAG
634
22
1





hsa-miR-19b-1-5p
AGUUUUGCAGGUUUGCAUCCAGC
387
23
1





hsa-miR-200a-3p
UAACACUGUCUGGUAACGAUGU
635
22
1





hsa-miR-204-3p
GCUGGGAAGGCAAAGGGACGU
636
21
1





hsa-miR-214-3p
ACAGCAGGCACAGACAGGCAGU
637
22
1





hsa-miR-29a-5p
ACUGAUUUCUUUUGGUGUUCAG
393
22
1





hsa-miR-3064-5p
UCUGGCUGUUGUGGUGUGCAA
638
21
1





hsa-miR-3116
UGCCUGGAACAUAGUAGGGACU
639
22
1





hsa-miR-3125
UAGAGGAAGCUGUGGAGAGA
640
20
1





hsa-miR-3127-3p
UCCCCUUCUGCAGGCCUGCUGG
641
22
1





hsa-miR-3130-3p
GCUGCACCGGAGACUGGGUAA
395
21
1





hsa-miR-3140-5p
ACCUGAAUUACCAAAAGCUUU
397
21
1





hsa-miR-3157-5p
UUCAGCCAGGCUAGUGCAGUCU
642
22
1





hsa-miR-3179
AGAAGGGGUGAAAUUUAAACGU
643
22
1





hsa-miR-3181
AUCGGGCCCUCGGCGCCGG
644
19
1





hsa-miR-3187-5p
CCUGGGCAGCGUGUGGCUGAAGG
645
23
1





hsa-miR-3190-5p
UCUGGCCAGCUACGUCCCCA
646
20
1





hsa-miR-3198
GUGGAGUCCUGGGGAAUGGAGA
647
22
1





hsa-miR-320b
AAAAGCUGGGUUGAGAGGGCAA
648
22
1





hsa-miR-323b-5p
AGGUUGUCCGUGGUGAGUUCGCA
401
23
1





hsa-miR-3591-5p
UUUAGUGUGAUAAUGGCGUUUGA
649
23
1





hsa-miR-3619-5p
UCAGCAGGCAGGCUGGUGCAGC
650
22
1





hsa-miR-3659
UGAGUGUUGUCUACGAGGGCA
651
21
1





hsa-miR-3674
AUUGUAGAACCUAAGAUUGGCC
652
22
1





hsa-miR-3679-3p
CUUCCCCCCAGUAAUCUUCAUC
653
22
1





hsa-miR-375
UUUGUUCGUUCGGCUCGCGUGA
654
22
1





hsa-miR-378b
ACUGGACUUGGAGGCAGAA
655
19
1





hsa-miR-3908
GAGCAAUGUAGGUAGACUGUUU
656
22
1





hsa-miR-3911
UGUGUGGAUCCUGGAGGAGGCA
657
22
1





hsa-miR-3913-5p
UUUGGGACUGAUCUUGAUGUCU
658
22
1





hsa-miR-3917
GCUCGGACUGAGCAGGUGGG
659
20
1





hsa-miR-3944-3p
UUCGGGCUGGCCUGCUGCUCCGG
660
23
1





hsa-miR-429
UAAUACUGUCUGGUAAAACCGU
661
22
1





hsa-miR-4421
ACCUGUCUGUGGAAAGGAGCUA
718
22
1





hsa-miR-4443
UUGGAGGCGUGGGUUUU
663
17
1





hsa-miR-4459
CCAGGAGGCGGAGGAGGUGGAG
664
22
1





hsa-miR-4473
CUAGUGCUCUCCGUUACAAGUA
665
22
1





hsa-miR-4479
CGCGCGGCCGUGCUCGGAGCAG
666
22
1





hsa-miR-4497
CUCCGGGACGGCUGGGC
232
17
1





hsa-miR-4504
UGUGACAAUAGAGAUGAACAUG
667
22
1





hsa-miR-4520b-3p
UUUGGACAGAAAACACGCAGGU
668
22
1





hsa-miR-452-5p
AACUGUUUGCAGAGGAAACUGA
669
22
1





hsa-miR-4636
AACUCGUGUUCAAAGCCUUUAG
670
22
1





hsa-miR-4659b-3p
UUUCUUCUUAGACAUGGCAGCU
671
22
1





hsa-miR-4664-3p
CUUCCGGUCUGUGAGCCCCGUC
672
22
1





hsa-miR-4665-5p
CUGGGGGACGCGUGAGCGCGAGC
673
23
1





hsa-miR-4666a-5p
AUACAUGUCAGAUUGUAUGCC
674
21
1





hsa-miR-4673
UCCAGGCAGGAGCCGGACUGGA
422
22
1





hsa-miR-4681
AACGGGAAUGCAGGCUGUAUCU
675
22
1





hsa-miR-4682
UCUGAGUUCCUGGAGCCUGGUCU
676
23
1





hsa-miR-4690-5p
GAGCAGGCGAGGCUGGGCUGAA
677
22
1





hsa-miR-4699-5p
AGAAGAUUGCAGAGUAAGUUCC
678
22
1





hsa-miR-4700-3p
CACAGGACUGACUCCUCACCCCAGUG
424
26
1





hsa-miR-4706
AGCGGGGAGGAAGUGGGCGCUGCUU
679
25
1





hsa-miR-4721
UGAGGGCUCCAGGUGACGGUGG
680
22
1





hsa-miR-4728-3p
CAUGCUGACCUCCCUCCUGCCCCAG
681
25
1





hsa-miR-4742-5p
UCAGGCAAAGGGAUAUUUACAGA
682
23
1





hsa-miR-4747-3p
AAGGCCCGGGCUUUCCUCCCAG
683
22
1





hsa-miR-4749-5p
UGCGGGGACAGGCCAGGGCAUC
684
22
1





hsa-miR-4755-3p
AGCCAGGCUCUGAAGGGAAAGU
685
22
1





hsa-miR-4763-5p
CGCCUGCCCAGCCCUCCUGCU
686
21
1





hsa-miR-4766-3p
AUAGCAAUUGCUCUUUUGGAA
687
21
1





hsa-miR-4781-3p
AAUGUUGGAAUCCUCGCUAGAG
688
22
1





hsa-miR-4793-3p
UCUGCACUGUGAGUUGGCUGGCU
689
23
1





hsa-miR-488-3p
UUGAAAGGCUAUUUCUUGGUC
690
21
1





hsa-miR-4999-5p
UGCUGUAUUGUCAGGUAGUGA
691
21
1





hsa-miR-5001-5p
AGGGCUGGACUCAGCGGCGGAGCU
692
24
1





hsa-miR-5002-5p
AAUUUGGUUUCUGAGGCACUUAGU
693
24
1





hsa-miR-5004-5p
UGAGGACAGGGCAAAUUCACGA
694
22
1





hsa-miR-5006-3p
UUUCCCUUUCCAUCCUGGCAG
695
21
1





hsa-miR-5088
CAGGGCUCAGGGAUUGGAUGGAG
696
23
1





hsa-miR-544a
AUUCUGCAUUUUUAGCAAGUUC
697
22
1





hsa-miR-548al
AACGGCAAUGACUUUUGUACCA
698
22
1





hsa-miR-548aq-5p
GAAAGUAAUUGCUGUUUUUGCC
699
22
1





hsa-miR-548at-5p
AAAAGUUAUUGCGGUUUUGGCU
700
22
1





hsa-miR-548au-5p
AAAAGUAAUUGCGGUUUUUGC
701
21
1





hsa-miR-548b-3p
CAAGAACCUCAGUUGCUUUUGU
702
22
1





hsa-miR-556-3p
AUAUUACCAUUAGCUCAUCUUU
703
22
1





hsa-miR-5582-3p
UAAAACUUUAAGUGUGCCUAGG
704
22
1





hsa-miR-5586-3p
CAGAGUGACAAGCUGGUUAAAG
705
22
1





hsa-miR-5588-5p
ACUGGCAUUAGUGGGACUUUU
706
21
1





hsa-miR-5683
UACAGAUGCAGAUUCUCUGACUUC
707
24
1





hsa-miR-5696
CUCAUUUAAGUAGUCUGAUGCC
708
22
1





hsa-miR-5701
UUAUUGUCACGUUCUGAUU
709
19
1





hsa-miR-5706
UUCUGGAUAACAUGCUGAAGCU
710
22
1





hsa-miR-592
UUGUGUCAAUAUGCGAUGAUGU
711
22
1





hsa-miR-603
CACACACUGCAAUUACUUUUGC
712
22
1





hsa-miR-624-3p
CACAAGGUAUUGGUAUUACCU
713
21
1





hsa-miR-885-5p
UCCAUUACACUACCCUGCCUCU
714
22
1





hsa-miR-933
UGUGCGCAGGGAGACCUCUCCC
715
22
1
















TABLE 6







Microvesicles EI











MICROVESICLES






CTX0E0307EI

SEQ ID
MIRNA
READ


MIRNA
MIRNA.SEQUENCE
NO:
LENGTH
COUNTS














hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
21
19
32723





hsa-miR-4492
GGGGCUGGGCGCGCGCC
34
17
16225





hsa-miR-4488
AGGGGGCGGGCUCCGGCG
61
18
12878





hsa-miR-4532
CCCCGGGGAGCCCGGCG
23
17
6746





hsa-miR-4508
GCGGGGCUGGGCGCGCG
135
17
531





hsa-miR-4516
GGGAGAAGGGUCGGGGC
110
17
500





hsa-miR-3676-5p
AGGAGAUCCUGGGUU
280
15
357





hsa-miR-4485
UAACGGCCGCGGUACCCUAA
67
20
44





hsa-miR-4497
CUCCGGGACGGCUGGGC
232
17
43





hsa-miR-21-5p
UAGCUUAUCAGACUGAUGUUGA
9
22
33





hsa-miR-3195
CGCGCCGGGCCCGGGUU
716
17
28





hsa-miR-3648
AGCCGCGGGGAUCGCCGAGGG
259
21
26





hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
180
22
24





hsa-miR-3656
GGCGGGUGCGGGGGUGG
251
17
19





hsa-miR-3687
CCCGGACAGGCGUUCGUGCGACGU
190
24
19





hsa-miR-4466
GGGUGCGGGCCGGCGGGG
264
18
19





hsa-miR-4792
CGGUGAGCGCUCGCUGGC
363
18
19





hsa-miR-99b-5p
CACCCGUAGAACCGACCUUGCG
4
22
18





hsa-let-7a-5p
UGAGGUAGUAGGUUGUAUAGUU
1
22
15





hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
375
19
7





hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
294
24
7





hsa-miR-182-5p
UUUGGCAAUGGUAGAACUCACACU
16
24
7





hsa-miR-5096
GUUUCACCAUGUUGGUCAGGC
220
21
7





hsa-miR-1273f
GGAGAUGGAGGUUGCAGUG
292
19
5





hsa-miR-26a-5p
UUCAAGUAAUCCAGGAUAGGCU
12
22
5





hsa-miR-4284
GGGCUCACAUCACCCCAU
191
18
5





hsa-miR-92b-3p
UAUUGCACUCGUCCCGGCCUCC
13
22
5





hsa-let-7b-5p
UGAGGUAGUAGGUUGUGUGGUU
28
22
4





hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
17
22
4





hsa-let-7f-5p
UGAGGUAGUAGAUUGUAUAGUU
11
22
4





hsa-miR-100-5p
AACCCGUAGAUCCGAACUUGUG
3
22
4





hsa-miR-1248
ACCUUCUUGUAUAAGCACUGUGCUAAA
269
27
4





hsa-miR-1973
ACCGUGCAAAGGUAGCAUA
171
19
4





hsa-miR-21-3p
CAACACCAGUCGAUGGGCUGU
20
21
4





hsa-miR-3654
GACUGGACAAGCUGAGGAA
325
19
4





hsa-miR-92a-3p
UAUUGCACUUGUCCCGGCCUGU
7
22
4





hsa-miR-1273g-3p
ACCACUGCACUCCAGCCUGAG
210
21
3





hsa-miR-23b-3p
AUCACAUUGCCAGGGAUUACC
59
21
3





hsa-miR-3609
CAAAGUGAUGAGUAAUACUGGCUG
216
24
3





hsa-miR-3615
UCUCUCGGCUCCUCGCGGCUC
323
21
3





hsa-miR-3653
CUAAGAAGUUGACUGAAG
544
18
3





hsa-miR-3960
GGCGGCGGCGGAGGCGGGGG
416
20
3





hsa-miR-4448
GGCUCCUUGGUCUAGGGGUA
231
20
3





hsa-let-7d-5p
AGAGGUAGUAGGUUGCAUAGUU
92
22
2





hsa-miR-16-5p
UAGCAGCACGUAAAUAUUGGCG
29
22
2





hsa-miR-181a-5p
AACAUUCAACGCUGUCGGUGAGU
15
23
2





hsa-miR-181b-5p
AACAUUCAUUGCUGUCGGUGGGU
38
23
2





hsa-miR-222-3p
AGCUACAUCUGGCUACUGGGU
36
21
2





hsa-miR-24-3p
UGGCUCAGUUCAGCAGGAACAG
119
22
2





hsa-miR-3196
CGGGGCGGCAGGGGCCUC
717
18
2





hsa-miR-4419b
GAGGCUGAAGGAAGAUGG
718
18
2





hsa-miR-4461
GAUUGAGACUAGUAGGGCUAGGC
129
23
2





hsa-miR-4486
GCUGGGCGAGGCUGGCA
719
17
2





hsa-miR-663a
AGGCGGGGCGCCGCGGGACCGC
365
22
2





hsa-miR-9-5p
UCUUUGGUUAUCUAGCUGUAUGA
58
23
2





hsa-let-7i-3p
CUGCGCAAGCUACUGCCUUGCU
483
22
1





hsa-let-7i-5p
UGAGGUAGUAGUUUGUGCUGUU
22
22
1





hsa-miR-1225-5p
GUGGGUACGGCCCAGUGGGGGG
720
22
1





hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
340
26
1





hsa-miR-125b-5p
UCCCUGAGACCCUAACUUGUGA
42
22
1





hsa-miR-1275
GUGGGGGAGAGGCUGUC
162
17
1





hsa-miR-1280
UCCCACCGCUGCCACCC
584
17
1





hsa-miR-134
UGUGACUGGUUGACCAGAGGGG
94
22
1





hsa-miR-149-5p
UCUGGCUCCGUGUCUUCACUCCC
121
23
1





hsa-miR-191-5p
CAACGGAAUCCCAAAAGCAGCUG
8
23
1





hsa-miR-221-3p
AGCUACAUUGUCUGCUGGGUUUC
79
23
1





hsa-miR-22-3p
AAGCUGCCAGUUGAAGAACUGU
33
22
1





hsa-miR-26b-5p
UUCAAGUAAUUCAGGAUAGGU
90
21
1





hsa-miR-30c-5p
UGUAAACAUCCUACACUCUCAGC
66
23
1





hsa-miR-30d-5p
UGUAAACAUCCCCGACUGGAAG
31
22
1





hsa-miR-3182
GCUUCUGUAGUGUAGUC
721
17
1





hsa-miR-320a
AAAAGCUGGGUUGAGAGGGCGA
97
22
1





hsa-miR-34a-5p
UGGCAGUGUCUUAGCUGGUUGU
101
22
1





hsa-miR-3607-3p
ACUGUAAACGCUUUCUGAUG
543
20
1





hsa-miR-361-5p
UUAUCAGAAUCUCCAGGGGUAC
70
22
1





hsa-miR-3652
CGGCUGGAGGUGUGAGGA
722
18
1





hsa-miR-409-3p
GAAUGUUGCUCGGUGAACCCCU
47
22
1





hsa-miR-423-3p
AGCUCGGUCUGAGGCCCCUCAGU
57
23
1





hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
41
23
1





hsa-miR-432-5p
UCUUGGAGUAGGUCAUUGGGUGG
95
23
1





hsa-miR-4417
GGUGGGCUUCCCGGAGGG
175
18
1





hsa-miR-4426
GAAGAUGGACGUACUUU
359
17
1





hsa-miR-4449
CGUCCCGGGGCUGCGCGAGGCA
155
22
1





hsa-miR-4800-3p
CAUCCGUCCGUCUGUCCAC
549
19
1





hsa-miR-484
UCAGGCUCAGUCCCCUCCCGAU
118
22
1





hsa-miR-486-5p
UCCUGUACUGAGCUGCCCCGAG
5
22
1





hsa-miR-493-3p
UGAAGGUCUACUGUGUGCCAGG
83
22
1





hsa-miR-5095
UUACAGGCGUGAACCACCGCG
723
21
1





hsa-miR-556-3p
AUAUUACCAUUAGCUCAUCUUU
703
22
1





hsa-miR-644b-5p
UGGGCUAAGGGAGAUGAUUGGGUA
724
24
1





hsa-miR-664-5p
ACUGGCUAGGGAAAAUGAUUGGAU
443
24
1





hsa-miR-760
CGGCUCUGGGUCUGUGGGGA
289
20
1





hsa-miR-941
CACCCGGCUGUGUGCACAUGUGC
60
23
1





hsa-miR-98
UGAGGUAGUAAGUUGUAUUGUU
10
22
1





hsa-miR-99a-5p
AACCCGUAGAUCCGAUCUUGUG
52
22
1
















TABLE 7







Exosomes EI











EXOSOMES






CTX0E03 07EI

SEQ ID
MIRNA
READ


MIRNA
MIRNA.SEQUENCE
NO:
LENGTH
COUNTS














hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
21
19
83958





hsa-miR-4492
GGGGCUGGGCGCGCGCC
34
17
22482





hsa-miR-4488
AGGGGGCGGGCUCCGGCG
61
18
20618





hsa-miR-4532
CCCCGGGGAGCCCGGCG
23
17
6419





hsa-miR-4516
GGGAGAAGGGUCGGGGC
110
17
904





hsa-miR-4508
GCGGGGCUGGGCGCGCG
135
17
723





hsa-miR-3676-5p
AGGAGAUCCUGGGUU
280
15
174





hsa-miR-4485
UAACGGCCGCGGUACCCUAA
67
20
43





hsa-miR-21-5p
UAGCUUAUCAGACUGAUGUUGA
9
22
41





hsa-miR-4497
CUCCGGGACGGCUGGGC
232
17
28





hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
180
22
26





hsa-miR-4792
CGGUGAGCGCUCGCUGGC
363
18
24





hsa-miR-4454
GGAUCCGAGUCACGGCACCA
299
20
22





hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
294
24
17





hsa-miR-26a-5p
UUCAAGUAAUCCAGGAUAGGCU
12
22
17





hsa-miR-3195
CGCGCCGGGCCCGGGUU
716
17
17





hsa-let-7a-5p
UGAGGUAGUAGGUUGUAUAGUU
1
22
15





hsa-miR-182-5p
UUUGGCAAUGGUAGAACUCACACU
16
24
15





hsa-miR-99b-5p
CACCCGUAGAACCGACCUUGCG
4
22
15





hsa-miR-5096
GUUUCACCAUGUUGGUCAGGC
220
21
14





hsa-miR-3648
AGCCGCGGGGAUCGCCGAGGG
259
21
13





hsa-miR-3654
GACUGGACAAGCUGAGGAA
325
19
13





hsa-miR-4466
GGGUGCGGGCCGGCGGGG
264
18
12





hsa-miR-3687
CCCGGACAGGCGUUCGUGCGACGU
190
24
11





hsa-miR-4284
GGGCUCACAUCACCCCAU
191
18
11





hsa-miR-3656
GGCGGGUGCGGGGGUGG
251
17
10





hsa-miR-3609
CAAAGUGAUGAGUAAUACUGGCUG
216
24
8





hsa-miR-644b-5p
UGGGCUAAGGGAGAUGAUUGGGUA
724
24
8





hsa-miR-664-5p
ACUGGCUAGGGAAAAUGAUUGGAU
443
24
8





hsa-miR-92a-3p
UAUUGCACUUGUCCCGGCCUGU
7
22
7





hsa-miR-92b-3p
UAUUGCACUCGUCCCGGCCUCC
13
22
7





hsa-let-7b-5p
UGAGGUAGUAGGUUGUGUGGUU
28
22
6





hsa-let-7f-5p
UGAGGUAGUAGAUUGUAUAGUU
11
22
6





hsa-miR-127-3p
UCGGAUCCGUCUGAGCUUGGCU
14
22
6





hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
375
19
6





hsa-miR-4449
CGUCCCGGGGCUGCGCGAGGCA
155
22
6





hsa-miR-4461
GAUUGAGACUAGUAGGGCUAGGC
129
23
6





hsa-miR-100-5p
AACCCGUAGAUCCGAACUUGUG
3
22
5





hsa-miR-1248
ACCUUCUUGUAUAAGCACUGUGCUAAA
269
27
5





hsa-miR-1973
ACCGUGCAAAGGUAGCAUA
171
19
5





hsa-miR-3653
CUAAGAAGUUGACUGAAG
544
18
5





hsa-miR-4417
GGUGGGCUUCCCGGAGGG
175
18
5





hsa-miR-125b-5p
UCCCUGAGACCCUAACUUGUGA
42
22
4





hsa-miR-151a-3p
CUAGACUGAAGCUCCUUGAGG
25
21
4





hsa-miR-16-5p
UAGCAGCACGUAAAUAUUGGCG
29
22
4





hsa-miR-21-3p
CAACACCAGUCGAUGGGCUGU
20
21
4





hsa-miR-23a-3p
AUCACAUUGCCAGGGAUUUCC
55
21
4





hsa-miR-4419b
GAGGCUGAAGGAAGAUGG
718
18
4





hsa-miR-1273f
GGAGAUGGAGGUUGCAGUG
292
19
3





hsa-miR-1273g-3p
ACCACUGCACUCCAGCCUGAG
210
21
3





hsa-miR-181b-5p
AACAUUCAUUGCUGUCGGUGGGU
38
23
3





hsa-miR-221-3p
AGCUACAUUGUCUGCUGGGUUUC
79
23
3





hsa-miR-3615
UCUCUCGGCUCCUCGCGGCUC
323
21
3





hsa-miR-9-5p
UCUUUGGUUAUCUAGCUGUAUGA
58
23
3





hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
17
22
2





hsa-let-7e-5p
UGAGGUAGGAGGUUGUAUAGUU
27
22
2





hsa-let-7i-5p
UGAGGUAGUAGUUUGUGCUGUU
22
22
2





hsa-miR-103a-3p
AGCAGCAUUGUACAGGGCUAUGA
62
23
2





hsa-miR-106b-5p
UAAAGUGCUGACAGUGCAGAU
170
21
2





hsa-miR-1273e
UUGCUUGAACCCAGGAAGUGGA
342
22
2





hsa-miR-221-5p
ACCUGGCAUACAAUGUAGAUUU
39
22
2





hsa-miR-222-3p
AGCUACAUCUGGCUACUGGGU
36
21
2





hsa-miR-30d-5p
UGUAAACAUCCCCGACUGGAAG
31
22
2





hsa-miR-3960
GGCGGCGGCGGAGGCGGGGG
416
20
2





hsa-let-7d-3p
CUAUACGACCUGCUGCCUUUCU
92
22
1





hsa-let-7d-5p
AGAGGUAGUAGGUUGCAUAGUU
53
22
1





hsa-let-7g-5p
UGAGGUAGUAGUUUGUACAGUU
43
22
1





hsa-let-7i-3p
CUGCGCAAGCUACUGCCUUGCU
483
22
1





hsa-miR-10a-5p
UACCCUGUAGAUCCGAAUUUGUG
2
23
1





hsa-miR-1181
CCGUCGCCGCCACCCGAGCCG
725
21
1





hsa-miR-1225-3p
UGAGCCCCUGUGCCGCCCCCAG
369
22
1





hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
340
26
1





hsa-miR-125a-5p
UCCCUGAGACCCUUUAACCUGUGA
35
24
1





hsa-miR-1296
UUAGGGCCCUGGCUCCAUCUCC
271
22
1





hsa-miR-1307-5p
UCGACCGGACCUCGACCGGCU
91
21
1





hsa-miR-146b-5p
UGAGAACUGAAUUCCAUAGGCU
19
22
1





hsa-miR-149-5p
UCUGGCUCCGUGUCUUCACUCCC
121
23
1





hsa-miR-151a-5p
UCGAGGAGCUCACAGUCUAGU
37
21
1





hsa-miR-15b-5p
UAGCAGCACAUCAUGGUUUACA
78
22
1





hsa-miR-181a-2-3p
ACCACUGACCGUUGACUGUACC
102
22
1





hsa-miR-181a-5p
AACAUUCAACGCUGUCGGUGAGU
15
23
1





hsa-miR-191-5p
CAACGGAAUCCCAAAAGCAGCUG
8
23
1





hsa-miR-198
GGUCCAGAGGGGAGAUAGGUUC
726
22
1





hsa-miR-204-5p
UUCCCUUUGUCAUCCUAUGCCU
89
22
1





hsa-miR-20a-5p
UAAAGUGCUUAUAGUGCAGGUAG
146
23
1





hsa-miR-219-5p
UGAUUGUCCAAACGCAAUUCU
527
21
1





hsa-miR-22-3p
AAGCUGCCAGUUGAAGAACUGU
33
22
1





hsa-miR-23b-3p
AUCACAUUGCCAGGGAUUACC
59
21
1





hsa-miR-26b-3p
CCUGUUCUCCAUUACUUGGCUC
391
22
1





hsa-miR-299-5p
UGGUUUACCGUCCCACAUACAU
319
22
1





hsa-miR-29a-3p
UAGCACCAUCUGAAAUCGGUUA
106
22
1





hsa-miR-30e-3p
CUUUCAGUCGGAUGUUUACAGC
71
22
1





hsa-miR-31-3p
UGCUAUGCCAACAUAUUGCCAU
172
22
1





hsa-miR-3198
GUGGAGUCCUGGGGAAUGGAGA
647
22
1





hsa-miR-323a-3p
CACAUUACACGGUCGACCUCU
158
21
1





hsa-miR-342-3p
UCUCACACAGAAAUCGCACCCGU
81
23
1





hsa-miR-3607-3p
ACUGUAAACGCUUUCUGAUG
543
20
1





hsa-miR-3651
CAUAGCCCGGUCGCUGGUACAUGA
727
24
1





hsa-miR-378a-3p
ACUGGACUUGGAGUCAGAAGG
65
21
1





hsa-miR-379-5p
UGGUAGACUAUGGAACGUAGG
18
21
1





hsa-miR-423-3p
AGCUCGGUCUGAGGCCCCUCAGU
57
23
1





hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
41
23
1





hsa-miR-425-5p
AAUGACACGAUCACUCCCGUUGA
111
23
1





hsa-miR-4258
CCCCGCCACCGCCUUGG
728
17
1





hsa-miR-4426
GAAGAUGGACGUACUUU
359
17
1





hsa-miR-4443
UUGGAGGCGUGGGUUUU
663
17
1





hsa-miR-4448
GGCUCCUUGGUCUAGGGGUA
231
20
1





hsa-miR-4697-3p
UGUCAGUGACUCCUGCCCCUUGGU
729
24
1





hsa-miR-4700-3p
CACAGGACUGACUCCUCACCCCAGUG
424
26
1





hsa-miR-4700-5p
UCUGGGGAUGAGGACAGUGUGU
730
22
1





hsa-miR-4797-3p
UCUCAGUAAGUGGCACUCUGU
731
21
1





hsa-miR-484
UCAGGCUCAGUCCCCUCCCGAU
118
22
1





hsa-miR-486-5p
UCCUGUACUGAGCUGCCCCGAG
5
22
1





hsa-miR-494
UGAAACAUACACGGGAAACCUC
240
22
1





hsa-miR-500a-5p
UAAUCCUUGCUACCUGGGUGAGA
303
23
1





hsa-miR-644b-3p
UUCAUUUGCCUCCCAGCCUACA
442
22
1





hsa-miR-663a
AGGCGGGGCGCCGCGGGACCGC
365
22
1
















TABLE 8







Microvesicles EH











MICROVESICLES






CTX0E03 07EH

SEQ ID
MIRNA
READ


MIRNA
MIRNA.SEQUENCE
NO:
LENGTH
COUNTS














hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
21
19
78791





hsa-miR-4492
GGGGCUGGGCGCGCGCC
34
17
6012





hsa-miR-4532
CCCCGGGGAGCCCGGCG
23
17
3410





hsa-miR-4488
AGGGGGCGGGCUCCGGCG
61
18
1737





hsa-miR-4485
UAACGGCCGCGGUACCCUAA
67
20
319





hsa-miR-4508
GCGGGGCUGGGCGCGCG
135
17
221





hsa-miR-4516
GGGAGAAGGGUCGGGGC
110
17
114





hsa-miR-4466
GGGUGCGGGCCGGCGGGG
264
18
61





hsa-miR-4497
CUCCGGGACGGCUGGGC
232
17
51





hsa-miR-3195
CGCGCCGGGCCCGGGUU
716
17
41





hsa-miR-1973
ACCGUGCAAAGGUAGCAUA
171
19
30





hsa-miR-21-5p
UAGCUUAUCAGACUGAUGUUGA
9
22
22





hsa-miR-4284
GGGCUCACAUCACCCCAU
191
18
20





hsa-miR-4792
CGGUGAGCGCUCGCUGGC
363
18
12





hsa-miR-92a-3p
UAUUGCACUUGUCCCGGCCUGU
7
22
12





hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
294
24
11





hsa-miR-3676-5p
AGGAGAUCCUGGGUU
280
15
10





hsa-miR-100-5p
AACCCGUAGAUCCGAACUUGUG
3
22
8





hsa-miR-3656
GGCGGGUGCGGGGGUGG
251
17
8





hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
180
22
8





hsa-let-7a-5p
UGAGGUAGUAGGUUGUAUAGUU
1
22
7





hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
375
19
7





hsa-miR-3687
CCCGGACAGGCGUUCGUGCGACGU
190
24
7





hsa-miR-4461
GAUUGAGACUAGUAGGGCUAGGC
52
23
6





hsa-miR-664-5p
ACUGGCUAGGGAAAAUGAUUGGAU
91
24
6





hsa-miR-92b-3p
UAUUGCACUCGUCCCGGCCUCC
13
22
6





hsa-miR-125b-5p
UCCCUGAGACCCUAACUUGUGA
42
22
5





hsa-miR-3653
CUAAGAAGUUGACUGAAG
544
18
5





hsa-let-7f-5p
UGAGGUAGUAGAUUGUAUAGUU
11
22
4





hsa-miR-16-5p
UAGCAGCACGUAAAUAUUGGCG
29
22
4





hsa-miR-181a-5p
AACAUUCAACGCUGUCGGUGAGU
15
23
4





hsa-miR-3609
CAAAGUGAUGAGUAAUACUGGCUG
216
24
4





hsa-miR-9-5p
UCUUUGGUUAUCUAGCUGUAUGA
58
23
4





hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
17
22
3





hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
59
26
3





hsa-miR-127-3p
UCGGAUCCGUCUGAGCUUGGCU
14
22
3





hsa-miR-181b-5p
AACAUUCAUUGCUGUCGGUGGGU
38
23
3





hsa-miR-21-3p
CAACACCAGUCGAUGGGCUGU
20
21
3





hsa-miR-26a-5p
UUCAAGUAAUCCAGGAUAGGCU
12
22
3





hsa-miR-30c-5p
UGUAAACAUCCUACACUCUCAGC
66
23
3





hsa-miR-3960
GGCGGCGGCGGAGGCGGGGG
416
20
3





hsa-miR-485-3p
GUCAUACACGGCUCUCCUCUCU
153
22
3





hsa-let-7b-5p
UGAGGUAGUAGGUUGUGUGGUU
28
22
2





hsa-let-7g-5p
UGAGGUAGUAGUUUGUACAGUU
43
22
2





hsa-miR-1273f
GGAGAUGGAGGUUGCAGUG
292
19
2





hsa-miR-151a-3p
CUAGACUGAAGCUCCUUGAGG
25
21
2





hsa-miR-182-5p
UUUGGCAAUGGUAGAACUCACACU
16
24
2





hsa-miR-191-5p
CAACGGAAUCCCAAAAGCAGCUG
8
23
2





hsa-miR-197-3p
UUCACCACCUUCUCCACCCAGC
122
22
2





hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
41
23
2





hsa-miR-4468
AGAGCAGAAGGAUGAGAU
732
18
2





hsa-miR-644b-5p
UGGGCUAAGGGAGAUGAUUGGGUA
724
24
2





hsa-miR-93-5p
CAAAGUGCUGUUCGUGCAGGUAG
116
23
2





hsa-let-7d-5p
AGAGGUAGUAGGUUGCAUAGUU
92
22
1





hsa-miR-1225-3p
UGAGCCCCUGUGCCGCCCCCAG
369
22
1





hsa-miR-1254
AGCCUGGAAGCUGGAGCCUGCAGU
270
24
1





hsa-miR-1273g-3p
ACCACUGCACUCCAGCCUGAG
210
21
1





hsa-miR-1275
GUGGGGGAGAGGCUGUC
162
17
1





hsa-miR-1296
UUAGGGCCCUGGCUCCAUCUCC
271
22
1





hsa-miR-1307-5p
UCGACCGGACCUCGACCGGCU
91
21
1





hsa-miR-134
UGUGACUGGUUGACCAGAGGGG
94
22
1





hsa-miR-15b-5p
UAGCAGCACAUCAUGGUUUACA
78
22
1





hsa-miR-17-5p
CAAAGUGCUUACAGUGCAGGUAG
145
23
1





hsa-miR-1972
UCAGGCCAGGCACAGUGGCUCA
733
22
1





hsa-miR-22-3p
AAGCUGCCAGUUGAAGAACUGU
33
22
1





hsa-miR-25-3p
CAUUGCACUUGUCUCGGUCUGA
63
22
1





hsa-miR-27b-3p
UUCACAGUGGCUAAGUUCUGC
6
21
1





hsa-miR-3065-5p
UCAACAAAAUCACUGAUGCUGGA
226
23
1





hsa-miR-30d-5p
UGUAAACAUCCCCGACUGGAAG
31
22
1





hsa-miR-320a
AAAAGCUGGGUUGAGAGGGCGA
97
22
1





hsa-miR-342-3p
UCUCACACAGAAAUCGCACCCGU
81
23
1





hsa-miR-3648
AGCCGCGGGGAUCGCCGAGGG
259
21
1





hsa-miR-3652
CGGCUGGAGGUGUGAGGA
722
18
1





hsa-miR-376c
AACAUAGAGGAAAUUCCACGU
185
21
1





hsa-miR-378a-3p
ACUGGACUUGGAGUCAGAAGG
65
21
1





hsa-miR-409-3p
GAAUGUUGCUCGGUGAACCCCU
47
22
1





hsa-miR-433
AUCAUGAUGGGCUCCUCGGUGU
174
22
1





hsa-miR-4417
GGUGGGCUUCCCGGAGGG
175
18
1





hsa-miR-4448
GGCUCCUUGGUCUAGGGGUA
231
20
1





hsa-miR-4454
GGAUCCGAGUCACGGCACCA
299
20
1





hsa-miR-454-3p
UAGUGCAAUAUUGCUUAUAGGGU
169
23
1





hsa-miR-4800-3p
CAUCCGUCCGUCUGUCCAC
549
19
1





hsa-miR-493-3p
UGAAGGUCUACUGUGUGCCAGG
83
22
1





hsa-miR-5095
UUACAGGCGUGAACCACCGCG
723
21
1





hsa-miR-574-3p
CACGCUCAUGCACACACCCACA
253
22
1





hsa-miR-665
ACCAGGAGGCUGAGGCCCCU
309
20
1





hsa-miR-720
UCUCGCUGGGGCCUCCA
84
17
1





hsa-miR-99a-5p
AACCCGUAGAUCCGAUCUUGUG
52
22
1





hsa-miR-99b-5p
CACCCGUAGAACCGACCUUGCG
4
22
1
















TABLE 9







Exosomes EH











EXOSOMES






CTX0E03 07EH

SEQ ID
MIRNA
READ


MIRNA
MIRNA.SEQUENCE
NO:
LENGTH
COUNTS














hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
21
19
111092





hsa-miR-4492
GGGGCUGGGCGCGCGCC
34
17
5188





hsa-miR-4532
CCCCGGGGAGCCCGGCG
23
17
3368





hsa-miR-4488
AGGGGGCGGGCUCCGGCG
61
18
1389





hsa-miR-4485
UAACGGCCGCGGUACCCUAA
67
20
386





hsa-miR-4508
GCGGGGCUGGGCGCGCG
135
17
188





hsa-miR-4516
GGGAGAAGGGUCGGGGC
110
17
135





hsa-miR-4497
CUCCGGGACGGCUGGGC
232
17
73





hsa-miR-1973
ACCGUGCAAAGGUAGCAUA
171
19
50





hsa-miR-3195
CGCGCCGGGCCCGGGUU
716
17
48





hsa-miR-4466
GGGUGCGGGCCGGCGGGG
264
18
43





hsa-let-7a-5p
UGAGGUAGUAGGUUGUAUAGUU
1
22
20





hsa-miR-99b-5p
CACCCGUAGAACCGACCUUGCG
4
22
19





hsa-miR-21-5p
UAGCUUAUCAGACUGAUGUUGA
9
22
18





hsa-miR-92a-3p
UAUUGCACUUGUCCCGGCCUGU
7
22
18





hsa-miR-3676-5p
AGGAGAUCCUGGGUU
280
15
17





hsa-miR-4792
CGGUGAGCGCUCGCUGGC
363
18
15





hsa-miR-664-5p
ACUGGCUAGGGAAAAUGAUUGGAU
443
24
13





hsa-miR-100-5p
AACCCGUAGAUCCGAACUUGUG
3
22
11





hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
294
24
10





hsa-miR-16-5p
UAGCAGCACGUAAAUAUUGGCG
29
22
10





hsa-miR-4284
GGGCUCACAUCACCCCAU
191
18
10





hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
180
22
9





hsa-miR-25-3p
CAUUGCACUUGUCUCGGUCUGA
63
22
8





hsa-miR-3656
GGCGGGUGCGGGGGUGG
251
17
8





hsa-miR-181a-5p
AACAUUCAACGCUGUCGGUGAGU
15
23
7





hsa-miR-26a-5p
UUCAAGUAAUCCAGGAUAGGCU
12
22
6





hsa-miR-3654
GACUGGACAAGCUGAGGAA
325
19
6





hsa-miR-644b-5p
UGGGCUAAGGGAGAUGAUUGGGUA
724
24
6





hsa-let-7b-5p
UGAGGUAGUAGGUUGUGUGGUU
28
22
5





hsa-let-7f-5p
UGAGGUAGUAGAUUGUAUAGUU
11
22
5





hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
375
19
5





hsa-miR-4426
GAAGAUGGACGUACUUU
359
17
5





hsa-miR-5096
GUUUCACCAUGUUGGUCAGGC
220
21
5





hsa-miR-125b-5p
UCCCUGAGACCCUAACUUGUGA
42
22
4





hsa-miR-1273f
GGAGAUGGAGGUUGCAGUG
292
19
4





hsa-miR-191-5p
CAACGGAAUCCCAAAAGCAGCUG
8
23
4





hsa-miR-22-3p
AAGCUGCCAGUUGAAGAACUGU
33
22
4





hsa-miR-3609
CAAAGUGAUGAGUAAUACUGGCUG
216
24
4





hsa-miR-3687
CCCGGACAGGCGUUCGUGCGACGU
190
24
4





hsa-miR-93-5p
CAAAGUGCUGUUCGUGCAGGUAG
116
23
4





hsa-miR-1248
ACCUUCUUGUAUAAGCACUGUGCUAAA
269
27
3





hsa-miR-1273g-3p
ACCACUGCACUCCAGCCUGAG
210
21
3





hsa-miR-151a-3p
CUAGACUGAAGCUCCUUGAGG
25
21
3





hsa-miR-182-5p
UUUGGCAAUGGUAGAACUCACACU
16
24
3





hsa-miR-221-3p
AGCUACAUUGUCUGCUGGGUUUC
79
23
3





hsa-miR-222-3p
AGCUACAUCUGGCUACUGGGU
36
21
3





hsa-miR-29a-3p
UAGCACCAUCUGAAAUCGGUUA
106
22
3





hsa-miR-4461
GAUUGAGACUAGUAGGGCUAGGC
129
23
3





hsa-miR-486-5p
UCCUGUACUGAGCUGCCCCGAG
5
22
3





hsa-miR-92b-3p
UAUUGCACUCGUCCCGGCCUCC
13
22
3





hsa-miR-9-5p
UCUUUGGUUAUCUAGCUGUAUGA
58
23
3





hsa-miR-98
UGAGGUAGUAAGUUGUAUUGUU
10
22
3





hsa-let-7d-5p
AGAGGUAGUAGGUUGCAUAGUU
53
22
2





hsa-miR-134
UGUGACUGGUUGACCAGAGGGG
94
22
2





hsa-miR-151a-5p
UCGAGGAGCUCACAGUCUAGU
37
21
2





hsa-miR-15b-5p
UAGCAGCACAUCAUGGUUUACA
78
22
2





hsa-miR-30a-5p
UGUAAACAUCCUCGACUGGAAG
30
22
2





hsa-miR-3124-3p
ACUUUCCUCACUCCCGUGAAGU
734
22
2





hsa-miR-3653
CUAAGAAGUUGACUGAAG
544
18
2





hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
17
22
1





hsa-let-7d-3p
CUAUACGACCUGCUGCCUUUCU
92
22
1





hsa-let-7g-5p
UGAGGUAGUAGUUUGUACAGUU
43
22
1





hsa-let-7i-5p
UGAGGUAGUAGUUUGUGCUGUU
22
22
1





hsa-miR-103a-3p
AGCAGCAUUGUACAGGGCUAUGA
62
23
1





hsa-miR-106b-5p
UAAAGUGCUGACAGUGCAGAU
170
21
1





hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
340
26
1





hsa-miR-128
UCACAGUGAACCGGUCUCUUU
109
21
1





hsa-miR-1285-3p
UCUGGGCAACAAAGUGAGACCU
464
22
1





hsa-miR-1307-3p
ACUCGGCGUGGCGUCGGUCGUG
124
22
1





hsa-miR-140-3p
UACCACAGGGUAGAACCACGG
138
21
1





hsa-miR-148b-3p
UCAGUGCAUCACAGAACUUUGU
48
22
1





hsa-miR-181b-5p
AACAUUCAUUGCUGUCGGUGGGU
38
23
1





hsa-miR-193a-3p
AACUGGCCUACAAAGUCCCAGU
386
22
1





hsa-miR-1972
UCAGGCCAGGCACAGUGGCUCA
733
22
1





hsa-miR-21-3p
CAACACCAGUCGAUGGGCUGU
20
21
1





hsa-miR-2277-3p
UGACAGCGCCCUGCCUGGCUC
735
21
1





hsa-miR-23a-3p
AUCACAUUGCCAGGGAUUUCC
55
21
1





hsa-miR-23b-3p
AUCACAUUGCCAGGGAUUACC
59
21
1





hsa-miR-24-3p
UGGCUCAGUUCAGCAGGAACAG
119
22
1





hsa-miR-27a-3p
UUCACAGUGGCUAAGUUCCGC
46
21
1





hsa-miR-27b-3p
UUCACAGUGGCUAAGUUCUGC
6
21
1





hsa-miR-299-3p
UAUGUGGGAUGGUAAACCGCUU
182
22
1





hsa-miR-30b-5p
UGUAAACAUCCUACACUCAGCU
96
22
1





hsa-miR-30c-5p
UGUAAACAUCCUACACUCUCAGC
66
23
1





hsa-miR-31-3p
UGCUAUGCCAACAUAUUGCCAU
172
22
1





hsa-miR-3196
CGGGGCGGCAGGGGCCUC
717
18
1





hsa-miR-3198
GUGGAGUCCUGGGGAAUGGAGA
647
22
1





hsa-miR-320a
AAAAGCUGGGUUGAGAGGGCGA
97
22
1





hsa-miR-329
AACACACCUGGUUAACCUCUUU
214
22
1





hsa-miR-339-5p
UCCCUGUCCUCCAGGAGCUCACG
402
23
1





hsa-miR-34a-5p
UGGCAGUGUCUUAGCUGGUUGU
101
22
1





hsa-miR-3607-5p
GCAUGUGAUGAAGCAAAUCAGU
249
22
1





hsa-miR-3648
AGCCGCGGGGAUCGCCGAGGG
259
21
1





hsa-miR-376c
AACAUAGAGGAAAUUCCACGU
185
21
1





hsa-miR-3960
GGCGGCGGCGGAGGCGGGGG
416
20
1





hsa-miR-411-3p
UAUGUAACACGGUCCACUAACC
482
22
1





hsa-miR-423-3p
AGCUCGGUCUGAGGCCCCUCAGU
57
23
1





hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
41
23
1





hsa-miR-4417
GGUGGGCUUCCCGGAGGG
175
18
1





hsa-miR-4444
CUCGAGUUGGAAGAGGCG
418
18
1





hsa-miR-4499
AAGACUGAGAGGAGGGA
736
17
1





hsa-miR-4521
GCUAAGGAAGUCCUGUGCUCAG
233
22
1





hsa-miR-4680-5p
AGAACUCUUGCAGUCUUAGAUGU
737
23
1





hsa-miR-4709-5p
ACAACAGUGACUUGCUCUCCAA
575
22
1





hsa-miR-501-3p
AAUGCACCCGGGCAAGGAUUCU
26
22
1





hsa-miR-644b-3p
UUCAUUUGCCUCCCAGCCUACA
442
22
1





hsa-miR-654-3p
UAUGUCUGCUGACCAUCACCUU
336
22
1





hsa-miR-9-3p
AUAAAGCUAGAUAACCGAAAGU
183
22
1





hsa-miR-940
AAGGCAGGGCCCCCGCUCCCC
366
21
1





hsa-miR-99a-5p
AACCCGUAGAUCCGAUCUUGUG
52
22
1









D) Identification of Top Ranking Coding and Non-Coding RNAs by GENCODE Analysis Performed in Exosomes, MV and Producer Cells

















CTX0E0307EH
CTX0E0307EH
CTX0E0307EH
CTX0E0307EI

CTX0E0307EI


cells
EXO
MV
cells
CTX0E0307EIEXO
MV







18741941
12678688
10876797
22116110
16311289
835970









Table 10: Total number of sequence reads identified by using GENCODE in each tested samples


Using GENCODE database analysis of the sequence results, seven putative novel miRNA sequences were identified in exosomes (EXO), microvesicles (MV) and producer cells, as shown in Table 11. (nb CTX0E03 07E1 MV reads are misrepresented due to the lower amount of starting material—see Table 10). These data are shown graphically in FIG. 16, which shows that these sequences are preferentially shuttled into exosomes and microvesicles compared to the cells.









TABLE 11





Identification of putative novel miRNA sequences using GENCODE in exosomes


(EXO), microvesicles (MV) and producer cells. CTX0E03 07EI MV reads are


misrepresented due to the lower amount of starting material (table 1). The


transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





AC079949.1
AC079949.1-201
57
Novel miRNA
2629
27006


AP000318.1
AP000318.1-201
64
Novel miRNA
1353
9379


AL161626.1
AL161626.1-201
57
Novel miRNA
471
4450


AC004943.1
AC004943.1-201
81
Novel miRNA
24
81


AL121897.1
AL121897.1-201
89
Novel miRNA
6
22

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





AC079949.1
AC079949.1-201
14873
2425
11433
848


AP000318.1
AP000318.1-201
11002
7469
2963
419


AL161626.1
AL161626.1-201
3712
291
1263
129


AC004943.1
AC004943.1-201
43
23
94
5


AL121897.1
AL121897.1-201
14
2
30
3










Validation and of Novel miRNAs


AC079949.1-201 (SEQ ID NO:738)


Gene: AC079949.1 ENSG00000239776











>12 dna:chromosome chromosome:GRCh37:12:



127650616:127650672:1



GGCCGCGCCCCGTTTCCCAGGACAAAGGGCACTCCGCACCGGACC



CTGGTCCCAGCG






For AC079949.1-201 putative mature miRNA, gaccaggguccggugcggagug (SEQ ID NO:745) was identified as the possible 5′ stem mature miRNA using http://mirna.imbb.forth.gr/MatureBayes.html, a tool for finding mature miRNA within a miRNA precursor sequence using a Naive Bays classifier. Its presence validation was performed using AGGGTCCGGTGCGGAGT (SEQ ID NO:746) primer sequence. This sequence was entered in mirbase (http://www.mirbase.org/) and the following miRNA was found with similar sequence: Bos taurus miR-2887-1 (Accession No. MIMAT0013845).


bta-miR-2887: 9-20 (SEQ ID NO:747)




embedded image


The presence of this novel miRNA was tested by qRT-PCR on purified exosomes retro transcribed miRNA.


The same analysis was performed using the 3′ stem of AC079949, sequence TGCGGAGTGCCCTTTGTCCT (SEQ ID NO:748), but in this case no similar miRNA was identified in mirbase.


AP000318.1-201 (SEQ ID NO:739)

Gene: AP000318.1 ENSG00000266007











>21 dna:chromosome chromosome:GRCh37:21:



35677430:35677493:1



CCCACTCCCTGGCGCCGCTTGTGGAGGGCCCAAGTCCTTCTGATT



GAGGCCCAACCCGTGGAAG






For AP000318.1-201 putative mature miRNA, ggagggcccaaguccuucugau (SEQ ID NO:744) was identified as the possible 5′ stem mature miRNA. Its presence validation was performed using GGAGGGCCCAAGTCCTTCTGAT (SEQ ID NO:749) primer sequence. Caenorhabditis remanei miR-55 stem-loop was identified as similar miRNA. Primer validation was again carried out by qRT-PCR.

    • crm-miR-55-5p: 4-17 (SEQ ID NO:750)




embedded image


AL161626.1-201 (SEQ ID NO:740)


Gene: AL161626.1 ENSG00000241781











>9 dna:chromosome chromosome:GRCh37:9:



79186731:79186787:1



CGCCGGGACCGGGGTCCGGGGCGGAGTGCCCTTCCTCCTGGGAAA



CGGGGTGCGGC






For AL161626.1-201 putative mature miRNA, ggcggagugcccuucuuccugg (SEQ ID NO:743) was identified as the possible 5′ stem mature miRNA. Its presence validation was performed using CGGAGTGCCCTTCTTCCT (SEQ ID NO:751) primer sequence. Zea mays miR164c stem-loop and Achypodium distachyon miR164f stem-loop were identified as similar miRNA. Primer validation was again carried out by qRT-PCR.

    • zma-miR164c-3p: 4-15 (SEQ ID NO:752)




embedded image


AC004943.1 (SEQ ID NO:741)


Gene: AC004943.1 ENSG00000265573











>16 dna:chromosome chromosome:GRCh37:16:



72821592:72821672:-1



GCTTCACGTCCCCACCGGCGGCGGCGGCGGTGGCAGTGGCGGCGG



CGGCGGCGGTGGCGGCGGCGGCGGCGGCGGCGGCTC






AL121897.1 (SEQ ID NO:742)


Gene: AL121897.1 ENSG00000264308











>20 dna:chromosome chromosome:GRCh37:20:



30865503:30865591:1



GCCGCCCCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCC



CGCTTTCGGCTCGGGCCTCAGGTGAGTCGGAGGGGCCGGGCGCC






Miscellaneous RNA (Misc_RNA), Including Novel Putative

Misc_RNA is short for miscellaneous RNA, a general term for a series of miscellaneous small RNA. Miscellaneous transcript feature are not defined by other RNA keys.


List of top ranking previously known and novel misc_RNAs identified using GENCODE sequence data set:









TABLE 12





Identification of misc_RNA, including putative novel misc_RNA, sequences using


GENCODE in exosomes (EXO), microvesicles (MV) and producer cells. (CTX0E03 07EI


MV reads are misrepresented due to the lower amount of starting material - Table


10). The transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





RPPH1
RPPH1-201
333
misc RNA
76
2229


RMRP
RMRP-201
264
misc RNA
139
1803


RPPH1
RPPH1-001
638
misc RNA
182
931


VTRNA1-1
VTRNA1-1-201
99
misc RNA
43
720


Y_RNA
Y_RNA.321-201
93
Novel misc RNtext missing or illegible when filed
159
196


Y_RNA
Y_RNA.725-201
95
Novel misc RNtext missing or illegible when filed
1092
18


Y_RNA
Y_RNA.125-201
96
Novel misc RNtext missing or illegible when filed
1079
15


Y_RNA
Y_RNA.118-201
99
Novel misc RNtext missing or illegible when filed
134
12


Y_RNA
Y_RNA.394-201
109
Novel misc RNtext missing or illegible when filed
9
9


Y_RNA
Y_RNA.687-201
111
Novel misc RNtext missing or illegible when filed
36
6


Y_RNA
Y_RNA.144-201
102
Novel misc RNtext missing or illegible when filed
129
5


Y_RNA
Y_RNA.337-201
105
Novel misc RNtext missing or illegible when filed
7
4


Y_RNA
Y_RNA.413-201
97
Novel misc RNtext missing or illegible when filed
136
4


Y_RNA
Y_RNA.30-201
103
Novel misc RNtext missing or illegible when filed
74
3

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





RPPH1
RPPH1-201
1785
0
1077
197


RMRP
RMRP-201
1443
191
659
87


RPPH1
RPPH1-001
1372
795
2017
157


VTRNA1-1
VTRNA1-1-201
52
247
210
9


Y_RNA
Y_RNA.321-201
661
960
903
217


Y_RNA
Y_RNA.725-201
74
1005
39
11


Y_RNA
Y_RNA.125-201
58
906
27
12


Y_RNA
Y_RNA.118-201
9
156
45
7


Y_RNA
Y_RNA.394-201
7
33
13
1


Y_RNA
Y_RNA.687-201
15
103
41
10


Y_RNA
Y_RNA.144-201
21
187
84
5


Y_RNA
Y_RNA.337-201
0
15
4
0


Y_RNA
Y_RNA.413-201
8
125
46
3


Y_RNA
Y_RNA.30-201
3
62
21
2






text missing or illegible when filed indicates data missing or illegible when filed







Among the misc_RNA the following sequences were found preferentially down or up shuttled in exosomes and MV: RPHI, RMRP, and VTRNA1-1 up shuttled and Y_RNA.725-201, and Y_RNA.125-201 down respectively. RPHI is a ribonuclease P RNA component H1. RMRP gene encodes the RNA component of mitochondrial RNA processing endoribonuclease, which cleaves mitochondrial RNA at a priming site of mitochondrial DNA replication. This RNA also interacts with the telomerase reverse transcriptase catalytic subunit to form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase activity and produces double-stranded RNAs that can be processed into small interfering RNA. VTRNA1-1 is vault RNA component 1. Vaults are large cytoplasmic ribonucleoproteins and they are composed of a major vault protein, MVP, 2 minor vault proteins, TEP1 and PARP4, and a non-translated RNA component, VTRNA1-1. Y_RNA.725-201, and Y_RNA.125-201 are novel misc_RNAs and their function is not defined.


Metazoa Miscellaneous RNA

The signal recognition particle RNA, also known as 7SL, 6S, ffs, or 4.5S RNA, is the RNA component of the signal recognition particle (SRP) ribonucleoprotein complex. SRP is a universally conserved ribonucleoprotein that directs the traffic of proteins within the cell and allows them to be secreted. The SRP RNA, together with one or more SRP proteins contributes to the binding and release of the signal peptide. The RNA and protein components of this complex are highly conserved but do vary between the different kingdoms of life.


List of top ranking Metazoa misc_RNAs identified using GENCODE sequence data set:









TABLE 13





Identification signal recognition particle RNA (misc_RNA) sequences using


GENCODE in exosomes (EXO), microvesicles (MV) and producer cells. The transcript


IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





Metazoa_SRP
Metazoa_SRP.791-201
288
Metazoan signal recogntext missing or illegible when filed
679
2324


Metazoa_SRP
Metazoa_SRP.561-201
294
Metazoan signal recogntext missing or illegible when filed
634
2006


Metazoa_SRP
Metazoa_SRP.864-201
297
Metazoan signal recogntext missing or illegible when filed
252
1884


Metazoa_SRP
Metazoa_SRP.824-201
297
Metazoan signal recogntext missing or illegible when filed
438
881


Metazoa_SRP
Metazoa_SRP.72-201
278
Metazoan signal recogntext missing or illegible when filed
441
630


Metazoa_SRP
Metazoa_SRP.151-201
307
Metazoan signal recogntext missing or illegible when filed
377
464


Metazoa_SRP
Metazoa_SRP.208-201
277
Metazoan signal recogntext missing or illegible when filed
382
410


Metazoa_SRP
Metazoa_SRP.501-201
280
Metazoan signal recogntext missing or illegible when filed
265
272


Metazoa_SRP
Metazoa_SRP.682-201
298
Metazoan signal recogntext missing or illegible when filed
12
52



















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI



Gene Symbol
Transcript ID
MV
cells
EXO
MV







Metazoa_SRP
Metazoa_SRP.791-201
2058
771
2698
465



Metazoa_SRP
Metazoa_SRP.561-201
1683
744
2147
432



Metazoa_SRP
Metazoa_SRP.864-201
1544
78
170
148



Metazoa_SRP
Metazoa_SRP.824-201
958
505
1860
342



Metazoa_SRP
Metazoa_SRP.72-201
631
494
2184
349



Metazoa_SRP
Metazoa_SRP.151-201
470
432
1431
265



Metazoa_SRP
Metazoa_SRP.208-201
431
422
1104
242



Metazoa_SRP
Metazoa_SRP.501-201
266
236
434
44



Metazoa_SRP
Metazoa_SRP.682-201
21
10
13
2








text missing or illegible when filed indicates data missing or illegible when filed







RRNA (Ribosomal RNA)

Ribosomal RNA (rRNA) forms part of the protein-synthesizing organelle known as a ribosome and that is exported to the cytoplasm to help translate the information in messenger RNA (mRNA) into protein. Eukaryotic ribosome (80S) rRNA components are: large unit (rRNA 5S, 5.8S, and 28S) small unit (rRNA 18S). Both rRNA 28S and 5.8S are selectively up-shuttled in exosomes and MV.


List of top ranking rRNA identified using GENCODE sequence data set:









TABLE 14





Identification rRNA sequences using GENCODE in exosomes (EXO), microvesicles (MV) and producer


cells. The transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





RNA5-8SP6
RNA5-8SP6-201
152
rRNA
205008
1148190


RNA28S5
RNA28S5-001
432
rRNA
86111
458585


RNA18S5
RNA18S5-001
599
rRNA
74634
52055


RNA5-8SP2
RNA5-8SP2-201
152
rRNA
6488
1719


RNA5-8SP5
RNA5-8SP5-201
152
rRNA
2794
7393

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





RNA5-8SP6
RNA5-8SP6-201
706558
213187
135909
14732


RNA28S5
RNA28S5-001
516754
62829
390237
47483


RNA18S5
RNA18S5-001
61639
116874
138484
14616


RNA5-8SP2
RNA5-8SP2-201
1540
9231
3112
149


RNA5-8SP5
RNA5-8SP5-201
3924
7314
3579
232










Small Nucleolar RNA: snoRNA


Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guides chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are two main classes of snoRNA, the C/D box snoRNAs which are associated with methylation, and the H/ACA box snoRNAs which are associated with pseudouridylation. List of top ranking snoRNA identified using GENCODE sequence data set:









TABLE 15





Identification of snoRNA sequences using GENCODE in exosomes (EXO), microvesicles (MV) and


producer cells. The transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





SNORD3A
SNORD3A-201
216
snoRNA
1433
2085


SNORD3C
SNORD3C-201
216
snoRNA
1169
1702


SNORD29
SNORD29-201
65
snoRNA
28130
1633


SNORD83B
SNORD83B-201
93
snoRNA
1835
675


SNORD30
SNORD30-201
70
snoRNA
29743
254

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





SNORD3A
SNORD3A-201
1621
906
1732
120


SNORD3C
SNORD3C-201
1220
639
1176
86


SNORD29
SNORD29-201
1070
36677
1752
45


SNORD83B
SNORD83B-201
487
638
575
29


SNORD30
SNORD30-201
244
29071
283
24










Small Nuclear RNA (snRNA)


Small nuclear ribonucleic acid (snRNA), also commonly referred to as U-RNA, is a class of small RNA molecules that make up the major spliceosome are named U1, U2, U4, U5, and U6, and participate in several RNA-RNA and RNA-protein interactions. Their primary function is in the processing of pre-mRNA (hnRNA) in the nucleus. They have also been shown to aide in the regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA), and maintaining the telomeres.


List of top ranking snRNA identified using GENCODE sequence data set:









TABLE 16A





Identification of snRNA sequences using GENCODE in exosomes (EXO), microvesicles (MV) and


producer cells. The transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





U2
U2.38-201
191
snRNA
1354
71596


U2
U2.6-201
192
snRNA
834
15561


U1
U1.81-201
164
snRNA
584
10901


U1
U1.90-201
167
snRNA
533
9927


U2
U2.7-201
191
snRNA
201
9267

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





U2
U2.38-201
49223
751
35290
1919


U2
U2.6-201
13594
303
8146
272


U1
U1.81-201
7307
91
3197
121


U1
U1.90-201
6689
48
2187
84


U2
U2.7-201
3109
288
6736
262










LincRNA and Novel lincRNA


Large intergenic non-coding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides.


List of top ranking previously known and novel lincRNAs identified using GENCODE sequence data set:









TABLE 16B





Identification of lincRNA and putative novel lincRNA sequences using


GENCODE in exosomes (EXO), microvesicles (MV) and producer cells. The


transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





RP11-108M9.3
RP11-108M9.3-0text missing or illegible when filed
1761
Novel lincRNA
244
159


RP11-329L6.1
RP11-329L6.1-001
507
Novel lincRNA
19
70


RP11-160E2.6
RP11-160E2.6-00text missing or illegible when filed
637
Novel lincRNA
228
67


AC004528.3
AC004528.3-001
107
Novel lincRNA
16
58


MALAT1
MALAT1-201
4585
lincRNA
150
308


GAS5
GAS5-007
2743
lincRNA
12024
215

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





RP11-108M9.3
RP11-108M9.3-0text missing or illegible when filed
240
539
324
45


RP11-329L6.1
RP11-329L6.1-001
41
29
84
2


RP11-160E2.6
RP11-160E2.6-00text missing or illegible when filed
115
489
74
6


AC004528.3
AC004528.3-001
46
14
55
4


MALAT1
MALAT1-201
234
26
182
12


GAS5
GAS5-007
120
46501
875
13






text missing or illegible when filed indicates data missing or illegible when filed







GAS5 lincRNA is highly expressed in cell producer compared to in exosomes and microvesicles (down shuttled in both exosomes and MV).


mRNA


Coding sequencing mRNA were also identified.









TABLE 17





Identification of mRNA sequences using GENCODE in exosomes (EXO), microvesicles (MV) and


producer cells. The transcript IDs are taken from the Ensembl database (www.ensembl.org).
























CTX0E0307EH
CTX0E0307EH


Gene Symbol
Transcript ID
Length
Type of RNA
cells
EXO





EEF2
EEF2-201
9407
mRNA
710
578


MTRNR2L8
MTRNR2L8-201
1290
mRNA
1383
548


NES
NES-001
8635
mRNA
668
406


VIM
VIM-001
8316
mRNA
563
911

















CTX0E0307EH
CTX0E0307EI
CTX0E0307EI
CTX0E0307EI


Gene Symbol
Transcript ID
MV
cells
EXO
MV





EEF2
EEF2-201
449
1155
471
33


MTRNR2L8
MTRNR2L8-201
642
1323
258
15


NES
NES-001
234
1448
267
20


VIM
VIM-001
501
1500
618
36









Example 12
Conclusion

The main scope of the deep sequence analysis was to identify their miRNA components in neural stem cell-derived vesicles (exosomes and microvesicles). This analysis identified a new set of known and novel miRNAs that are preferentially shuttled into both exosomes and MV. Among the identified miRNAs already included in mirbase database were hsa-miR-1246, hsa-miR-4488, hsa-miR-4492, hsa-miR-4508, hsa-miR-4516, hsa-miR-4532, and among the novel miRNAs were AC079949.1, AP000318.1, AL161626.1, AC004943.1, AL121897.1. Top ranking shuttled miRNAs, including novel ones were validated by qRT-PCR in exosomes.


The size distribution of shuttle RNA, as shown here, is mostly in the range of 20 to 200 nt and other RNA species are released by cells into the extracellular space. By deep sequencing and GENCODE sequence set analysis we found a greater complexity and diversity of non-coding RNA transcripts. We extended this analysis with detailed evaluation and this led to the discovery of preferentially up (defined as log 2 fold change 2) and down (defined as log 2 fold change ≦−2) shuttle of other non-coding RNAs in both exosomes and microvesicles. Differentially shuttled non coding RNA were found in almost all the non-coding RNA subtypes, ribosomal RNA (rRNA), small nucleolar (snoRNA), small nuclear RNA (snRNA), microRNA (miRNA), miscellaneous other RNA (mist RNA, e.g. RMRP, vault RNA, metazoa SRP, and RNY), and large intergenic non-coding RNAs (lincRNAs).


The unequal distribution of the detected RNA species over cellular and shuttle RNA, combined with increasing evidence for their role in gene regulation strongly suggest that cells specifically release these RNAs to modify the function of target cells.


Example 13
Proteomic Analysis
Methods

Exosomes and microvesicle fractions were prepared from a CTX0E03 cell Integra culture (week 2), using differential ultracentrifugation. Exosomes and microvesicles were disrupted in modified RIPA buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1% SDS, 0.1% Triton X100, 10 mM DTT, 1× Complete protease inhibitor (Roche) and 1× PhosStop phosphatase inhibitor (Roche)) and subjected to manual shearing using a 1 mL tuberculin syringe and 25 gauge needle. Samples were re-quantitated post disruption using the Qubit fluorometer (Invitrogen). 20 μg of each sample was loaded onto a 4-12% SDS-PAGE gel (Novex, Invitrogen). The gel was excised into forty segments per lane and gel slices were processed using a robot (ProGest, DigiLab) with the following protocol:

    • a) wash with 25 mM ammonium bicarbonate followed by acetonitrile;
    • b) reduce with 10 mM dithiothreitol at 60° C. followed by alkylation with 50 mM iodoacetamide at room temperature;
    • c) digest with trypsin (Promega) at 37° C. for 4 h;
    • d) quench with formic acid;
    • e) the supernatant was analysed by mass spectrometry directly without further processing.


Mass Spectrometry

Each gel digest was analysed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded on a trapping column and eluted over a 75 μm analytical column at 350 nL/min; both columns were packed with Jupiter Proteo resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70,000 FWHM and 17,500 FWHM resolution, respectively.


Exosomes

2572 proteins were identified by Mass spectrometry in exosomes purified by ultracentrifugation. The exosomes were isolated from the initial stages of an Integra culture (week 2). The gene names and corresponding SWISSPROT accession numbers (in brackets) of all 2572 proteins are listed in Table 18 (in alphabetical order of gene name) and the 100 most abundant proteins are listed in Table 19, in order of decreasing abundance. The characteristic exosome markers CD9, CD81 and Alix (also known as PDCD6IP) are present in the most abundant 100 proteins.









TABLE 18





Gene names and SWISSPROT accession numbers of all 2572 proteins identified in


CTX0E03 exosomes (listed in alphabetical order of gene name).















A1BG (P04217), A2M (P01023), AACS (Q86V21), AAMP (Q13685), AARS (P49588), AARSD1


(Q9BTE6), AASDHPPT (Q9NRN7), ABCA3 (Q99758), ABCE1 (P61221), ABCF1 (Q8NE71),


ABCF3 (Q9NUQ8), ABHD10 (Q9NUJ1), ABHD14B (Q96IU4), ABI1 (Q8IZP0), ABR (Q12979),


ACAA2 (P42765), ACACA (Q13085), ACADVL (P49748), ACAP2 (Q15057), ACAT1 (P24752),


ACAT2 (Q9BWD1), ACBD7 (Q8N6N7), ACLY (P53396), ACO1 (P21399), ACO2 (Q99798),


ACOT1 (Q86TX2), ACOT13 (Q9NPJ3), ACOT7 (O00154), ACP1 (P24666), ACSL1 (P33121),


ACSL3 (O95573), ACSL4 (O60488), ACSS2 (Q9NR19), ACTC1 (P68032), ACTG1 (P63261),


ACTL6A (O96019), ACTN1 (P12814), ACTN4 (O43707), ACTR10 (Q9NZ32), ACTR1A


(P61163), ACTR1B (P42025), ACTR2 (P61160), ACTR3 (P61158), ADAM10 (O14672),


ADAM12 (O43184), ADAMTS15 (Q8TE58), ADAMTS16 (Q8TE57), ADAR (P55265), ADAT2


(Q7Z6V5), ADH5 (P11766), ADI1 (Q9BV57), ADK (P55263), ADRBK1 (P25098), ADRM1


(Q16186), ADSL (P30566), ADSS (P30520), AEBP1 (Q8IUX7), AFM (P43652), AGL (P35573),


AGRN (O00468), AGT (P01019), AHCY (P23526), AHCYL1 (O43865), AHNAK (Q09666),


AHSA1 (O95433), AHSG (P02765), AIDA (Q96BJ3), AIFM1 (O95831), AIMP1 (Q12904),


AIMP2 (Q13155), AIP (O00170), AK1 (P00568), AK3 (Q9UIJ7), AK4 (P27144), AKAP12


(Q02952), AKAP9 (Q99996), AKR1A1 (P14550), AKR1B1 (P15121), AKR1C1 (Q04828),


AKR7A2 (O43488), AKR7A3 (O95154), AKT1 (P31749), ALCAM (Q13740), ALDH16A1


(Q8IZ83), ALDH3A1 (P30838), ALDH7A1 (P49419), ALDH9A1 (P49189), ALDOA (P04075),


ALDOC (P09972), ALKBH2 (Q6NS38), ALKBH4 (Q9NXW9), AMBP (P02760), AMDHD2


(Q9Y303), AMPD2 (Q01433), AMZ2 (Q86W34), ANAPC1 (Q9H1A4), ANAPC4 (Q9UJX5),


ANAPC5 (Q9UJX4), ANAPC7 (Q9UJX3), ANKFY1 (Q9P2R3), ANKRD28 (O15084), ANP32A


(P39687), ANP32B (Q92688), ANP32E (Q9BTT0), ANXA1 (P04083), ANXA2 (P07355), ANXA4


(P09525), ANXA5 (P08758), ANXA6 (P08133), ANXA7 (P20073), AP1B1 (Q10567), AP1G1


(O43747), AP1M1 (Q9BXS5), AP1S1 (P61966), AP1S2 (P56377), AP2A1 (O95782), AP2A2


(O94973), AP2B1 (P63010), AP2M1 (Q96CW1), AP2S1 (P53680), AP3B1 (O00203), AP3D1


(O14617), AP3M1 (Q9Y2T2), AP3S1 (Q92572), AP3S2 (P59780), AP4S1 (Q9Y587), APEH


(P13798), APEX1 (P27695), API5 (Q9BZZ5), APIP (Q96GX9), APOA1 (P02647), APOA1BP


(Q8NCW5), APOA2 (P02652), APOBEC3C (Q9NRW3), APOC2 (P02655), APOD (P05090),


APOH (P02749), APOM (O95445), APPL1 (Q9UKG1), APRT (P07741), AQR (O60306),


ARCN1 (P48444), ARF1 (P84077), ARF4 (P18085), ARF5 (P84085), ARF6 (P62330), ARFIP1


(P53367), ARFIP2 (P53365), ARHGAP1 (Q07960), ARHGAP12 (Q8IWW6), ARHGDIA


(P52565), ARHGEF1 (Q92888), ARHGEF10 (O15013), ARHGEF7 (Q14155), ARIH1 (Q9Y4X5),


ARIH2 (O95376), ARL1 (P40616), ARL2 (P36404), ARL3 (P36405), ARL6IP1 (Q15041),


ARL8B (Q9NVJ2), ARMC10 (Q8N2F6), ARMC6 (Q6NXE6), ARMC8 (Q8IUR7), ARMC9


(Q7Z3E5), ARMCX3 (Q9UH62), ARPC1A (Q92747), ARPC1B (O15143), ARPC2 (O15144),


ARPC3 (O15145), ARPC4 (P59998), ARPC5 (O15511), ARPC5L (Q9BPX5), ARRDC1


(Q8N5I2), ASB6 (Q9NWX5), ASCC1 (Q8N9N2), ASCC2 (Q9H1I8), ASCC3 (Q8N3C0), ASF1A


(Q9Y294), ASH2L (Q9UBL3), ASMTL (O95671), ASNA1 (O43681), ASNS (P08243), ASS1


(P00966), ATG16L1 (Q676U5), ATG3 (Q9NT62), ATG4B (Q9Y4P1), ATG7 (O95352), ATIC


(P31939), ATL3 (Q6DD88), ATM (Q13315), ATOX1 (O00244), ATP1A1 (P05023), ATP1B1


(P05026), ATP1B3 (P54709), ATP2B1 (P20020), ATP2B4 (P23634), ATP5B (P06576), ATP5E


(P56381), ATP5I (P56385), ATP6AP2 (O75787), ATP6V0D1 (P61421), ATP6V1A (P38606),


ATP6V1B2 (P21281), ATP6V1C1 (P21283), ATP6V1D (Q9Y5K8), ATP6V1E1 (P36543),


ATP6V1G1 (O75348), ATP6V1H (Q9UI12), ATR (Q13535), ATRN (O75882), ATXN10


(Q9UBB4), B2M (P61769), B3GAT3 (O94766), B3GNT1 (O43505), B4GALT7 (Q9UBV7), BAG2


(O95816), BAIAP2 (Q9UQB8), BANF1 (O75531), BAT1 (Q13838), BAT3 (P46379), BBOX1


(O75936), BCAS2 (O75934), BCAT1 (P54687), BCCIP (Q9P287), BCL2L13 (Q9BXK5),


BCLAF1 (Q9NYF8), BDH2 (Q9BUT1), BICD2 (Q8TD16), BLOC1S1 (P78537), BLVRA


(P53004), BLVRB (P30043), BMP1 (P13497), BOLA2 (Q9H3K6), BPGM (P07738), BPHL


(Q86WA6), BPNT1 (O95861), BRCC3 (P46736), BRE (Q9NXR7), BROX (Q5VW32), BRP16L


(P0CB43), BSG (P35613), BST1 (Q10588), BTAF1 (O14981), BUB3 (O43684), BUD31


(P41223), BYSL (Q13895), BZW1 (Q7L1Q6), BZW2 (Q9Y6E2), C10orf119 (Q9BTE3), C10orf58


(Q9BRX8), C10orf76 (Q5T2E6), C11orf54 (Q9H0W9), C11orf68 (Q9H3H3), C12orf10


(Q9HB07), C14orf149 (Q96EM0), C14orf166 (Q9Y224), C15orf58 (Q6ZNW5), C16orf13


(Q96S19), C16orf80 (Q9Y6A4), C1D (Q13901), C1orf123 (Q9NWV4), C1orf50 (Q9BV19),


C1orf57 (Q9BSD7), C1RL (Q9NZP8), C20orf11 (Q9NWU2), C20orf27 (Q9GZN8), C20orf4


(Q9Y312), C21orf59 (P57076), C22orf25 (Q6ICL3), C22orf28 (Q9Y3I0), C2orf29 (Q9UKZ1),


C2orf79 (Q6GMV3), C3orf10 (Q8WUW1), C3orf26 (Q9BQ75), C3orf75 (Q0PNE2), C4orf27


(Q9NWY4), C4orf41 (Q7Z392), C5orf32 (Q9H1C7), C6orf130 (Q9Y530), C6orf211 (Q9H993),


C7orf25 (Q9BPX7), C7orf28B (P86790), C7orf41 (Q8N3F0), C7orf59 (Q0VGL1), C9orf142


(Q9BUH6), C9orf23 (Q8N5L8), C9orf41 (Q8N4J0), C9orf64 (Q5T6V5), CA11 (O75493), CAB39


(Q9Y376), CACNA2D1 (P54289), CACYBP (Q9HB71), CAD (P27708), CADM1 (Q9BY67),


CADM4 (Q8NFZ8), CALB1 (P05937), CALD1 (Q05682), CALM1 (P62158), CAMK2D (Q13557),


CAND1 (Q86VP6), CAP1 (Q01518), CAPN1 (P07384), CAPN2 (P17655), CAPN5 (O15484),


CAPNS1 (P04632), CAPS (Q13938), CAPZA1 (P52907), CAPZA2 (P47755), CAPZB (P47756),


CARHSP1 (Q9Y2V2), CARKD (Q8IW45), CARM1 (Q86X55), CARS (P49589), CASK


(O14936), CASP3 (P42574), CASP6 (P55212), CAT (P04040), CBFB (Q13951), CBR1


(P16152), CBR3 (O75828), CBS (P35520), CBWD2 (Q8IUF1), CBX1 (P83916), CBX3


(Q13185), CBX5 (P45973), CC2D1A (Q6P1N0), CC2D1B (Q5T0F9), CCAR1 (Q8IX12), CCBL1


(Q16773), CCBL2 (Q6YP21), CCDC22 (O60826), CCDC25 (Q86WR0), CCDC53 (Q9Y3C0),


CCDC56 (Q9Y2R0), CCDC93 (Q567U6), CCNC (P24863), CCND2 (P30279), CCNH (P51946),


CCT2 (P78371), CCT3 (P49368), CCT4 (P50991), CCT5 (P48643), CCT6A (P40227), CCT7


(Q99832), CCT8 (P50990), CD109 (Q6YHK3), CD151 (P48509), CD276 (Q5ZPR3), CD44


(P16070), CD47 (Q08722), CD59 (P13987), CD63 (P08962), CD81 (P60033), CD9 (P21926),


CD99 (P14209), CDC16 (Q13042), CDC23 (Q9UJX2), CDC27 (P30260), CDC34 (P49427),


CDC37 (Q16543), CDC40 (O60508), CDC42 (P60953), CDCSL (Q99459), CDCP1 (Q9H5V8),


CDH2 (P19022), CDK1 (P06493), CDK2 (P24941), CDK2AP2 (O75956), CDK4 (P11802),


CDK5 (Q00535), CDK5RAP3 (Q96JB5), CDK7 (P50613), CDKN2A (P42771), CDKN2AIP


(Q9NXV6), CELSR1 (Q9NYQ6), CELSR2 (Q9HCU4), CEP57 (Q86XR8), CFL1 (P23528), CFL2


(Q9Y281), CHAC2 (Q8WUX2), CHAF1B (Q13112), CHD4 (Q14839), CHEK2 (O96017),


CHERP (Q8IWX8), CHID1 (Q9BWS9), CHML (P26374), CHMP1B (Q7LBR1), CHMP2A


(O43633), CHMP4A (Q9BY43), CHMP4B (Q9H444), CHMP6 (Q96FZ7), CHORDC1 (Q9UHD1),


CHP (Q99653), CHRAC1 (Q9NRG0), CHST14 (Q8NCH0), CHST3 (Q7LGC8), CHURC1


(Q8WUH1), CIAO1 (O76071), CIAPIN1 (Q6FI81), CIRH1A (Q969X6), CKAP5 (Q14008), CKB


(P12277), CLASP1 (Q7Z460), CLDN3 (O15551), CLEC18B (Q6UXF7), CLIC1 (O00299), CLIC4


(Q9Y696), CLLD6 (Q5W111), CLNS1A (P54105), CLP1 (Q92989), CLPB (Q9H078), CLTA


(P09496), CLTC (Q00610), CLU (P10909), CMAS (Q8NFW8), CMBL (Q96DG6), CMPK1


(P30085), CNBP (P62633), CNDP2 (Q96KP4), CNN2 (Q99439), CNN3 (Q15417), CNOT1


(A5YKK6), CNOT10 (Q9H9A5), CNOT6L (Q96LI5), CNOT7 (Q9UIV1), CNP (P09543), COASY


(Q13057), COBRA1 (Q8WX92), COG1 (Q8WTW3), COG2 (Q14746), COG3 (Q96JB2), COG4


(Q9H9E3), COG5 (Q9UP83), COG6 (Q9Y2V7), COG7 (P83436), COG8 (Q96MW5), COL11A1


(P12107), COL14A1 (Q05707), COL6A1 (P12109), COMMD1 (Q8N668), COMMD10


(Q9Y6G5), COMMD2 (Q86X83), COMMD3 (Q9UBI1), COMMD4 (Q9H0A8), COMMD5


(Q9GZQ3), COMMD6 (Q7Z4G1), COMMD7 (Q86VX2), COMMD8 (Q9NX08), COMMD9


(Q9P000), COMT (P21964), COPA (P53621), COPB1 (P53618), COPB2 (P35606), COPE


(O14579), COPG (Q9Y678), COPG2 (Q9UBF2), COPS2 (P61201), COPS3 (Q9UNS2), COPS4


(Q9BT78), COPS5 (Q92905), COPS6 (Q7L5N1), COPS7A (Q9UBW8), COPS7B (Q9H9Q2),


COPS8 (Q99627), COPZ1 (P61923), CORO1A (P31146), CORO1B (Q9BR76), CORO1C


(Q9ULV4), CORO2B (Q9UQ03), CORO7 (P57737), COTL1 (Q14019), COX5A (P20674),


COX5B (P10606), COX6C (P09669), COX7A2 (P14406), CP (P00450), CPD (O75976), CPN2


(P22792), CPNE1 (Q99829), CPNE3 (O75131), CPNE7 (Q9UBL6), CPSF1 (Q10570), CPSF2


(Q9P2I0), CPSF3 (Q9UKF6), CPSF7 (Q8N684), CPXM1 (Q96SM3), CRIP2 (P52943), CRK


(P46108), CRLF3 (Q8IUI8), CRTAP (O75718), CRYAB (P02511), CRYM (Q14894), CRYZ


(Q08257), CRYZL1 (O95825), CS (O75390), CSDE1 (O75534), CSE1L (P55060), CSK


(P41240), CSNK1A1 (P48729), CSNK2A1 (P68400), CSNK2B (P67870), CSRP1 (P21291),


CSRP2 (Q16527), CSTB (P04080), CSTF1 (Q05048), CSTF2T (Q9H0L4), CSTF3 (Q12996),


CTBP1 (Q13363), CTBP2 (P56545), CTNNA1 (P35221), CTNNB1 (P35222), CTNNBL1


(Q8WYA6), CTNND1 (O60716), CTPS (P17812), CTPS2 (Q9NRF8), CTR9 (Q6PD62), CTSC


(P53634), CTSD (P07339), CTSF (Q9UBX1), CTSL2 (O60911), CTU1 (Q7Z7A3), CTU2


(Q2VPK5), CUL1 (Q13616), CUL2 (Q13617), CUL3 (Q13618), CUL4A (Q13619), CUL4B


(Q13620), CUL5 (Q93034), CWF19L1 (Q69YN2), CXADR (P78310), CXorf26 (Q9BVG4),


CYB5A (P00167), CYCS (P99999), CYFIP1 (Q7L576), CYFIP2 (Q96F07), CYR61 (O00622),


DAG1 (Q14118), DAK (Q3LXA3), DARS (P14868), DAZAP1 (Q96EP5), DBI (P07108), DBN1


(Q16643), DBNL (Q9UJU6), DBR1 (Q9UK59), DCAF7 (P61962), DCAF8 (Q5TAQ9), DCD


(P81605), DCK (P27707), DCLK1 (O15075), DCPS (Q96C86), DCTD (P32321), DCTN1


(Q14203), DCTN2 (Q13561), DCTN3 (O75935), DCTN4 (Q9UJW0), DCTN5 (Q9BTE1), DCTN6


(O00399), DCUN1D1 (Q96GG9), DCUN1D5 (Q9BTE7), DCXR (Q7Z4W1), DDA1 (Q9BW61),


DDAH2 (O95865), DDB1 (Q16531), DDB2 (Q92466), DDI2 (Q5TDH0), DDR1 (Q08345), DDT


(P30046), DDX1 (Q92499), DDX17 (Q92841), DDX19A (Q9NUU7), DDX21 (Q9NR30), DDX23


(Q9BUQ8), DDX39 (O00148), DDX3X (O00571), DDX5 (P17844), DDX51 (Q8N8A6), DDX6


(P26196), DECR1 (Q16698), DEF (Q68CQ4), DEFA1 (P59665), DENR (O43583), DERA


(Q9Y315), DFFA (O00273), DHFR (P00374), DHPS (P49366), DHRS1 (Q96LJ7), DHRS11


(Q6UWP2), DHRS4 (Q9BTZ2), DHX15 (O43143), DHX16 (O60231), DHX29 (Q7Z478), DHX36


(Q9H2U1), DHX9 (Q08211), DIAPH1 (O60610), DIAPH2 (O60879), DIMT1L (Q9UNQ2), DIP2B


(Q9P265), DIP2C (Q9Y2E4), DIS3 (Q9Y2L1), DIS3L2 (Q8IYB7), DKC1 (O60832), DLG1


(Q12959), DNAH17 (Q9UFH2), DNAJA1 (P31689), DNAJA2 (O60884), DNAJB1 (P25685),


DNAJB4 (Q9UDY4), DNAJC13 (O75165), DNAJC3 (Q13217), DNAJC7 (Q99615), DNASE1L1


(P49184), DNM1 (Q05193), DNM1L (O00429), DNM2 (P50570), DNPEP (Q9ULA0), DOCK1


(Q14185), DOCK4 (Q8N1I0), DOCK5 (Q9H7D0), DOCK7 (Q96N67), DOHH (Q9BU89), DOM3Z


(O77932), DPCD (Q9BVM2), DPH1 (Q9BZG8), DPH2 (Q9BQC3), DPH5 (Q9H2P9), DPM1


(O60762), DPP3 (Q9NY33), DPP9 (Q86TI2), DPY30 (Q9C005), DPYSL2 (Q16555), DPYSL3


(Q14195), DPYSL4 (O14531), DPYSL5 (Q9BPU6), DRG1 (Q9Y295), DRG2 (P55039), DSG1


(Q02413), DSP (P15924), DST (Q03001), DSTN (P60981), DTD1 (Q8TEA8), DTYMK


(P23919), DUS2L (Q9NX74), DUSP12 (Q9UNI6), DUSP23 (Q9BVJ7), DUSP3 (P51452), DYM


(Q7RTS9), DYNC1H1 (Q14204), DYNC1I2 (Q13409), DYNC1LI1 (Q9Y6G9), DYNC1LI2


(O43237), DYNC2H1 (Q8NCM8), DYNLL1 (P63167), DYNLL2 (Q96FJ2), DYNLRB1 (Q9NP97),


DYNLT1 (P63172), ECHDC1 (Q9NTX5), ECHDC3 (Q96DC8), ECHS1 (P30084), ECM29


(Q5VYK3), EDC4 (Q6P2E9), EEA1 (Q15075), EEF1A1 (P68104), EEF1B2 (P24534), EEF1D


(P29692), EEF1E1 (O43324), EEF1G (P26641), EEF2 (P13639), EEFSEC (P57772), EFEMP2


(O95967), EFHD2 (Q96C19), EFNB2 (P52799), EFTUD1 (Q7Z2Z2), EFTUD2 (Q15029), EGFR


(P00533), EHD1 (Q9H4M9), EHD2 (Q9NZN4), EHD4 (Q9H223), EIF1 (P41567), EIF1AX


(P47813), EIF2A (Q9BY44), EIF2AK2 (P19525), EIF2B1 (Q14232), EIF2B2 (P49770), EIF2B3


(Q9NR50), EIF2B4 (Q9UI10), EIF2B5 (Q13144), EIF2C2 (Q9UKV8), EIF2S1 (P05198), EIF2S2


(P20042), EIF2S3 (P41091), EIF3A (Q14152), EIF3B (P55884), EIF3C (Q99613), EIF3D


(O15371), EIF3E (P60228), EIF3F (O00303), EIF3G (O75821), EIF3H (O15372), EIF3I


(Q13347), EIF3J (O75822), EIF3K (Q9UBQ5), EIF3L (Q9Y262), EIF3M (Q7L2H7), EIF4A1


(P60842), EIF4A2 (Q14240), EIF4A3 (P38919), EIF4E (P06730), EIF4E2 (O60573), EIF4G1


(Q04637), EIF4G2 (P78344), EIF4G3 (O43432), EIF4H (Q15056), EIF5 (P55010), EIF5A


(P63241), EIF5B (O60841), EIF6 (P56537), ELAC2 (Q9BQ52), ELAVL1 (Q15717), ELMO2


(Q96JJ3), ELP2 (Q6IA86), ELP3 (Q9H9T3), EMG1 (Q92979), EMILIN1 (Q9Y6C2), EML1


(O00423), EML2 (O95834), EML3 (Q32P44), EML4 (Q9HC35), ENAH (Q8N8S7), ENO1


(P06733), ENO2 (P09104), ENOPH1 (Q9UHY7), ENY2 (Q9NPA8), EPB41L2 (O43491),


EPB41L3 (Q9Y2J2), EPHA2 (P29317), EPHB3 (P54753), EPHX1 (P07099), EPM2AIP1


(Q7L775), EPRS (P07814), ERH (P84090), ERI1 (Q8IV48), ERI3 (O43414), ERP44 (Q9BS26),


ESD (P10768), ESYT1 (Q9BSJ8), ETF1 (P62495), ETFA (P13804), ETFB (P38117), EXOC1


(Q9NV70), EXOC2 (Q96KP1), EXOC3 (O60645), EXOC4 (Q96A65), EXOC5 (O00471),


EXOC6 (Q8TAG9), EXOC7 (Q9UPT5), EXOC8 (Q8IYI6), EXOSC1 (Q9Y3B2), EXOSC2


(Q13868), EXOSC3 (Q9NQT5), EXOSC4 (Q9NPD3), EXOSC5 (Q9NQT4), EXOSC6


(Q5RKV6), EXOSC7 (Q15024), EXOSC8 (Q96B26), EXOSC9 (Q06265), EXTL3 (O43909),


EYA3 (Q99504), EZR (P15311), F3 (P13726), F8 (P00451), F8A1 (P23610), FABP5 (Q01469),


FABP7 (O15540), FADD (Q13158), FAF1 (Q9UNN5), FAH (P16930), FAHD2A (Q96GK7),


FAM114A2 (Q9NRY5), FAM115A (Q9Y4C2), FAM120A (Q9NZB2), FAM125A (Q96EY5),


FAM127A (A6ZKI3), FAM129B (Q96TA1), FAM136A (Q96C01), FAM168A (Q92567),


FAM175B (Q15018), FAM188A (Q9H8M7), FAM3A (P98173), FAM3C (Q92520), FAM45B


(Q6NSW5), FAM49B (Q9NUQ9), FAM82B (Q96DB5), FAM84B (Q96KN1), FAM98A (Q8NCA5),


FAM98B (Q52LJ0), FARP1 (Q9Y4F1), FARP2 (O94887), FARSA (Q9Y285), FARSB


(Q9NSD9), FASN (P49327), FAT1 (Q14517), FBL (P22087), FBLN2 (P98095), FBN1 (P35555),


FBN2 (P35556), FBXL18 (Q96ME1), FBXO21 (O94952), FBXO22 (Q8NEZ5), FDFT1 (P37268),


FDPS (P14324), FEN1 (P39748), FERMT1 (Q9BQL6), FERMT2 (Q96AC1), FGF1 (P05230),


FGFRL1 (Q8N441), FGGY (Q96C11), FH (P07954), FHL1 (Q13642), FHL2 (Q14192), FHL3


(Q13643), FIS1 (Q9Y3D6), FKBP1A (P62942), FKBP3 (Q00688), FKBP4 (Q02790), FKBP5


(Q13451), FLII (Q13045), FLNA (P21333), FLNB (O75369), FLNC (Q14315), FLOT1 (O75955),


FMNL2 (Q96PY5), FN3K (Q9H479), FN3KRP (Q9HA64), FNTA (P49354), FNTB (P49356),


FOLR1 (P15328), FREM2 (Q5SZK8), FRMD8 (Q9BZ67), FSCN1 (Q16658), FSD1 (Q9BTV5),


FTH1 (P02794), FTL (P02792), FTO (Q9C0B1), FTSJD2 (Q8N1G2), FUBP1 (Q96AE4), FUCA2


(Q9BTY2), FUK (Q8N0W3), FXR1 (P51114), G3BP1 (Q13283), G3BP2 (Q9UN86), G6PD


(P11413), GAA (P10253), GALK1 (P51570), GALK2 (Q01415), GALNT1 (Q10472), GALNT2


(Q10471), GANAB (Q14697), GAP43 (P17677), GAPDH (P04406), GAPVD1 (Q14C86), GAR1


(Q9NY12), GARS (P41250), GART (P22102), GATSL2 (A6NHX0), GBA (P04062), GBE1


(Q04446), GCLM (P48507), GCN1L1 (Q92616), GDI1 (P31150), GDI2 (P50395), GEMIN5


(Q8TEQ6), GEMIN6 (Q8WXD5), GET4 (Q7L5D6), GFAP (P14136), GFPT1 (Q06210), GFPT2


(O94808), GGCT (O75223), GGPS1 (O95749), GINS1 (Q14691), GINS4 (Q9BRT9), GIPC1


(O14908), GIT1 (Q9Y2X7), GLA (P06280), GLB1 (P16278), GLB1L2 (Q8IW92), GLG1


(Q92896), GLIPR2 (Q9H4G4), GLMN (Q92990), GLO1 (Q04760), GLOD4 (Q9HC38), GLRX


(P35754), GLRX3 (O76003), GLT25D1 (Q8NBJ5), GLTP (Q9NZD2), GLTPD1 (Q5TA50),


GLUD1 (P00367), GLUL (P15104), GMDS (O60547), GMFB (P60983), GMPPA (Q96IJ6),


GMPPB (Q9Y5P6), GMPR (P36959), GMPR2 (Q9P2T1), GMPS (P49915), GNA11 (P29992),


GNA13 (Q14344), GNAI2 (P04899), GNAI3 (P08754), GNAQ (P50148), GNAS (Q5JWF2),


GNB1 (P62873), GNB2 (P62879), GNB2L1 (P63244), GNB4 (Q9HAV0), GNE (Q9Y223),


GNG12 (Q9UBI6), GNG4 (P50150), GNG5 (P63218), GNPDA1 (P46926), GNPNAT1


(Q96EK6), GOLGA7 (Q7Z5G4), GOLGB1 (Q14789), GOLIM4 (O00461), GOLM1 (Q8NBJ4),


GOLPH3 (Q9H4A6), GORASP2 (Q9H8Y8), GPC1 (P35052), GPC4 (O75487), GPC6


(Q9Y625), GPD1L (Q8N335), GPI (P06744), GPLD1 (P80108), GPM6A (P51674), GPM6B


(Q13491), GPN1 (Q9HCN4), GPR56 (Q9Y653), GPS1 (Q13098), GPX1 (P07203), GPX4


(P36969), GRB2 (P62993), GRHPR (Q9UBQ7), GRP (Q3ZCW2), GRPEL1 (Q9HAV7), GRWD1


(Q9BQ67), GSK3A (P49840), GSK3B (P49841), GSN (P06396), GSPT1 (P15170), GSS


(P48637), GSTK1 (Q9Y2Q3), GSTM2 (P28161), GSTM3 (P21266), GSTM4 (Q03013), GSTO1


(P78417), GSTP1 (P09211), GSTT2 (P0CG29), GSTZ1 (O43708), GTF2F2 (P13984), GTF2H2


(Q13888), GTF2I (P78347), GTF3C1 (Q12789), GTF3C2 (Q8WUA4), GTF3C4 (Q9UKN8),


GTPBP1 (O00178), GUK1 (Q16774), GYG1 (P46976), GYS1 (P13807), H2AFY (O75367),


H2AFZ (P0C0S5), HADH (Q16836), HAGH (Q16775), HARS (P12081), HAT1 (O14929),


HAUS3 (Q68CZ6), HAUS4 (Q9H6D7), HBA1 (P69905), HBB (P68871), HCFC1 (P51610),


HDAC1 (Q13547), HDAC2 (Q92769), HDAC3 (O15379), HDHD2 (Q9H0R4), HDLBP (Q00341),


HEATR1 (Q9H583), HEATR2 (Q86Y56), HEBP1 (Q9NRV9), HECTD3 (Q5T447), HEG1


(Q9ULI3), HELZ (P42694), HERC4 (Q5GLZ8), HEXB (P07686), HGS (O14964), HHIP


(Q96QV1), HIBCH (Q6NVY1), HIF1AN (Q9NWT6), HINT1 (P49773), HIP1R (O75146),


HIST1H1B (P16401), HIST1H1C (P16403), HIST1H2BM (Q99879), HIST1H2BO (P23527),


HIST1H4A (P62805), HIST2H2AA3 (Q6FI13), HIST2H3A (Q71DI3), HK1 (P19367), HK2


(P52789), HLA-A (P30443), HLA-A (P01892), HLCS (P50747), HMGA1 (P17096), HMGB1


(P09429), HMGCL (P35914), HMGCS1 (Q01581), HMGN2 (P05204), HNRNPA1 (P09651),


HNRNPA2B1 (P22626), HNRNPA3 (P51991), HNRNPAB (Q99729), HNRNPC (P07910),


HNRNPD (Q14103), HNRNPF (P52597), HNRNPH1 (P31943), HNRNPH2 (P55795),


HNRNPH3 (P31942), HNRNPK (P61978), HNRNPL (P14866), HNRNPM (P52272), HNRNPR


(O43390), HNRNPU (Q00839), HNRNPUL2 (Q1KMD3), HNRPDL (O14979), HNRPLL


(Q8WVV9), HOOK3 (Q86VS8), HP (P00738), HP1BP3 (Q5SSJ5), HPCAL1 (P37235), HPRT1


(P00492), HPX (P02790), HRAS (P01112), HS6ST2 (Q96MM7), HSD17B10 (Q99714),


HSD17B4 (P51659), HSP90AA1 (P07900), HSP90AB1 (P08238), HSP90B1 (P14625),


HSPA12A (O43301), HSPA14 (Q0VDF9), HSPA1A (P08107), HSPA2 (P54652), HSPA4


(P34932), HSPA4L (O95757), HSPA5 (P11021), HSPA8 (P11142), HSPA9 (P38646), HSPB1


(P04792), HSPB11 (Q9Y547), HSPBP1 (Q9NZL4), HSPD1 (P10809), HSPE1 (P61604),


HSPG2 (P98160), HSPH1 (Q92598), HTATIP2 (Q9BUP3), HTRA1 (Q92743), HTT (P42858),


HUWE1 (Q7Z6Z7), HYOU1 (Q9Y4L1), IARS (P41252), ICAM1 (P05362), IDE (P14735), IDH1


(O75874), IDH2 (P48735), IDI1 (Q13907), IDUA (P35475), IFI16 (Q16666), IFI35 (P80217),


IFIT5 (Q13325), IFITM3 (Q01628), IGF1R (P08069), IGF2BP2 (Q9Y6M1), IGF2BP3 (O00425),


IGF2R (P11717), IGFBP3 (P17936), IGSF3 (O75054), IGSF8 (Q969P0), IKBKAP (O95163),


IL1RAP (Q9NPH3), ILF2 (Q12905), ILF3 (Q12906), ILK (Q13418), ILKAP (Q9H0C8), IMP4


(Q96G21), IMPA1 (P29218), IMPA2 (O14732), IMPAD1 (Q9NX62), IMPDH2 (P12268), INF2


(Q27J81), INPP1 (P49441), INPPL1 (O15357), INTS1 (Q8N201), INTS10 (Q9NVR2), INTS3


(Q68E01), INTS5 (Q6P9B9), IPO11 (Q9UI26), IPO13 (O94829), IPO4 (Q8TEX9), IPO5


(O00410), IPO7 (O95373), IPO8 (O15397), IPO9 (Q96P70), IQGAP1 (P46940), IRF2BP2


(Q7Z5L9), IRF3 (Q14653), IRGQ (Q8WZA9), ISG15 (P05161), ISOC1 (Q96CN7), ISPD


(A4D126), ISYNA1 (Q9NPH2), ITFG3 (Q9H0X4), ITGA2 (P17301), ITGA3 (P26006), ITGA4


(P13612), ITGA5 (P08648), ITGA6 (P23229), ITGA7 (Q13683), ITGAV (P06756), ITGB1


(P05556), ITGB4 (P16144), ITGB8 (P26012), ITPA (Q9BY32), JAM3 (Q9BX67), JUP (P14923),


KARS (Q15046), KBTBD4 (Q9NVX7), KBTBD6 (Q86V97), KCTD12 (Q96CX2), KDM1A


(O60341), KEAP1 (Q14145), KHDRBS1 (Q07666), KHSRP (Q92945), KIAA0174 (P53990),


KIAA0196 (Q12768), KIAA0319L (Q8IZA0), KIAA0664 (O75153), KIAA0776 (O94874),


KIAA1033 (Q2M389), KIAA1279 (Q96EK5), KIAA1468 (Q9P260), KIAA1598 (A0MZ66),


KIAA1797 (Q5VW36), KIAA1967 (Q8N163), KIF1A (Q12756), KIF3A (Q9Y496), KIF5B


(P33176), KIF5C (O60282), KLC1 (Q07866), KLC2 (Q9H0B6), KLC4 (Q9NSK0), KLHDC3


(Q9BQ90), KLHL13 (Q9P2N7), KNG1 (P01042), KNTC1 (P50748), KPNA1 (P52294), KPNA2


(P52292), KPNA3 (O00505), KPNA4 (O00629), KPNA6 (O60684), KPNB1 (Q14974), KPRP


(Q5T749), KRAS (P01116), KRIT1 (O00522), KRT13 (P13646), KRT14 (P02533), KRT71


(Q3SY84), KTN1 (Q86UP2), L1CAM (P32004), LAGE3 (Q14657), LAMA4 (Q16363), LAMA5


(O15230), LAMB1 (P07942), LAMC1 (P11047), LAMP1 (P11279), LAMP2 (P13473), LANCL1


(O43813), LANCL2 (Q9NS86), LAP3 (P28838), LARP1 (Q6PKG0), LARS (Q9P2J5), LASP1


(Q14847), LCAT (P04180), LCMT1 (Q9UIC8), LDHA (P00338), LDHB (P07195), LDLR


(P01130), LEFTY2 (O00292), LEPRE1 (Q32P28), LFNG (Q8NES3), LGALS1 (P09382),


LGALS3 (P17931), LGALS3BP (Q08380), LHFP (Q9Y693), LIMA1 (Q9UHB6), LIMS1


(P48059), LIN7C (Q9NUP9), LIPG (Q9Y5X9), LLGL1 (Q15334), LMCD1 (Q9NZU5), LMNA


(P02545), LMNB1 (P20700), LOXL4 (Q96JB6), LPL (P06858), LRBA (P50851), LRCH3


(Q96II8), LRG1 (P02750), LRP1 (Q07954), LRRC20 (Q8TCA0), LRRC40 (Q9H9A6), LRRC47


(Q8N1G4), LRRC57 (Q8N9N7), LRSAM1 (Q6UWE0), LRWD1 (Q9UFC0), LSM1 (O15116),


LSM12 (Q3MHD2), LSM2 (Q9Y333), LSM3 (P62310), LSM4 (Q9Y4Z0), LSM6 (P62312), LSM7


(Q9UK45), LSS (P48449), LTA4H (P09960), LTBP2 (Q14767), LTBP3 (Q9NS15), LUM


(P51884), LYPLA1 (O75608), LYPLA2 (O95372), LYPLAL1 (Q5VWZ2), M6PR (P20645),


MACF1 (Q9UPN3), MAD1L1 (Q9Y6D9), MAD2L1 (Q13257), MAEA (Q7L5Y9), MAGEE1


(Q9HCI5), MAGOHB (Q96A72), MALT1 (Q9UDY8), MAN1B1 (Q9UKM7), MAN2A1 (Q16706),


MANBA (O00462), MAP1B (P46821), MAP1S (Q66K74), MAP2K1 (Q02750), MAP2K2


(P36507), MAP2K3 (P46734), MAP3K4 (Q9Y6R4), MAP4 (P27816), MAP4K4 (O95819),


MAPK1 (P28482), MAPK12 (P53778), MAPK3 (P27361), MAPK9 (P45984), MAPKAPK2


(P49137), MAPKSP1 (Q9UHA4), MAPRE1 (Q15691), MAPRE3 (Q9UPY8), MARCKS


(P29966), MARCKSL1 (P49006), MARK2 (Q7KZI7), MARS (P56192), MAT2A (P31153),


MAT2B (Q9NZL9), MATR3 (P43243), MBD3 (O95983), MBNL1 (Q9NR56), MCAM (P43121),


MCAT (Q8IVS2), MCM2 (P49736), MCM3 (P25205), MCM4 (P33991), MCM5 (P33992), MCM6


(Q14566), MCM7 (P33993), MCTS1 (Q9ULC4), MDH1 (P40925), MDH2 (P40926), MDK


(P21741), MDN1 (Q9NU22), ME1 (P48163), ME2 (P23368), MED1 (Q15648), MED16


(Q9Y2X0), MED17 (Q9NVC6), MED18 (Q9BUE0), MED20 (Q9H944), MED22 (Q15528),


MED23 (Q9ULK4), MED27 (Q6P2C8), MED30 (Q96HR3), MED31 (Q9Y3C7), MEMO1


(Q9Y316), MERIT40 (Q9NWV8), METAP1 (P53582), METAP2 (P50579), METT10D (Q86W50),


METTL1 (Q9UBP6), METTL11A (Q9BV86), METTL13 (Q8N6R0), METTL2B (Q6P1Q9),


METTL5 (Q9NRN9), MFAP2 (P55001), MFAP4 (P55083), MFGE8 (Q08431), MFI2 (P08582),


MGAT4B (Q9UQ53), MGAT5 (Q09328), MGEA5 (O60502), MICAL1 (Q8TDZ2), MIF (P14174),


MIF4GD (A9UHW6), MINA (Q8IUF8), MINK1 (Q8N4C8), MIOS (Q9NXC5), MIS12 (Q9H081),


MKLN1 (Q9UL63), MLTK (Q9NYL2), MMP14 (P50281), MMS19 (Q96T76), MOB2 (Q70IA6),


MOBKL1B (Q9H8S9), MOBKL2A (Q96BX8), MOBKL3 (Q9Y3A3), MOCS2 (O96033), MON2


(Q7Z3U7), MORC2 (Q9Y6X9), MOV10 (Q9HCE1), MOXD1 (Q6UVY6), MPI (P34949), MPP6


(Q9NZW5), MPRIP (Q6WCQ1), MPST (P25325), MPZL1 (O95297), MRC2 (Q9UBG0), MRI1


(Q9BV20), MRTO4 (Q9UKD2), MSH2 (P43246), MSN (P26038), MSTO1 (Q9BUK6), MTA1


(Q13330), MTA2 (O94776), MTAP (Q13126), MTHFD1 (P11586), MTHFS (P49914), MTM1


(Q13496), MTMR1 (Q13613), MTMR6 (Q9Y217), MTMR9 (Q96QG7), MTOR (P42345), MTPN


(P58546), MTR (Q99707), MVD (P53602), MVK (Q03426), MVP (Q14764), MYADM (Q96S97),


MYBBP1A (Q9BQG0), MYCBP (Q99417), MYD88 (Q99836), MYH10 (P35580), MYH9


(P35579), MYL12B (O14950), MYL6 (P60660), MYO18A (Q92614), MYO1B (O43795), MYO1C


(O00159), MYO1E (Q12965), MYO6 (Q9UM54), MYOF (Q9NZM1), MZT1 (Q08AG7), NAA10


(P41227), NAA15 (Q9BXJ9), NAA16 (Q6N069), NAA20 (P61599), NAA30 (Q147X3), NAA38


(O95777), NAA50 (Q9GZZ1), NACA (Q13765), NADSYN1 (Q6IA69), NAE1 (Q13564), NAGK


(Q9UJ70), NAGLU (P54802), NAMPT (P43490), NANS (Q9NR45), NAP1L1 (P55209), NAP1L4


(Q99733), NAPA (P54920), NAPG (Q99747), NAPRT1 (Q6XQN6), NARS (O43776), NASP


(P49321), NCAM1 (P13591), NCAPD2 (Q15021), NCAPG (Q9BPX3), NCBP1 (Q09161),


NCBP2 (P52298), NCDN (Q9UBB6), NCKAP1 (Q9Y2A7), NCKIPSD (Q9NZQ3), NCL (P19338),


NCS1 (P62166), NCSTN (Q92542), NDRG3 (Q9UGV2), NDRG4 (Q9ULP0), NDUFA2


(O43678), NDUFA3 (O95167), NDUFA5 (Q16718), NDUFAB1 (O14561), NDUFS6 (O75380),


NEDD4L (Q96PU5), NEFL (P07196), NEK9 (Q8TD19), NES (P48681), NF1 (P21359), NFIC


(P08651), NFIX (Q14938), NFKB2 (Q00653), NHLRC2 (Q8NBF2), NHP2L1 (P55769), NID1


(P14543), NIP7 (Q9Y221), NIT1 (Q86X76), NIT2 (Q9NQR4), NLE1 (Q9NVX2), NLGN4X


(Q8N0W4), NLN (Q9BYT8), NMD3 (Q96D46), NME1 (P15531), NME2 (P22392), NME3


(Q13232), NME7 (Q9Y5B8), NMT1 (P30419), NNMT (P40261), NOB1 (Q9ULX3), NOL11


(Q9H8H0), NOL6 (Q9H6R4), NOMO2 (Q5JPE7), NONO (Q15233), NOP10 (Q9NPE3), NOP2


(P46087), NOTCH1 (P46531), NOTCH3 (Q9UM47), NOVA2 (Q9UNW9), NPEPPS (P55786),


NPLOC4 (Q8TAT6), NPM1 (P06748), NPM3 (O75607), NPTN (Q9Y639), NPW (Q8N729),


NQO1 (P15559), NQO2 (P16083), NR2C2AP (Q86WQ0), NRAS (P01111), NRBP1 (Q9UHY1),


NRBP2 (Q9NSY0), NRD1 (O43847), NRP2 (O60462), NSF (P46459), NSMAF (Q92636),


NSMCE1 (Q8WV22), NSUN2 (Q08J23), NT5C (Q8TCD5), NT5DC1 (Q5TFE4), NTN1


(O95631), NUBP1 (P53384), NUBP2 (Q9Y5Y2), NUCB1 (Q02818), NUDC (Q9Y266), NUDCD1


(Q96RS6), NUDCD2 (Q8WVJ2), NUDT1 (P36639), NUDT10 (Q8NFP7), NUDT12 (Q9BQG2),


NUDT16 (Q96DE0), NUDT16L1 (Q9BRJ7), NUDT2 (P50583), NUDT21 (O43809), NUDT4


(Q9NZJ9), NUDT5 (Q9UKK9), NUMA1 (Q14980), NUP188 (Q5SRE5), NUP37 (Q8NFH4),


NUP43 (Q8NFH3), NUP54 (Q7Z3B4), NUP88 (Q99567), NUP93 (Q8N1F7), NUTF2 (P61970),


NXN (Q6DKJ4), OBFC2B (Q9BQ15), OCRL (Q01968), ODZ2 (Q9NT68), ODZ3 (Q9P273),


OGFOD1 (Q8N543), OGT (O15294), OLA1 (Q9NTK5), OLFML3 (Q9NRN5), OPA1 (O60313),


OPLAH (O14841), OSBP (P22059), OSBPL1A (Q9BXW6), OSGEP (Q9NPF4), OTUB1


(Q96FW1), OVCA2 (Q8WZ82), OXCT1 (P55809), OXSR1 (O95747), P4HB (P07237), PA2G4


(Q9UQ80), PAAF1 (Q9BRP4), PABPC1 (P11940), PABPC4 (Q13310), PABPN1 (Q86U42),


PACSIN2 (Q9UNF0), PACSIN3 (Q9UKS6), PAF1 (Q8N7H5), PAFAH1B1 (P43034), PAFAH1B2


(P68402), PAFAH1B3 (Q15102), PAICS (P22234), PAIP1 (Q9H074), PAK2 (Q13177), PALD


(Q9ULE6), PALLD (Q8WX93), PANK4 (Q9NVE7), PAPOLA (P51003), PAPSS1 (O43252),


PARF (Q3YEC7), PARK7 (Q99497), PARN (O95453), PARP1 (P09874), PARP4 (Q9UKK3),


PARVA (Q9NVD7), PBK (Q96KB5), PBLD (P30039), PCBP1 (Q15365), PCBP2 (Q15366),


PCDHB2 (Q9Y5E7), PCDHGB4 (Q9UN71), PCDHGC3 (Q9UN70), PCID2 (Q5JVF3), PCMT1


(P22061), PCNA (P12004), PCOLCE2 (Q9UKZ9), PCYT2 (Q99447), PDCD10 (Q9BUL8),


PDCD2L (Q9BRP1), PDCD4 (Q53EL6), PDCD5 (O14737), PDCD6 (O75340), PDCD6IP


(Q8WUM4), PDCL3 (Q9H2J4), PDDC1 (Q8NB37), PDE12 (Q6L8Q7), PDE6D (O43924),


PDGFC (Q9NRA1), PDIA3 (P30101), PDIA6 (Q15084), PDLIM1 (O00151), PDLIM4 (P50479),


PDLIM5 (Q96HC4), PDLIM7 (Q9NR12), PDRG1 (Q9NUG6), PDRO (Q6IAA8), PDS5A


(Q29RF7), PDXK (O00764), PDXP (Q96GD0), PEA15 (Q15121), PEBP1 (P30086), PEF1


(Q9UBV8), PELO (Q9BRX2), PELP1 (Q8IZL8), PEPD (P12955), PFAS (O15067), PFDN2


(Q9UHV9), PFDN5 (Q99471), PFDN6 (O15212), PFKL (P17858), PFKM (P08237), PFKP


(Q01813), PFN1 (P07737), PFN2 (P35080), PGAM1 (P18669), PGAM5 (Q96HS1), PGD


(P52209), PGGT1B (P53609), PGK1 (P00558), PGLS (O95336), PGLYRP2 (Q96PD5), PGM1


(P36871), PGM2L1 (Q6PCE3), PGM3 (O95394), PGP (A6NDG6), PGRMC1 (O00264),


PGRMC2 (O15173), PHF5A (Q7RTV0), PHGDH (O43175), PHKB (Q93100), PHLDA3


(Q9Y5J5), PHPT1 (Q9NRX4), PIK3CB (P42338), PIK3R4 (Q99570), PIN1 (Q13526), PIP4K2A


(P48426), PIPOX (Q9P0Z9), PITPNB (P48739), PKM2 (P14618), PKP1 (Q13835), PLAA


(Q9Y263), PLCD3 (Q8N3E9), PLCG1 (P19174), PLD3 (Q8IV08), PLEC (Q15149), PLEKHB2


(Q96CS7), PLIN3 (O60664), PLOD1 (Q02809), PLOD2 (O00469), PLOD3 (O60568), PLRG1


(O43660), PLS1 (Q14651), PLS3 (P13797), PLSCR3 (Q9NRY6), PLTP (P55058), PLXNA1


(Q9UIW2), PLXNB2 (O15031), PLXND1 (Q9Y4D7), PM20D2 (Q8IYS1), PML (P29590), PMM2


(O15305), PMPCA (Q10713), PMPCB (O75439), PMVK (Q15126), PNMA2 (Q9UL42), PNO1


(Q9NRX1), PNP (P00491), PODXL (O00592), POLA1 (P09884), POLD1 (P28340), POLD2


(P49005), POLE3 (Q9NRF9), POLR1A (O95602), POLR1B (Q9H9Y6), POLR1C (O15160),


POLR1D (Q9Y2S0), POLR1E (Q9GZS1), POLR2A (P24928), POLR2B (P30876), POLR2C


(P19387), POLR2E (P19388), POLR2G (P62487), POLR2H (P52434), POLR2J (P52435),


POLR2L (P62875), POLR3A (O14802), POLR3B (Q9NW08), POLR3C (Q9BUI4), POLR3F


(Q9H1D9), POP1 (Q99575), POP4 (O95707), POP5 (Q969H6), POP7 (O75817), PPA1


(Q15181), PPA2 (Q9H2U2), PPAT (Q06203), PPCS (Q9HAB8), PPIA (P62937), PPIB


(P23284), PPID (Q08752), PPIF (P30405), PPIH (O43447), PPIL1 (Q9Y3C6), PPM1A


(P35813), PPM1F (P49593), PPM1G (O15355), PPME1 (Q9Y570), PPP1CA (P62136),


PPP1CB (P62140), PPP1CC (P36873), PPP1R7 (Q15435), PPP1R8 (Q12972), PPP2CA


(P67775), PPP2CB (P62714), PPP2R1A (P30153), PPP2R2A (P63151), PPP2R4 (Q15257),


PPP2R5C (Q13362), PPP2R5D (Q14738), PPP2R5E (Q16537), PPP3CA (Q08209), PPP4C


(P60510), PPP4R1 (Q8TF05), PPP5C (P53041), PPP6C (O00743), PPP6R3 (Q5H9R7),


PPPDE2 (Q6ICB0), PPT1 (P50897), PPWD1 (Q96BP3), PRCP (P42785), PRDX1 (Q06830),


PRDX2 (P32119), PRDX3 (P30048), PRDX5 (P30044), PRDX6 (P30041), PREP (P48147),


PREPL (Q4J6C6), PRIM1 (P49642), PRIM2 (P49643), PRKACA (P17612), PRKACB (P22694),


PRKAG1 (P54619), PRKAR1A (P10644), PRKAR2A (P13861), PRKAR2B (P31323), PRKDC


(P78527), PRMT1 (Q99873), PRMT3 (O60678), PRMT5 (O14744), PROM1 (O43490), PROSC


(O94903), PRPF19 (Q9UMS4), PRPF31 (Q8WWY3), PRPF4 (O43172), PRPF4B (Q13523),


PRPF8 (Q6P2Q9), PRPS1 (P60891), PRPS2 (P11908), PRPSAP1 (Q14558), PRPSAP2


(O60256), PRSS23 (O95084), PRTFDC1 (Q9NRG1), PSAT1 (Q9Y617), PSMA1 (P25786),


PSMA2 (P25787), PSMA3 (P25788), PSMA4 (P25789), PSMA5 (P28066), PSMA6 (P60900),


PSMA7 (O14818), PSMB1 (P20618), PSMB2 (P49721), PSMB3 (P49720), PSMB4 (P28070),


PSMB5 (P28074), PSMB6 (P28072), PSMB7 (Q99436), PSMB8 (P28062), PSMC1 (P62191),


PSMC2 (P35998), PSMC3 (P17980), PSMC4 (P43686), PSMC5 (P62195), PSMC6 (P62333),


PSMD1 (Q99460), PSMD10 (O75832), PSMD11 (O00231), PSMD12 (O00232), PSMD13


(Q9UNM6), PSMD14 (O00487), PSMD2 (Q13200), PSMD3 (O43242), PSMD4 (P55036),


PSMD5 (Q16401), PSMD6 (Q15008), PSMD7 (P51665), PSMD8 (P48556), PSMD9 (O00233),


PSME1 (Q06323), PSME2 (Q9UL46), PSME3 (P61289), PSME4 (Q14997), PSMF1 (Q92530),


PSMG1 (O95456), PSMG2 (Q969U7), PSMG3 (Q9BT73), PSPC1 (Q8WXF1), PSPH (P78330),


PTBP1 (P26599), PTGES3 (Q15185), PTGFRN (Q9P2B2), PTGR1 (Q14914), PTGR2


(Q8N8N7), PTK2 (Q05397), PTK7 (Q13308), PTN (P21246), PTP4A1 (Q93096), PTPN1


(P18031), PTPN11 (Q06124), PTPN23 (Q9H3S7), PTPRA (P18433), PTPRG (P23470),


PTPRZ1 (P23471), PUF60 (Q9UHX1), PUM1 (Q14671), PURB (Q96QR8), PUS7 (Q96PZ0),


PVR (P15151), PWP1 (Q13610), PXDN (Q92626), PXK (Q7Z7A4), PYCR1 (P32322), PYCRL


(Q53H96), PYGB (P11216), PYGL (P06737), QARS (P47897), QDPR (P09417), QKI


(Q96PU8), QRICH1 (Q2TAL8), QSOX2 (Q6ZRP7), QTRT1 (Q9BXR0), RAB10 (P61026),


RAB11A (P62491), RAB11FIP1 (Q6WKZ4), RAB12 (Q6IQ22), RAB13 (P51153), RAB14


(P61106), RAB18 (Q9NP72), RAB1A (P62820), RAB1B (Q9H0U4), RAB21 (Q9UL25), RAB22A


(Q9UL26), RAB23 (Q9ULC3), RAB27A (P51159), RAB2A (P61019), RAB34 (Q9BZG1), RAB35


(Q15286), RAB3A (P20336), RAB3GAP1 (Q15042), RAB3GAP2 (Q9H2M9), RAB4A (P20338),


RAB5A (P20339), RAB5B (P61020), RAB5C (P51148), RAB6A (P20340), RAB6B (Q9NRW1),


RAB7A (P51149), RAB8A (P61006), RAB8B (Q92930), RABAC1 (Q9UI14), RABGAP1


(Q9Y3P9), RABGGTA (Q92696), RABGGTB (P53611), RABIF (P47224), RAC1 (P63000),


RAD1 (O60671), RAD50 (Q92878), RAE1 (P78406), RAI14 (Q9P0K7), RALA (P11233), RALB


(P11234), RALY (Q9UKM9), RAN (P62826), RANBP1 (P43487), RANBP2 (P49792), RANBP6


(O60518), RANBP9 (Q96S59), RANGAP1 (P46060), RAP1A (P62834), RAP1B (P61224),


RAP1GDS1 (P52306), RAP2B (P61225), RARS (P54136), RASA1 (P20936), RBBP4 (Q09028),


RBBP5 (Q15291), RBBP7 (Q16576), RBBP9 (O75884), RBM12 (Q9NTZ6), RBM15 (Q96T37),


RBM17 (Q96I25), RBM22 (Q9NW64), RBM4 (Q9BWF3), RBMX (P38159), RBP1 (P09455),


RBPJ (Q06330), RBX1 (P62877), RCC1 (P18754), RCC2 (Q9P258), RCL (O43598), RDX


(P35241), RECQL (P46063), REEP5 (Q00765), REEP6 (Q96HR9), REPS1 (Q96D71), RFC4


(P35249), RFC5 (P40937), RFTN1 (Q14699), RHEB (Q15382), RHOA (P61586), RHOB


(P62745), RHOC (P08134), RHOF (Q9HBH0), RHOG (P84095), RIC8A (Q9NPQ8), RMND5A


(Q9H871), RNASEH2A (O75792), RNASEH2C (Q8TDP1), RNF123 (Q5XPI4), RNF20


(Q5VTR2), RNF213 (Q63HN8), RNF7 (Q9UBF6), RNGTT (O60942), RNH1 (P13489), RNMT


(O43148), RNPEP (Q9H4A4), ROBLD3 (Q9Y2Q5), ROCK1 (Q13464), ROCK2 (O75116),


ROR1 (Q01973), RP2 (O75695), RPA1 (P27694), RPA2 (P15927), RPA3 (P35244), RPE


(Q96AT9), RPF2 (Q9H7B2), RPL10 (P27635), RPL10A (P62906), RPL11 (P62913), RPL12


(P30050), RPL13 (P26373), RPL13A (P40429), RPL14 (P50914), RPL15 (P61313), RPL17


(P18621), RPL18 (Q07020), RPL18A (Q02543), RPL19 (P84098), RPL21 (P46778), RPL22


(P35268), RPL22L1 (Q6P5R6), RPL23 (P62829), RPL23A (P62750), RPL24 (P83731), RPL26


(P61254), RPL27 (P61353), RPL27A (P46776), RPL28 (P46779), RPL3 (P39023), RPL30


(P62888), RPL31 (P62899), RPL32 (P62910), RPL34 (P49207), RPL35 (P42766), RPL35A


(P18077), RPL36 (Q9Y3U8), RPL36A (P83881), RPL36AL (Q969Q0), RPL37A (P61513),


RPL38 (P63173), RPL4 (P36578), RPL5 (P46777), RPL6 (Q02878), RPL7 (P18124), RPL7A


(P62424), RPL8 (P62917), RPL9 (P32969), RPLP0 (P05388), RPLP1 (P05386), RPLP2


(P05387), RPP30 (P78346), RPP40 (O75818), RPRD1A (Q96P16), RPS10 (P46783), RPS11


(P62280), RPS12 (P25398), RPS13 (P62277), RPS14 (P62263), RPS15 (P62841), RPS15A


(P62244), RPS16 (P62249), RPS17 (P08708), RPS18 (P62269), RPS19 (P39019), RPS2


(P15880), RPS20 (P60866), RPS21 (P63220), RPS23 (P62266), RPS24 (P62847), RPS25


(P62851), RPS26 (P62854), RPS27 (P42677), RPS27A (P62979), RPS27L (Q71UM5), RPS28


(P62857), RPS29 (P62273), RPS3 (P23396), RPS3A (P61247), RPS4X (P62701), RPS4Y1


(P22090), RPS5 (P46782), RPS6 (P62753), RPS6KA3 (P51812), RPS7 (P62081), RPS8


(P62241), RPS9 (P46781), RPSA (P08865), RQCD1 (Q92600), RRAGA (Q7L523), RRAS


(P10301), RRAS2 (P62070), RRBP1 (Q9P2E9), RRM1 (P23921), RRM2 (P31350), RRM2B


(Q7LG56), RRP12 (Q5JTH9), RRP9 (O43818), RSL1D1 (O76021), RSU1 (Q15404), RTCD1


(O00442), RTN3 (O95197), RTN4 (Q9NQC3), RUVBL1 (Q9Y265), RUVBL2 (Q9Y230),


RWDD2B (P57060), S100A10 (P60903), S100A11 (P31949), S100A13 (Q99584), S100A16


(Q96FQ6), S100A4 (P26447), S100A6 (P06703), S100A8 (P05109), SAAL1 (Q96ER3), SACS


(Q9NZJ4), SAE1 (Q9UBE0), SAFB2 (Q14151), SAMHD1 (Q9Y3Z3), SAP18 (O00422), SAR1A


(Q9NR31), SARM1 (Q6SZW1), SARS (P49591), SART3 (Q15020), SBDS (Q9Y3A5), SBF1


(O95248), SCARB1 (Q8WTV0), SCARB2 (Q14108), SCFD1 (Q8WVM8), SCLY (Q96I15),


SCP2 (P22307), SCPEP1 (Q9HB40), SCRG1 (O75711), SCRIB (Q14160), SCRN1 (Q12765),


SCRN2 (Q96FV2), SCYL1 (Q96KG9), SCYL2 (Q6P3W7), SDC1 (P18827), SDC2 (P34741),


SDCBP (O00560), SDF4 (Q9BRK5), SDHA (P31040), SDK1 (Q7Z5N4), SDSL (Q96GA7),


SEC11A (P67812), SEC13 (P55735), SEC22B (O75396), SEC23A (Q15436), SEC23B


(Q15437), SEC23IP (Q9Y6Y8), SEC24A (O95486), SEC24B (O95487), SEC24C (P53992),


SEC24D (O94855), SEC31A (O94979), SEH1L (Q96EE3), SELH (Q8IZQ5), SEMA3A


(Q14563), SEPSECS (Q9HD40), 40787 (Q9NVA2), 37500 (Q15019), 38596 (Q99719), 39326


(Q16181), 39692 (Q92599), 40057 (Q9UHD8), SERBP1 (Q8NC51), SERPINA1 (P01009),


SERPINA3 (P01011), SERPINA7 (P05543), SERPINB6 (P35237), SERPINB8 (P50452),


SERPINE1 (P05121), SERPINE2 (P07093), SERPING1 (P05155), SERPINH1 (P50454),


SETD3 (Q86TU7), SETD7 (Q8WTS6), SF3A1 (Q15459), SF3A2 (Q15428), SF3A3 (Q12874),


SF3B1 (O75533), SF3B14 (Q9Y3B4), SF3B2 (Q13435), SF3B3 (Q15393), SF3B4 (Q15427),


SF3B5 (Q9BWJ5), SFPQ (P23246), SFRP4 (Q6FHJ7), SGTA (O43765), SH3BP4 (Q9P0V3),


SH3GL1 (Q99961), SH3GLB1 (Q9Y371), SHBG (P04278), SHC1 (P29353), SHMT1 (P34896),


SHMT2 (P34897), SHOC2 (Q9UQ13), SHPK (Q9UHJ6), SKIV2L (Q15477), SKIV2L2 (P42285),


SKP1 (P63208), SLC16A1 (P53985), SLC1A3 (P43003), SLC1A5 (Q15758), SLC29A1


(Q99808), SLC2A1 (P11166), SLC31A1 (O15431), SLC3A2 (P08195), SLC44A2 (Q8IWA5),


SLC5A3 (P53794), SLC7A5 (Q01650), SLC9A3R1 (O14745), SLC9A3R2 (Q15599), SLIRP


(Q9GZT3), SMAD4 (Q13485), SMARCA4 (P51532), SMARCA5 (O60264), SMARCC1


(Q92922), SMARCC2 (Q8TAQ2), SMARCD1 (Q96GM5), SMARCD2 (Q92925), SMARCE1


(Q969G3), SMC1A (Q14683), SMC2 (O95347), SMC3 (Q9UQE7), SMC4 (Q9NTJ3), SMC5


(Q8IY18), SMC6 (Q96SB8), SMCHD1 (A6NHR9), SMEK1 (Q6IN85), SMS (P52788), SMU1


(Q2TAY7), SMYD5 (Q6GMV2), SNAP23 (O00161), SNAPIN (O95295), SND1 (Q7KZF4), SNF8


(Q96H20), SNRNP200 (O75643), SNRNP40 (Q96DI7), SNRPA1 (P09661), SNRPB (P14678),


SNRPD1 (P62314), SNRPD2 (P62316), SNRPD3 (P62318), SNRPE (P62304), SNRPF


(P62306), SNRPG (P62308), SNTB1 (Q13884), SNUPN (O95149), SNX1 (Q13596), SNX12


(Q9UMY4), SNX17 (Q15036), SNX18 (Q96RF0), SNX2 (O60749), SNX27 (Q96L92), SNX3


(O60493), SNX5 (Q9Y5X3), SNX6 (Q9UNH7), SNX8 (Q9Y5X2), SNX9 (Q9Y5X1), SOD1


(P00441), SORD (Q00796), SORT1 (Q99523), SPAG9 (O60271), SPC24 (Q8NBT2), SPC25


(Q9HBM1), SPG21 (Q9NZD8), SPR (P35270), SPRYD4 (Q8WW59), SPTAN1 (Q13813),


SPTBN1 (Q01082), SPTBN2 (O15020), SRGAP2 (O75044), SRI (P30626), SRM (P19623),


SRP14 (P37108), SRP19 (P09132), SRP54 (P61011), SRP68 (Q9UHB9), SRP72 (O76094),


SRP9 (P49458), SRPX (P78539), SRPX2 (O60687), SRR (Q9GZT4), SRRT (Q9BXP5), SRSF1


(Q07955), SRSF11 (Q05519), SRSF2 (Q01130), SRSF3 (P84103), SRSF6 (Q13247), SRSF7


(Q16629), SRSF9 (Q13242), SRXN1 (Q9BYN0), SSB (P05455), SSBP1 (Q04837), SSRP1


(Q08945), SSSCA1 (O60232), ST13 (P50502), STAG2 (Q8N3U4), STAM (Q92783), STAMBP


(O95630), STAT1 (P42224), STAT3 (P40763), STIP1 (P31948), STK24 (Q9Y6E0), STK25


(O00506), STK38L (Q9Y2H1), STOM (P27105), STON2 (Q8WXE9), STRAP (Q9Y3F4), STUB1


(Q9UNE7), STX12 (Q86Y82), STX4 (Q12846), STX5 (Q13190), STX7 (O15400), STXBP1


(P61764), STXBP3 (O00186), STYX (Q8WUJ0), SUB1 (P53999), SUDS3 (Q9H7L9), SUGT1


(Q9Y2Z0), SUMO1 (P63165), SUPT16H (Q9Y5B9), SUPT4H1 (P63272), SUPT5H (O00267),


SUPT6H (Q7KZ85), SVEP1 (Q4LDE5), SWAP70 (Q9UH65), SYMPK (Q92797), SYNCRIP


(O60506), SYNE1 (Q8NF91), SYNE2 (Q8WXH0), SYNGR2 (O43760), SYNJ2BP (P57105),


TAB1 (Q15750), TAF9 (Q9Y3D8), TAF9 (Q16594), TAGLN (Q01995), TAGLN2 (P37802),


TALDO1 (P37837), TARDBP (Q13148), TARS (P26639), TATDN1 (Q6P1N9), TAX1BP3


(O14907), TBC1D13 (Q9NVG8), TBC1D15 (Q8TC07), TBC1D23 (Q9NUY8), TBC1D24


(Q9ULP9), TBC1D4 (O60343), TBC1D9B (Q66K14), TBCA (O75347), TBCB (Q99426), TBCD


(Q9BTW9), TBCE (Q15813), TBL1XR1 (Q9BZK7), TCEA1 (P23193), TCEB1 (Q15369), TCEB2


(Q15370), TCERG1 (O14776), TCP1 (P17987), TDP2 (O95551), TEP1 (Q99973), TEX10


(Q9NXF1), TF (P02787), TFCP2 (Q12800), TFG (Q92734), TFRC (P02786), TGFB1 (P01137),


TGFB2 (P61812), TGFBI (Q15582), TGM1 (P22735), TH1L (Q8IXH7), THBS1 (P07996),


THBS3 (P49746), THG1L (Q9NWX6), THOC2 (Q8NI27), THOC3 (Q96J01), THOC5 (Q13769),


THOC6 (Q86W42), THOC7 (Q6I9Y2), THOP1 (P52888), THUMPD1 (Q9NXG2), THY1


(P04216), THYN1 (Q9P016), TIA1 (P31483), TIGAR (Q9NQ88), TIMM13 (Q9Y5L4), TIMM50


(Q3ZCQ8), TIMM8B (Q9Y5J9), TIMM9 (Q9Y5J7), TIMP1 (P01033), TIPRL (O75663), TKT


(P29401), TLN1 (Q9Y490), TLN2 (Q9Y4G6), TM9SF2 (Q99805), TM9SF3 (Q9HD45), TMED10


(P49755), TMED2 (Q15363), TMED7 (Q9Y3B3), TMED9 (Q9BVK6), TMEM167A (Q8TBQ9),


TMEM2 (Q9UHN6), TMEM50B (P56557), TMEM87A (Q8NBN3), TMOD3 (Q9NYL9), TNC


(P24821), TNPO1 (Q92973), TNPO2 (O14787), TNPO3 (Q9Y5L0), TOLLIP (Q9H0E2),


TOMM20 (Q15388), TOMM22 (Q9NS69), TOMM34 (Q15785), TOMM5 (Q8N4H5), TOMM70A


(O94826), TOP1 (P11387), TOP2B (Q02880), TOR1B (O14657), TP53BP1 (Q12888), TP53RK


(Q96S44), TPI1 (P60174), TPM3 (P06753), TPM3L (A6NL28), TPM4 (P67936), TPMT


(P51580), TPP1 (O14773), TPP2 (P29144), TPR (P12270), TPRG1L (Q5T0D9), TPRKB


(Q9Y3C4), TPT1 (P13693), TRAF2 (Q12933), TRAP1 (Q12931), TRAPPC1 (Q9Y5R8),


TRAPPC2L (Q9UL33), TRAPPC3 (O43617), TRAPPC4 (Q9Y296), TRAPPC5 (Q8IUR0),


TRAPPC6A (O75865), TRAPPC6B (Q86SZ2), TRIM22 (Q8IYM9), TRIM25 (Q14258), TRIM28


(Q13263), TRIP12 (Q14669), TRIP13 (Q15645), TRIP6 (Q15654), TRMT1 (Q9NXH9),


TRMT112 (Q9UI30), TRMT5 (Q32P41), TRMT6 (Q9UJA5), TRMT61A (Q96FX7), TRNT1


(Q96Q11), TROVE2 (P10155), TRRAP (Q9Y4A5), TSG101 (Q99816), TSKU (Q8WUA8),


TSPAN14 (Q8NG11), TSPAN4 (O14817), TSPAN5 (P62079), TSPAN6 (O43657), TSPAN9


(O75954), TSSC1 (Q53HC9), TSTA3 (Q13630), TTC1 (Q99614), TTC37 (Q6PGP7), TTC38


(Q5R3I4), TTC5 (Q8N0Z6), TTC9C (Q8N5M4), TTL (Q8NG68), TTLL12 (Q14166), TTN


(Q8WZ42), TTR (P02766), TTYH1 (Q9H313), TTYH2 (Q9BSA4), TTYH3 (Q9C0H2), TUBA1B


(P68363), TUBA1C (Q9BQE3), TUBB (P07437), TUBB2A (Q13885), TUBB2B (Q9BVA1),


TUBB2C (P68371), TUBB3 (Q13509), TUBB4 (P04350), TUBB6 (Q9BUF5), TUBG1 (P23258),


TUBGCP2 (Q9BSJ2), TUBGCP3 (Q96CW5), TWF1 (Q12792), TWF2 (Q6IBS0), TXN (P10599),


TXNDC17 (Q9BRA2), TXNDC9 (O14530), TXNL1 (O43396), TXNL4B (Q9NX01), TXNRD1


(Q16881), TYMS (P04818), U2AF1 (Q01081), U2AF2 (P26368), UAP1 (Q16222), UBA1


(P22314), UBA2 (Q9UBT2), UBA3 (Q8TBC4), UBA5 (Q9GZZ9), UBA6 (A0AVT1), UBE2D1


(P51668), UBE2D3 (P61077), UBE2E1 (P51965), UBE2G2 (P60604), UBE2I (P63279),


UBE2J2 (Q8N2K1), UBE2K (P61086), UBE2L3 (P68036), UBE2M (P61081), UBE2N (P61088),


UBE2O (Q9C0C9), UBE2V1 (Q13404), UBE2V2 (Q15819), UBE2Z (Q9H832), UBE3A


(Q05086), UBE4A (Q14139), UBE4B (O95155), UBL3 (O95164), UBL4A (P11441), UBL5


(Q9BZL1), UBR1 (Q8IWV7), UBR4 (Q5T4S7), UBTD1 (Q9HAC8), UBXN1 (Q04323), UCHL1


(P09936), UCHL3 (P15374), UCHL5 (Q9Y5K5), UCK2 (Q9BZX2), UFC1 (Q9Y3C8), UFD1L


(Q92890), UFSP2 (Q9NUQ7), UGDH (O60701), UGP2 (Q16851), UMPS (P11172), UNC119B


(A6NIH7), UNC45A (Q9H3U1), UPF1 (Q92900), UPP1 (Q16831), UROD (P06132), UROS


(P10746), USO1 (O60763), USP10 (Q14694), USP11 (P51784), USP14 (P54578), USP15


(Q9Y4E8), USP24 (Q9UPU5), USP39 (Q53GS9), USP5 (P45974), USP7 (Q93009), USP9X


(Q93008), UTP15 (Q8TED0), UXS1 (Q8NBZ7), UXT (Q9UBK9), VAC14 (Q08AM6), VAMP3


(Q15836), VAMP5 (O95183), VAPA (Q9P0L0), VAPB (O95292), VARS (P26640), VASN


(Q6EMK4), VASP (P50552), VAT1 (Q99536), VAV2 (P52735), VBP1 (P61758), VCAN


(P13611), VCL (P18206), VCP (P55072), VIM (P08670), VPRBP (Q9Y4B6), VPS11 (Q9H270),


VPS13C (Q709C8), VPS16 (Q9H269), VPS18 (Q9P253), VPS24 (Q9Y3E7), VPS25 (Q9BRG1),


VPS26A (O75436), VPS26B (Q4G0F5), VPS28 (Q9UK41), VPS29 (Q9UBQ0), VPS33A


(Q96AX1), VPS33B (Q9H267), VPS35 (Q96QK1), VPS36 (Q86VN1), VPS37B (Q9H9H4),


VPS39 (Q96JC1), VPS45 (Q9NRW7), VPS4A (Q9UN37), VPS4B (O75351), VPS53 (Q5VIR6),


VRK1 (Q99986), VTA1 (Q9NP79), VWA1 (Q6PCB0), VWA5A (O00534), WARS (P23381),


WASF1 (Q92558), WASL (O00401), WDFY1 (Q8IWB7), WDR1 (O75083), WDR11 (Q9BZH6),


WDR12 (Q9GZL7), WDR18 (Q9BV38), WDR26 (Q9H7D7), WDR33 (Q9C0J8), WDR4


(P57081), WDR43 (Q15061), WDR45L (Q5MNZ6), WDR48 (Q8TAF3), WDR5 (P61964),


WDR54 (Q9H977), WDR55 (Q9H6Y2), WDR59 (Q6PJI9), WDR6 (Q9NNW5), WDR61


(Q9GZS3), WDR73 (Q6P4I2), WDR77 (Q9BQA1), WDR82 (Q6UXN9), WDR91 (A4D1P6),


WDR92 (Q96MX6), WNK1 (Q9H4A3), XPNPEP1 (Q9NQW7), XPO1 (O14980), XPO4


(Q9C0E2), XPO5 (Q9HAV4), XPO6 (Q96QU8), XPO7 (Q9UIA9), XPOT (O43592), XRCC1


(P18887), XRCC5 (P13010), XRCC6 (P12956), XRN2 (Q9H0D6), YARS (P54577), YBX1


(P67809), YEATS4 (O95619), YES1 (P07947), YIPF4 (Q9BSR8), YKT6 (O15498), YPEL5


(P62699), YRDC (Q86U90), YTHDF2 (Q9Y5A9), YWHAB (P31946), YWHAE (P62258),


YWHAG (P61981), YWHAH (Q04917), YWHAQ (P27348), YWHAZ (P63104), ZC3HAV1L


(Q96H79), ZCCHC3 (Q9NUD5), ZER1 (Q7Z7L7), ZFPL1 (O95159), ZFR (Q96KR1), ZMAT2


(Q96NC0), ZNF259 (O75312), ZW10 (O43264), ZWILCH (Q9H900), ZYG11B (Q9C0D3), ZYX


(Q15942), ZZEF1 (O43149).
















TABLE 19







100 most abundant proteins (name and SwissProt accession number)


observed in CTX0E03 exosomes








Identified proteins
Accession number





Actin, cytoplasmic 2
P63261


Glyceraldehyde-3-phosphate dehydrogenase
P04406


Histone H4
P62805


Pyruvate kinase isozymes M1/M2
P14618


Alpha-enolase
P06733


Heat shock protein HSP 90-beta
P08238


Ubiquitin-40S ribosomal protein S27a
P62979


Heat shock cognate 71 kDa protein
P11142


Haptoglobin
P00738


Heat shock protein HSP 90-alpha
P07900


Phosphoglycerate kinase 1
P00558


Actin, alpha cardiac muscle 1
P68032


40S ribosomal protein S3
P23396


Elongation factor 1-alpha 1
P68104


GTP-binding nuclear protein Ran
P62826


Histone H2B type 1-M
Q99879


Peptidyl-prolyl cis-trans isomerase A
P62937


Profilin-1
P07737


Elongation factor 2
P13639


Fatty acid synthase
P49327


Tubulin beta-2C chain
P68371


Tubulin alpha-1B chain
P68363


Tubulin beta chain
P07437


40S ribosomal protein S11
P62280


Eukaryotic initiation factor 4A-I
P60842


T-complex protein 1 subunit theta
P50990


14-3-3 protein theta
P27348


40S ribosomal protein S18
P62269


Tubulin beta-3 chain
Q13509


T-complex protein 1 subunit beta
P78371


40S ribosomal protein S16
P62249


Heat shock 70 kDa protein 1A/1B
P08107


Histone H3.2
Q71DI3


Transketolase
P29401


40S ribosomal protein SA
P08865


Clusterin
P10909


Fatty acid-binding protein, brain
O15540


Hemopexin
P02790


T-complex protein 1 subunit gamma
P49368


Tubulin beta-2B chain
Q9BVA1


Adenosylhomocysteinase
P23526


T-complex protein 1 subunit eta
Q99832


40S ribosomal protein S15a
P62244


T-complex protein 1 subunit delta
P50991


Vimentin
P08670


Guanine nucleotide-binding protein subunit beta-2-
P63244


like 1


Dihydropyrimidinase-related protein 3
Q14195


Elongation factor 1-gamma
P26641


Fascin
Q16658


Creatine kinase B-type
P12277


X-ray repair cross-complementing protein 5
P13010


40S ribosomal protein S2
P15880


Histone H2A type 2-A
Q6FI13


40S ribosomal protein S4, X isoform
P62701


14-3-3 protein zeta/delta
P63104


Heterogeneous nuclear ribonucleoprotein A1
P09651


CD81 antigen
P60033


Keratin, type I cytoskeletal 14
P02533


ATP-citrate synthase
P53396


40S ribosomal protein S9
P46781


Transgelin-2
P37802


Fructose-bisphosphate aldolase A
P04075


Ubiquitin-like modifier-activating enzyme 1
P22314


Peroxiredoxin-1
Q06830


40S ribosomal protein S5
P46782


T-complex protein 1 subunit epsilon
P48643


60S ribosomal protein L30
P62888


T-complex protein 1 subunit alpha
P17987


60S ribosomal protein L12
P30050


Cofilin-1
P23528


Heterogeneous nuclear ribonucleoproteins A2/B1
P22626


Eukaryotic translation initiation factor 5A-1
P63241


Phosphoglycerate mutase 1
P18669


Clathrin heavy chain 1
Q00610


Dihydropyrimidinase-related protein 2
Q16555


60S ribosomal protein L35a
P18077


T-complex protein 1 subunit zeta
P40227


Carbonyl reductase [NADPH] 1
P16152


40S ribosomal protein S3a
P61247


Ferritin heavy chain
P02794


Annexin A2
P07355


Myosin light polypeptide 6
P60660


Major vault protein
Q14764


Heterogeneous nuclear ribonucleoprotein D0
Q14103


60S acidic ribosomal protein P0
P05388


X-ray repair cross-complementing protein 6
P12956


40S ribosomal protein S20
P60866


Protein arginine N-methyltransferase 1
Q99873


40S ribosomal protein S10
P46783


Transaldolase
P37837


Histone H2B type 1-
P23527


Triosephosphate isomerase
P60174


Protein S100-A6
P06703


40S ribosomal protein S17
P08708


CD9 antigen
P21926


Filamin-A
P21333


Peptidyl-prolyl cis-trans isomerase FKBP4
Q02790


Programmed cell death 6-interacting protein
Q8WUM4


Glutathione S-transferase P
P09211


14-3-3 protein epsilon
P62258









Microvesicles

2940 proteins were identified by Mass spectrometry in Microvesicles isolated from the initial stages of an Integra culture (week 2) and purified by centrifugation at 10,000×g. The gene names and corresponding SWISSPROT accession numbers (in brackets) of all 2940 proteins are listed in Table 20 (in alphabetical order of gene name) and the 100 most abundant proteins are listed in Table 21, in order of decreasing abundance.









TABLE 20





Gene names and SWISSPROT accession numbers of all 2940 proteins identified in


CTX0E03 microvesicles (listed in alphabetical order of gene name).















A1BG (P04217), AACS (Q86V21), AAMP (Q13685), AARS (P49588), AARSD1 (Q9BTE6),


AASDHPPT (Q9NRN7), ABCA3 (Q99758), ABCC1 (P33527), ABCC4 (O15439), ABCE1


(P61221), ABCF1 (Q8NE71), ABCF2 (Q9UG63), ABCF3 (Q9NUQ8), ABHD14B (Q96IU4), ABI1


(Q8IZP0), ABR (Q12979), ACAA1 (P09110), ACAA2 (P42765), ACACA (Q13085), ACADM


(P11310), ACADVL (P49748), ACAT1 (P24752), ACAT2 (Q9BWD1), ACBD6 (Q9BR61),


ACBD7 (Q8N6N7), ACLY (P53396), ACO1 (P21399), ACO2 (Q99798), ACOT1 (Q86TX2),


ACOT13 (Q9NPJ3), ACOT7 (O00154), ACOX1 (Q15067), ACOX3 (O15254), ACP1 (P24666),


ACSL1 (P33121), ACSL3 (O95573), ACSL4 (O60488), ACSS2 (Q9NR19), ACTC1 (P68032),


ACTG1 (P63261), ACTL6A (O96019), ACTN1 (P12814), ACTN4 (O43707), ACTR10 (Q9NZ32),


ACTR1A (P61163), ACTR1B (P42025), ACTR2 (P61160), ACTR3 (P61158), ACY1 (Q03154),


ADAM10 (O14672), ADAM9 (Q13443), ADAMTS15 (Q8TE58), ADAMTS16 (Q8TE57), ADAR


(P55265), ADD1 (P35611), ADD3 (Q9UEY8), ADH5 (P11766), ADK (P55263), ADO (Q96SZ5),


ADPRH (P54922), ADRBK1 (P25098), ADRM1 (Q16186), ADSL (P30566), ADSS (P30520),


AEBP1 (Q8IUX7), AFM (P43652), AGL (P35573), AGPS (O00116), AGRN (O00468), AHCY


(P23526), AHCYL1 (O43865), AHNAK (Q09666), AHNAK2 (Q8IVF2), AHSA1 (O95433), AHSG


(P02765), AIDA (Q96BJ3), AIFM1 (O95831), AIMP1 (Q12904), AIMP2 (Q13155), AIP


(O00170), AK1 (P00568), AK2 (P54819), AK3 (Q9UIJ7), AK4 (P27144), AKAP12 (Q02952),


AKAP9 (Q99996), AKR1A1 (P14550), AKR1B1 (P15121), AKR1C1 (Q04828), AKR7A2


(O43488), AKR7A3 (O95154), AKT1 (P31749), ALCAM (Q13740), ALDH16A1 (Q8IZ83),


ALDH18A1 (P54886), ALDH2 (P05091), ALDH3A1 (P30838), ALDH7A1 (P49419), ALDH9A1


(P49189), ALDOA (P04075), ALDOC (P09972), ALKBH2 (Q6NS38), ALOX12B (O75342),


AMDHD2 (Q9Y303), AMPD2 (Q01433), ANAPC1 (Q9H1A4), ANAPC4 (Q9UJX5), ANAPC5


(Q9UJX4), ANAPC7 (Q9UJX3), ANKFY1 (Q9P2R3), ANKRD17 (O75179), ANKRD28


(O15084), ANKRD52 (Q8NB46), ANP32A (P39687), ANP32B (Q92688), ANP32E (Q9BTT0),


ANXA1 (P04083), ANXA11 (P50995), ANXA2 (P07355), ANXA3 (P12429), ANXA4 (P09525),


ANXA5 (P08758), ANXA6 (P08133), ANXA7 (P20073), AP1B1 (Q10567), AP1G1 (O43747),


AP1M1 (Q9BXS5), AP1S2 (P56377), AP2A1 (O95782), AP2A2 (O94973), AP2B1 (P63010),


AP2M1 (Q96CW1), AP2S1 (P53680), AP3B1 (O00203), AP3D1 (O14617), AP3M1 (Q9Y2T2),


AP3S1 (Q92572), AP4S1 (Q9Y587), APEH (P13798), APEX1 (P27695), API5 (Q9BZZ5), APIP


(Q96GX9), APMAP (Q9HDC9), APOA2 (P02652), APOBEC3C (Q9NRW3), APOH (P02749),


APOL2 (Q9BQE5), APPL1 (Q9UKG1), APRT (P07741), AQR (O60306), ARAF (P10398),


ARCN1 (P48444), ARF1 (P84077), ARF4 (P18085), ARF6 (P62330), ARFGAP2 (Q8N6H7),


ARFIP1 (P53367), ARFIP2 (P53365), ARG1 (P05089), ARHGAP1 (Q07960), ARHGAP5


(Q13017), ARHGDIA (P52565), ARHGEF1 (Q92888), ARHGEF10 (O15013), ARHGEF6


(Q15052), ARHGEF7 (Q14155), ARIH1 (Q9Y4X5), ARIH2 (O95376), ARL1 (P40616), ARL2


(P36404), ARL3 (P36405), ARL6IP1 (Q15041), ARL8A (Q96BM9), ARL8B (Q9NVJ2), ARMC10


(Q8N2F6), ARMC6 (Q6NXE6), ARMC8 (Q8IUR7), ARMC9 (Q7Z3E5), ARPC1A (Q92747),


ARPC1B (O15143), ARPC2 (O15144), ARPC3 (O15145), ARPC4 (P59998), ARPC5 (O15511),


ARPC5L (Q9BPX5), ASAH1 (Q13510), ASCC1 (Q8N9N2), ASCC3 (Q8N3C0), ASMTL


(O95671), ASNA1 (O43681), ASNS (P08243), ASPSCR1 (Q9BZE9), ASS1 (P00966), ATAD3A


(Q9NVI7), ATE1 (O95260), ATG101 (Q9BSB4), ATG16L1 (Q676U5), ATG3 (Q9NT62), ATG4B


(Q9Y4P1), ATG7 (O95352), ATIC (P31939), ATL3 (Q6DD88), ATM (Q13315), ATOX1


(O00244), ATP1A1 (P05023), ATP1B1 (P05026), ATP1B3 (P54709), ATP2A2 (P16615),


ATP2B1 (P20020), ATP2B4 (P23634), ATP5A1 (P25705), ATP5B (P06576), ATP5C1


(P36542), ATP5E (P56381), ATP5F1 (P24539), ATP5H (O75947), ATP5I (P56385), ATP5L


(O75964), ATP5O (P48047), ATP6AP1 (Q15904), ATP6AP2 (O75787), ATP6V0A1 (Q93050),


ATP6V0D1 (P61421), ATP6V1A (P38606), ATP6V1B2 (P21281), ATP6V1C1 (P21283),


ATP6V1D (Q9Y5K8), ATP6V1E1 (P36543), ATP6V1G1 (O75348), ATP6V1H (Q9UI12), ATR


(Q13535), ATRN (O75882), ATXN10 (Q9UBB4), B2M (P61769), B3GAT3 (O94766), B3GNT1


(O43505), BAG2 (O95816), BAG5 (Q9UL15), BAIAP2 (Q9UQB8), BANF1 (O75531), BAT1


(Q13838), BAT3 (P46379), BCAM (P50895), BCAS2 (O75934), BCAT1 (P54687), BCCIP


(Q9P287), BCL2L12 (Q9HB09), BDH2 (Q9BUT1), BICD2 (Q8TD16), BLMH (Q13867), BLVRA


(P53004), BLVRB (P30043), BMP1 (P13497), BOLA2 (Q9H3K6), BOP1 (Q14137), BPGM


(P07738), BPNT1 (O95861), BRCC3 (P46736), BRE (Q9NXR7), BRIX1 (Q8TDN6), BROX


(Q5VW32), BRP16L (P0CB43), BSG (P35613), BST1 (Q10588), BTAF1 (O14981), BUB3


(O43684), BUD31 (P41223), BYSL (Q13895), BZW1 (Q7L1Q6), BZW2 (Q9Y6E2), C10orf119


(Q9BTE3), C10orf58 (Q9BRX8), C10orf76 (Q5T2E6), C11orf54 (Q9H0W9), C11orf68


(Q9H3H3), C12orf10 (Q9HB07), C12orf57 (Q99622), C14orf149 (Q96EM0), C14orf166


(Q9Y224), C14orf21 (Q86U38), C15orf58 (Q6ZNW5), C16orf13 (Q96S19), C16orf61


(Q9NRP2), C16orf80 (Q9Y6A4), C18orf21 (Q32NC0), C18orf8 (Q96DM3), C1orf123


(Q9NWV4), C1orf128 (Q9GZP4), C1orf57 (Q9BSD7), C20orf11 (Q9NWU2), C20orf4 (Q9Y312),


C21orf33 (P30042), C21orf59 (P57076), C22orf28 (Q9Y3I0), C3orf10 (Q8WUW1), C3orf26


(Q9BQ75), C3orf75 (Q0PNE2), C4orf27 (Q9NWY4), C4orf41 (Q7Z392), C4orf43 (Q96EY4),


C5orf33 (Q4G0N4), C6orf211 (Q9H993), C7orf28B (P86790), C7orf50 (Q9BRJ6), C7orf59


(Q0VGL1), C8orf33 (Q9H7E9), C9orf142 (Q9BUH6), C9orf23 (Q8N5L8), C9orf41 (Q8N4J0),


C9orf64 (Q5T6V5), CA11 (O75493), CA12 (O43570), CA2 (P00918), CAB39 (Q9Y376),


CACNA2D1 (P54289), CACYBP (Q9HB71), CAD (P27708), CADM1 (Q9BY67), CADM4


(Q8NFZ8), CALB1 (P05937), CALD1 (Q05682), CALM1 (P62158), CALR (P27797), CALU


(O43852), CAMK1 (Q14012), CAMK2D (Q13557), CAMKV (Q8NCB2), CAND1 (Q86VP6),


CANX (P27824), CAP1 (Q01518), CAPN1 (P07384), CAPN2 (P17655), CAPN5 (O15484),


CAPN7 (Q9Y6W3), CAPNS1 (P04632), CAPRIN1 (Q14444), CAPS (Q13938), CAPZA1


(P52907), CAPZA2 (P47755), CAPZB (P47756), CARHSP1 (Q9Y2V2), CARKD (Q8IW45),


CARM1 (Q86X55), CARS (P49589), CASK (O14936), CASP14 (P31944), CASP3 (P42574),


CASP7 (P55210), CAT (P04040), CBFB (Q13951), CBR1 (P16152), CBR3 (O75828), CBS


(P35520), CBX1 (P83916), CBX3 (Q13185), CBX5 (P45973), CC2D1A (Q6P1N0), CCAR1


(Q8IX12), CCBL2 (Q6YP21), CCDC102B (Q68D86), CCDC22 (O60826), CCDC25 (Q86WR0),


CCDC93 (Q567U6), CCND2 (P30279), CCNY (Q8ND76), CCT2 (P78371), CCT3 (P49368),


CCT4 (P50991), CCT5 (P48643), CCT6A (P40227), CCT7 (Q99832), CCT8 (P50990), CD109


(Q6YHK3), CD151 (P48509), CD276 (Q5ZPR3), CD44 (P16070), CD46 (P15529), CD47


(Q08722), CD58 (P19256), CD59 (P13987), CD63 (P08962), CD81 (P60033), CD9 (P21926),


CD97 (P48960), CD99 (P14209), CDC123 (O75794), CDC16 (Q13042), CDC23 (Q9UJX2),


CDC34 (P49427), CDC37 (Q16543), CDC40 (O60508), CDC42 (P60953), CDC42BPB


(Q9Y5S2), CDC5L (Q99459), CDCP1 (Q9H5V8), CDH2 (P19022), CDK1 (P06493), CDK2


(P24941), CDK4 (P11802), CDK5 (Q00535), CDK5RAP3 (Q96JB5), CDK7 (P50613), CDKN2A


(P42771), CDKN2AIP (Q9NXV6), CECR5 (Q9BXW7), CELF1 (Q92879), CELSR1 (Q9NYQ6),


CELSR2 (Q9HCU4), CFL1 (P23528), CFL2 (Q9Y281), CHCHD3 (Q9NX63), CHD4 (Q14839),


CHEK2 (O96017), CHERP (Q8IWX8), CHID1 (Q9BWS9), CHMP1A (Q9HD42), CHMP1B


(Q7LBR1), CHMP2A (O43633), CHMP4A (Q9BY43), CHMP4B (Q9H444), CHMP5 (Q9NZZ3),


CHMP6 (Q96FZ7), CHN1 (P15882), CHORDC1 (Q9UHD1), CHP (Q99653), CHRAC1


(Q9NRG0), CHST3 (Q7LGC8), CIAO1 (O76071), CIAPIN1 (Q6FI81), CIRBP (Q14011),


CIRH1A (Q969X6), CISD2 (Q8N5K1), CKAP4 (Q07065), CKAP5 (Q14008), CKB (P12277),


CLASP1 (Q7Z460), CLIC1 (O00299), CLIC4 (Q9Y696), CLLD6 (Q5W111), CLNS1A (P54105),


CLPB (Q9H078), CLTA (P09496), CLTC (Q00610), CLTCL1 (P53675), CLU (P10909), CMBL


(Q96DG6), CMC1 (Q7Z7K0), CMPK1 (P30085), CMTM6 (Q9NX76), CNBP (P62633), CNDP2


(Q96KP4), CNN2 (Q99439), CNN3 (Q15417), CNNM3 (Q8NE01), CNOT1 (A5YKK6), CNOT10


(Q9H9A5), CNOT6L (Q96LI5), CNP (P09543), COASY (Q13057), COBRA1 (Q8WX92), COG1


(Q8WTW3), COG3 (Q96JB2), COG4 (Q9H9E3), COG5 (Q9UP83), COG6 (Q9Y2V7), COL11A1


(P12107), COL14A1 (Q05707), COL18A1 (P39060), COL6A1 (P12109), COMMD10 (Q9Y6G5),


COMMD2 (Q86X83), COMMD3 (Q9UBI1), COMMD5 (Q9GZQ3), COMMD8 (Q9NX08),


COMMD9 (Q9P000), COMT (P21964), COPA (P53621), COPB1 (P53618), COPB2 (P35606),


COPE (O14579), COPG (Q9Y678), COPG2 (Q9UBF2), COPS2 (P61201), COPS3 (Q9UNS2),


COPS4 (Q9BT78), COPS5 (Q92905), COPS6 (Q7L5N1), COPS7A (Q9UBW8), COPS7B


(Q9H9Q2), COPS8 (Q99627), CORO1B (Q9BR76), CORO1C (Q9ULV4), CORO2B (Q9UQ03),


CORO7 (P57737), COTL1 (Q14019), COX4NB (O43402), COX5A (P20674), COX5B (P10606),


COX6C (P09669), CP (P00450), CPD (O75976), CPNE1 (Q99829), CPNE2 (Q96FN4), CPNE3


(O75131), CPNE4 (Q96A23), CPNE7 (Q9UBL6), CPOX (P36551), CPSF1 (Q10570), CPSF2


(Q9P2I0), CPSF3 (Q9UKF6), CPSF3L (Q5TA45), CPSF6 (Q16630), CPSF7 (Q8N684), CPXM1


(Q96SM3), CRABP2 (P29373), CRIP2 (P52943), CRK (P46108), CRLF3 (Q8IUI8), CRNKL1


(Q9BZJ0), CRTAP (O75718), CRYAB (P02511), CRYM (Q14894), CRYZ (Q08257), CRYZL1


(O95825), CS (O75390), CSDE1 (O75534), CSE1L (P55060), CSK (P41240), CSNK1A1


(P48729), CSNK2A1 (P68400), CSNK2A2 (P19784), CSNK2B (P67870), CSRP1 (P21291),


CSRP2 (Q16527), CSTB (P04080), CSTF1 (Q05048), CSTF2T (Q9H0L4), CSTF3 (Q12996),


CTBP1 (Q13363), CTBP2 (P56545), CTNNA1 (P35221), CTNNAL1 (Q9UBT7), CTNNB1


(P35222), CTNNBL1 (Q8WYA6), CTNND1 (O60716), CTPS (P17812), CTPS2 (Q9NRF8),


CTR9 (Q6PD62), CTSC (P53634), CTSD (P07339), CTSF (Q9UBX1), CTSL2 (O60911), CTTN


(Q14247), CTU1 (Q7Z7A3), CUL1 (Q13616), CUL2 (Q13617), CUL3 (Q13618), CUL4A


(Q13619), CUL4B (Q13620), CUL5 (Q93034), CUL7 (Q14999), CXADR (P78310), CXCL14


(O95715), CXorf26 (Q9BVG4), CXorf38 (Q8TB03), CYB5R3 (P00387), CYC1 (P08574), CYCS


(P99999), CYFIP1 (Q7L576), CYFIP2 (Q96F07), CYR61 (O00622), DAB1 (O75553), DAD1


(P61803), DAG1 (Q14118), DAK (Q3LXA3), DAPK3 (O43293), DARS (P14868), DAZAP1


(Q96EP5), DBI (P07108), DBN1 (Q16643), DBNL (Q9UJU6), DCAF7 (P61962), DCAF8


(Q5TAQ9), DCBLD2 (Q96PD2), DCK (P27707), DCLK1 (O15075), DCPS (Q96C86), DCTD


(P32321), DCTN1 (Q14203), DCTN2 (Q13561), DCTN3 (O75935), DCTN4 (Q9UJW0), DCTN5


(Q9BTE1), DCTN6 (O00399), DCUN1D1 (Q96GG9), DCUN1D3 (Q8IWE4), DCUN1D5


(Q9BTE7), DCXR (Q7Z4W1), DDA1 (Q9BW61), DDAH1 (O94760), DDAH2 (O95865), DDB1


(Q16531), DDB2 (Q92466), DDI2 (Q5TDH0), DDOST (P39656), DDR1 (Q08345), DDT


(P30046), DDX1 (Q92499), DDX17 (Q92841), DDX18 (Q9NVP1), DDX19A (Q9NUU7), DDX20


(Q9UHI6), DDX21 (Q9NR30), DDX23 (Q9BUQ8), DDX24 (Q9GZR7), DDX27 (Q96GQ7),


DDX39 (O00148), DDX3X (O00571), DDX46 (Q7L014), DDX47 (Q9H0S4), DDX49 (Q9Y6V7),


DDX5 (P17844), DDX50 (Q9BQ39), DDX51 (Q8N8A6), DDX52 (Q9Y2R4), DDX54 (Q8TDD1),


DDX55 (Q8NHQ9), DDX56 (Q9NY93), DDX6 (P26196), DECR1 (Q16698), DECR2 (Q9NUI1),


DEF (Q68CQ4), DEK (P35659), DENR (O43583), DERA (Q9Y315), DFFA (O00273), DFFB


(O76075), DHCR24 (Q15392), DHCR7 (Q9UBM7), DHFR (P00374), DHPS (P49366), DHRS11


(Q6UWP2), DHRS4 (Q9BTZ2), DHX15 (O43143), DHX16 (O60231), DHX29 (Q7Z478), DHX30


(Q7L2E3), DHX32 (Q7L7V1), DHX36 (Q9H2U1), DHX37 (Q8IY37), DHX38 (Q92620), DHX9


(Q08211), DIAPH1 (O60610), DIAPH2 (O60879), DIMT1L (Q9UNQ2), DIP2A (Q14689), DIP2B


(Q9P265), DIP2C (Q9Y2E4), DIS3 (Q9Y2L1), DIS3L2 (Q8IYB7), DKC1 (O60832), DLAT


(P10515), DLD (P09622), DLG1 (Q12959), DLGAP4 (Q9Y2H0), DLST (P36957), DMD


(P11532), DNAJA1 (P31689), DNAJA2 (O60884), DNAJB1 (P25685), DNAJB11 (Q9UBS4),


DNAJB4 (Q9UDY4), DNAJB6 (O75190), DNAJC13 (O75165), DNAJC2 (Q99543), DNAJC3


(Q13217), DNAJC7 (Q99615), DNASE1L1 (P49184), DNM1 (Q05193), DNM1L (O00429),


DNM2 (P50570), DNMT1 (P26358), DNPEP (Q9ULA0), DOCK1 (Q14185), DOCK4 (Q8N1I0),


DOCK5 (Q9H7D0), DOCK7 (Q96N67), DOCK9 (Q9BZ29), DOHH (Q9BU89), DPCD


(Q9BVM2), DPH2 (Q9BQC3), DPH5 (Q9H2P9), DPM1 (O60762), DPM3 (Q9P2X0), DPP3


(Q9NY33), DPP9 (Q86TI2), DPY30 (Q9C005), DPYSL2 (Q16555), DPYSL3 (Q14195), DPYSL4


(O14531), DPYSL5 (Q9BPU6), DRG1 (Q9Y295), DRG2 (P55039), DSC1 (Q08554), DSG1


(Q02413), DSP (P15924), DST (Q03001), DSTN (P60981), DTD1 (Q8TEA8), DTNA (Q9Y4J8),


DTYMK (P23919), DUS2L (Q9NX74), DUS3L (Q96G46), DUSP12 (Q9UNI6), DUSP3 (P51452),


DYM (Q7RTS9), DYNC1H1 (Q14204), DYNC1I2 (Q13409), DYNC1LI1 (Q9Y6G9), DYNC1LI2


(O43237), DYNC2H1 (Q8NCM8), DYNLL1 (P63167), DYNLL2 (Q96FJ2), DYNLRB1 (Q9NP97),


DYNLT1 (P63172), EBNA1BP2 (Q99848), ECE1 (P42892), ECHDC1 (Q9NTX5), ECHS1


(P30084), ECM29 (Q5VYK3), EDC3 (Q96F86), EDC4 (Q6P2E9), EEA1 (Q15075), EEF1A1


(P68104), EEF1B2 (P24534), EEF1D (P29692), EEF1E1 (O43324), EEF1G (P26641), EEF2


(P13639), EEF2K (O00418), EEFSEC (P57772), EFEMP2 (O95967), EFHD2 (Q96C19),


EFTUD1 (Q7Z2Z2), EFTUD2 (Q15029), EGFR (P00533), EHD1 (Q9H4M9), EHD2 (Q9NZN4),


EHD3 (Q9NZN3), EHD4 (Q9H223), EIF1AX (P47813), EIF2A (Q9BY44), EIF2AK2 (P19525),


EIF2AK4 (Q9P2K8), EIF2B1 (Q14232), EIF2B2 (P49770), EIF2B3 (Q9NR50), EIF2B4


(Q9UI10), EIF2B5 (Q13144), EIF2C1 (Q9UL18), EIF2C2 (Q9UKV8), EIF2S1 (P05198), EIF2S2


(P20042), EIF2S3 (P41091), EIF3A (Q14152), EIF3B (P55884), EIF3C (Q99613), EIF3D


(O15371), EIF3E (P60228), EIF3F (O00303), EIF3G (O75821), EIF3H (O15372), EIF3I


(Q13347), EIF3J (O75822), EIF3K (Q9UBQ5), EIF3L (Q9Y262), EIF3M (Q7L2H7), EIF4A1


(P60842), EIF4A2 (Q14240), EIF4A3 (P38919), EIF4E (P06730), EIF4G1 (Q04637), EIF4G2


(P78344), EIF4H (Q15056), EIF5 (P55010), EIF5A (P63241), EIF5B (O60841), EIF6 (P56537),


ELAC2 (Q9BQ52), ELAVL1 (Q15717), ELMO2 (Q96JJ3), ELP2 (Q6IA86), ELP3 (Q9H9T3),


EMD (P50402), EMG1 (Q92979), EML1 (O00423), EML2 (O95834), EML3 (Q32P44), EML4


(Q9HC35), ENAH (Q8N8S7), ENC1 (O14682), ENO1 (P06733), ENO2 (P09104), ENOPH1


(Q9UHY7), ENY2 (Q9NPA8), EPB41L2 (O43491), EPB41L3 (Q9Y2J2), EPDR1 (Q9UM22),


EPHA2 (P29317), EPHB2 (P29323), EPHB3 (P54753), EPHB4 (P54760), EPHX1 (P07099),


EPM2AIP1 (Q7L775), EPN1 (Q9Y6I3), EPRS (P07814), ERBB2IP (Q96RT1), ERGIC1


(Q969X5), ERH (P84090), ERI1 (Q8IV48), ERI3 (O43414), ERLIN2 (O94905), ERO1L


(Q96HE7), ERP29 (P30040), ERP44 (Q9BS26), ESD (P10768), ESYT1 (Q9BSJ8), ETF1


(P62495), ETFA (P13804), ETFB (P38117), EXOC1 (Q9NV70), EXOC2 (Q96KP1), EXOC3


(O60645), EXOC4 (Q96A65), EXOC5 (O00471), EXOC6 (Q8TAG9), EXOC6B (Q9Y2D4),


EXOC7 (Q9UPT5), EXOC8 (Q8IYI6), EXOSC1 (Q9Y3B2), EXOSC10 (Q01780), EXOSC2


(Q13868), EXOSC3 (Q9NQT5), EXOSC4 (Q9NPD3), EXOSC5 (Q9NQT4), EXOSC6


(Q5RKV6), EXOSC7 (Q15024), EXOSC8 (Q96B26), EXOSC9 (Q06265), EZR (P15311), F11R


(Q9Y624), F8 (P00451), F8A1 (P23610), FABP5 (Q01469), FABP7 (O15540), FADD (Q13158),


FAH (P16930), FAHD1 (Q6P587), FAHD2A (Q96GK7), FAM115A (Q9Y4C2), FAM120A


(Q9NZB2), FAM125A (Q96EY5), FAM127A (A6ZKI3), FAM129A (Q9BZQ8), FAM129B


(Q96TA1), FAM136A (Q96C01), FAM175B (Q15018), FAM3C (Q92520), FAM45B (Q6NSW5),


FAM49B (Q9NUQ9), FAM82B (Q96DB5), FAM84B (Q96KN1), FAM96B (Q9Y3D0), FAM98A


(Q8NCA5), FAM98B (Q52LJ0), FANCI (Q9NVI1), FAR1 (Q8WVX9), FARP1 (Q9Y4F1), FARP2


(O94887), FARSA (Q9Y285), FARSB (Q9NSD9), FAS (P25445), FASN (P49327), FAT1


(Q14517), FAU (P62861), FBL (P22087), FBLN2 (P98095), FBN1 (P35555), FBN2 (P35556),


FBXL18 (Q96ME1), FBXO21 (O94952), FBXO22 (Q8NEZ5), FBXW11 (Q9UKB1), FCF1


(Q9Y324), FDFT1 (P37268), FDPS (P14324), FDXR (P22570), FEN1 (P39748), FERMT1


(Q9BQL6), FERMT2 (Q96AC1), FFR (Q9UID3), FGFBP3 (Q8TAT2), FH (P07954), FHL1


(Q13642), FHL2 (Q14192), FHL3 (Q13643), FIBP (O43427), FKBP10 (Q96AY3), FKBP1A


(P62942), FKBP2 (P26885), FKBP3 (Q00688), FKBP4 (Q02790), FKBP5 (Q13451), FLG


(P20930), FLG2 (Q5D862), FLII (Q13045), FLNA (P21333), FLNB (O75369), FLNC (Q14315),


FLOT1 (O75955), FLOT2 (Q14254), FMNL2 (Q96PY5), FN3K (Q9H479), FN3KRP (Q9HA64),


FNTA (P49354), FNTB (P49356), FOLR1 (P15328), FREM2 (Q5SZK8), FRG1 (Q14331),


FRMD5 (Q7Z6J6), FRMD8 (Q9BZ67), FRYL (O94915), FSCN1 (Q16658), FSD1 (Q9BTV5),


FTH1 (P02794), FTL (P02792), FTO (Q9C0B1), FTSJD2 (Q8N1G2), FUBP1 (Q96AE4), FUBP3


(Q96I24), FUCA2 (Q9BTY2), FUK (Q8N0W3), FUS (P35637), FXR1 (P51114), FXR2 (P51116),


FYCO1 (Q9BQS8), FYN (P06241), G3BP1 (Q13283), G3BP2 (Q9UN86), G6PD (P11413), GAA


(P10253), GALK1 (P51570), GALK2 (Q01415), GALNT1 (Q10472), GALNT2 (Q10471),


GALNT7 (Q86SF2), GAN (Q9H2C0), GANAB (Q14697), GAP43 (P17677), GAPDH (P04406),


GAPVD1 (Q14C86), GAR1 (Q9NY12), GARS (P41250), GART (P22102), GATSL2 (A6NHX0),


GBA (P04062), GBE1 (Q04446), GBF1 (Q92538), GCDH (Q92947), GCLC (P48506), GCLM


(P48507), GCN1L1 (Q92616), GDI1 (P31150), GDI2 (P50395), GEMIN4 (P57678), GEMIN5


(Q8TEQ6), GEMIN6 (Q8WXD5), GET4 (Q7L5D6), GFAP (P14136), GFM1 (Q96RP9), GFPT1


(Q06210), GFPT2 (O94808), GGCT (O75223), GGPS1 (O95749), GINS1 (Q14691), GINS2


(Q9Y248), GINS4 (Q9BRT9), GIPC1 (O14908), GIT1 (Q9Y2X7), GLA (P06280), GLB1L2


(Q8IW92), GLE1 (Q53GS7), GLG1 (Q92896), GLIPR2 (Q9H4G4), GLMN (Q92990), GLO1


(Q04760), GLOD4 (Q9HC38), GLRX (P35754), GLRX3 (O76003), GLT25D1 (Q8NBJ5),


GLT25D2 (Q8IYK4), GLTP (Q9NZD2), GLUD1 (P00367), GLUL (P15104), GMDS (O60547),


GMFB (P60983), GMPPA (Q96IJ6), GMPPB (Q9Y5P6), GMPR (P36959), GMPR2 (Q9P2T1),


GMPS (P49915), GNA11 (P29992), GNA12 (Q03113), GNA13 (Q14344), GNAI1 (P63096),


GNAI2 (P04899), GNAI3 (P08754), GNAQ (P50148), GNAS (Q5JWF2), GNB1 (P62873),


GNB1L (Q9BYB4), GNB2 (P62879), GNB2L1 (P63244), GNB4 (Q9HAV0), GNE (Q9Y223),


GNG10 (P50151), GNG12 (Q9UBI6), GNG4 (P50150), GNG5 (P63218), GNL3 (Q9BVP2),


GNPDA1 (P46926), GNPNAT1 (Q96EK6), GOLGA7 (Q7Z5G4), GOLM1 (Q8NBJ4), GOLPH3


(Q9H4A6), GORASP2 (Q9H8Y8), GOT1 (P17174), GOT2 (P00505), GPC1 (P35052), GPC4


(O75487), GPC6 (Q9Y625), GPD1L (Q8N335), GPHN (Q9NQX3), GPI (P06744), GPM6A


(P51674), GPN1 (Q9HCN4), GPR50 (Q13585), GPR56 (Q9Y653), GPS1 (Q13098), GPSM1


(Q86YR5), GPX1 (P07203), GPX4 (P36969), GRB2 (P62993), GRHPR (Q9UBQ7), GRP


(Q3ZCW2), GRWD1 (Q9BQ67), GSDMA (Q96QA5), GSK3A (P49840), GSK3B (P49841), GSN


(P06396), GSPT1 (P15170), GSR (P00390), GSS (P48637), GSTK1 (Q9Y2Q3), GSTM2


(P28161), GSTM3 (P21266), GSTM4 (Q03013), GSTO1 (P78417), GSTP1 (P09211), GSTT2


(POCG29), GSTZ1 (O43708), GTF2E2 (P29084), GTF2F2 (P13984), GTF2H3 (Q13889), GTF2I


(P78347), GTF3C2 (Q8WUA4), GTF3C3 (Q9Y5Q9), GTF3C4 (Q9UKN8), GTPBP1 (O00178),


GTPBP4 (Q9BZE4), GUK1 (Q16774), GYG1 (P46976), GYS1 (P13807), H1F0 (P07305), H1FX


(Q92522), H2AFX (P16104), H2AFY (O75367), H2AFZ (P0C0S5), HADH (Q16836), HADHA


(P40939), HARS (P12081), HAT1 (O14929), HAUS3 (Q68CZ6), HAUS4 (Q9H6D7), HBA1


(P69905), HBB (P68871), HBS1L (Q9Y450), HBXIP (O43504), HCFC1 (P51610), HDAC1


(Q13547), HDAC2 (Q92769), HDDC2 (Q7Z4H3), HDGF (P51858), HDGFRP2 (Q7Z4V5),


HDHD2 (Q9H0R4), HDLBP (Q00341), HEATR1 (Q9H583), HEATR2 (Q86Y56), HEBP1


(Q9NRV9), HECTD3 (Q5T447), HERC4 (Q5GLZ8), HEXB (P07686), HGS (O14964), HHIP


(Q96QV1), HINT1 (P49773), HINT2 (Q9BX68), HINT3 (Q9NQE9), HIP1R (O75146), HIST1H1B


(P16401), HIST1H1C (P16403), HIST1H1D (P16402), HIST1H1E (P10412), HIST1H2AD


(P20671), HIST1H2BJ (P06899), HIST1H2BM (Q99879), HIST1H2BO (P23527), HIST1H4A


(P62805), HIST2H2AA3 (Q6FI13), HIST2H2AB (Q8IUE6), HIST2H2BE (Q16778), HIST2H3A


(Q71DI3), HIST3H2BB (Q8N257), HK1 (P19367), HK2 (P52789), HLA-A (P30443), HLA-A


(P01892), HLA-B (P03989), HMGA1 (P17096), HMGB1 (P09429), HMGB2 (P26583), HMGCL


(P35914), HMGCS1 (Q01581), HMGN1 (P05114), HMGN2 (P05204), HMGN4 (O00479),


HNRNPA0 (Q13151), HNRNPA1 (P09651), HNRNPA2B1 (P22626), HNRNPA3 (P51991),


HNRNPAB (Q99729), HNRNPC (P07910), HNRNPD (Q14103), HNRNPF (P52597), HNRNPH1


(P31943), HNRNPH2 (P55795), HNRNPH3 (P31942), HNRNPK (P61978), HNRNPL (P14866),


HNRNPM (P52272), HNRNPR (O43390), HNRNPU (Q00839), HNRNPUL1 (Q9BUJ2),


HNRNPUL2 (Q1KMD3), HNRPDL (O14979), HNRPLL (Q8WVV9), HOOK3 (Q86VS8), HP


(P00738), HP1BP3 (Q5SSJ5), HPCAL1 (P37235), HPRT1 (P00492), HPX (P02790), HRAS


(P01112), HRNR (Q86YZ3), HSD17B10 (Q99714), HSD17B12 (Q53GQ0), HSD17B4 (P51659),


HSDL2 (Q6YN16), HSP90AA1 (P07900), HSP90AB1 (P08238), HSP90B1 (P14625), HSPA12A


(O43301), HSPA14 (Q0VDF9), HSPA1A (P08107), HSPA4 (P34932), HSPA4L (O95757),


HSPA5 (P11021), HSPA8 (P11142), HSPA9 (P38646), HSPB1 (P04792), HSPBP1 (Q9NZL4),


HSPD1 (P10809), HSPE1 (P61604), HSPG2 (P98160), HSPH1 (Q92598), HTRA1 (Q92743),


HTT (P42858), HUWE1 (Q7Z6Z7), HYOU1 (Q9Y4L1), IARS (P41252), ICAM1 (P05362), IDE


(P14735), IDH1 (O75874), IDH2 (P48735), IDH3A (P50213), IDI1 (Q13907), IFI16 (Q16666),


IFIT5 (Q13325), IFITM3 (Q01628), IFRD2 (Q12894), IFT172 (Q9UG01), IGF1R (P08069),


IGF2BP2 (Q9Y6M1), IGF2BP3 (O00425), IGF2R (P11717), IGFBP3 (P17936), IGFBP5


(P24593), IGHG1 (P01857), IGHG2 (P01859), IGSF3 (O75054), IGSF8 (Q969P0), IKBKAP


(O95163), IKBKB (O14920), IL1RAP (Q9NPH3), ILF2 (Q12905), ILF3 (Q12906), ILK (Q13418),


ILKAP (Q9H0C8), IMMT (Q16891), IMP3 (Q9NV31), IMPA1 (P29218), IMPA2 (O14732),


IMPAD1 (Q9NX62), IMPDH1 (P20839), IMPDH2 (P12268), INA (Q16352), INF2 (Q27J81),


INPP1 (P49441), INPPL1 (O15357), INTS10 (Q9NVR2), INTS3 (Q68E01), INTS7 (Q9NVH2),


INTS8 (Q75QN2), IPO11 (Q9UI26), IPO4 (Q8TEX9), IPO5 (O00410), IPO7 (O95373), IPO8


(O15397), IPO9 (Q96P70), IQGAP1 (P46940), IRF2BP2 (Q7Z5L9), IRF3 (Q14653), IRGQ


(Q8WZA9), ISOC1 (Q96CN7), ISYNA1 (Q9NPH2), ITFG3 (Q9H0X4), ITGA2 (P17301), ITGA3


(P26006), ITGA4 (P13612), ITGA5 (P08648), ITGA6 (P23229), ITGA7 (Q13683), ITGAV


(P06756), ITGB1 (P05556), ITGB1BP1 (O14713), ITGB3 (P05106), ITGB4 (P16144), ITGB5


(P18084), ITGB8 (P26012), ITPA (Q9BY32), JAM3 (Q9BX67), JUP (P14923), KARS (Q15046),


KATNB1 (Q9BVA0), KBTBD6 (Q86V97), KCTD21 (Q4G0X4), KDM1A (O60341), KEAP1


(Q14145), KHDRBS1 (Q07666), KHSRP (Q92945), KIAA0020 (Q15397), KIAA0090 (Q8N766),


KIAA0174 (P53990), KIAA0196 (Q12768), KIAA0664 (O75153), KIAA0776 (O94874),


KIAA1033 (Q2M389), KIAA1279 (Q96EK5), KIAA1598 (A0MZ66), KIAA1797 (Q5VW36),


KIAA1949 (Q6NYC8), KIAA1967 (Q8N163), KIDINS220 (Q9ULH0), KIF1A (Q12756), KIF2A


(O00139), KIF5B (P33176), KIF5C (O60282), KLC1 (Q07866), KLHDC4 (Q8TBB5), KLHL13


(Q9P2N7), KLHL22 (Q53GT1), KLHL26 (Q53HC5), KNTC1 (P50748), KPNA1 (P52294),


KPNA2 (P52292), KPNA3 (O00505), KPNA4 (O00629), KPNA6 (O60684), KPNB1 (Q14974),


KPRP (Q5T749), KRAS (P01116), KRIT1 (O00522), KRT13 (P13646), KRT14 (P02533),


KRT71 (Q3SY84), KTN1 (Q86UP2), L1CAM (P32004), LACTB2 (Q53H82), LAMA1 (P25391),


LAMA4 (Q16363), LAMA5 (O15230), LAMB1 (P07942), LAMB2 (P55268), LAMC1 (P11047),


LAMP1 (P11279), LAMP2 (P13473), LANCL1 (O43813), LANCL2 (Q9NS86), LAP3 (P28838),


LARP1 (Q6PKG0), LARS (Q9P2J5), LAS1L (Q9Y4W2), LASP1 (Q14847), LBR (Q14739),


LCMT1 (Q9UIC8), LDHA (P00338), LDHB (P07195), LDLR (P01130), LEFTY2 (O00292),


LEPRE1 (Q32P28), LGALS1 (P09382), LGALS3 (P17931), LGALS3BP (Q08380), LGALS7


(P47929), LIMA1 (Q9UHB6), LIMS1 (P48059), LIN7C (Q9NUP9), LIPG (Q9Y5X9), LLGL1


(Q15334), LMAN1 (P49257), LMAN2 (Q12907), LMCD1 (Q9NZU5), LMNA (P02545), LMNB1


(P20700), LMNB2 (Q03252), LNPEP (Q9UIQ6), LOH12CR1 (Q969J3), LONP1 (P36776), LOR


(P23490), LOXL4 (Q96JB6), LPHN2 (O95490), LPL (P06858), LRBA (P50851), LRG1


(P02750), LRP1 (Q07954), LRPPRC (P42704), LRRC1 (Q9BTT6), LRRC40 (Q9H9A6),


LRRC47 (Q8N1G4), LRRC57 (Q8N9N7), LRRC59 (Q96AG4), LRRC8A (Q8IWT6), LRSAM1


(Q6UWE0), LSM1 (O15116), LSM12 (Q3MHD2), LSM2 (Q9Y333), LSM4 (Q9Y4Z0), LSM6


(P62312), LSM7 (Q9UK45), LSS (P48449), LTA4H (P09960), LTBP2 (Q14767), LTBP3


(Q9NS15), LTN1 (O94822), LUC7L (Q9NQ29), LUC7L2 (Q9Y383), LUC7L3 (O95232), LYAR


(Q9NX58), LYPLA1 (O75608), LYPLA2 (O95372), LYPLAL1 (Q5VWZ2), LZTR1 (Q8N653),


M6PR (P20645), MACF1 (Q9UPN3), MACF1 (Q96PK2), MACROD1 (Q9BQ69), MAD1L1


(Q9Y6D9), MAD2L1 (Q13257), MAGEE1 (Q9HCI5), MAK16 (Q9BXY0), MALT1 (Q9UDY8),


MAN1A2 (O60476), MAN1B1 (Q9UKM7), MAN2C1 (Q9NTJ4), MAP1B (P46821), MAP1LC3A


(Q9H492), MAP1LC3B2 (A6NCE7), MAP2K1 (Q02750), MAP2K2 (P36507), MAP2K3


(P46734), MAP2K4 (P45985), MAP2K7 (O14733), MAP4 (P27816), MAP4K4 (O95819),


MAPK1 (P28482), MAPK14 (Q16539), MAPK3 (P27361), MAPKSP1 (Q9UHA4), MAPRE1


(Q15691), MAPRE3 (Q9UPY8), MARCKS (P29966), MARCKSL1 (P49006), MARK2 (Q7KZI7),


MARS (P56192), MAT2A (P31153), MAT2B (Q9NZL9), MATR3 (P43243), MBD3 (O95983),


MBLAC2 (Q68D91), MBNL1 (Q9NR56), MBNL2 (Q5VZF2), MCAM (P43121), MCM2 (P49736),


MCM3 (P25205), MCM4 (P33991), MCM5 (P33992), MCM6 (Q14566), MCM7 (P33993),


MCTS1 (Q9ULC4), MDH1 (P40925), MDH2 (P40926), MDK (P21741), MDN1 (Q9NU22), ME1


(P48163), ME2 (P23368), MED1 (Q15648), MED10 (Q9BTT4), MED11 (Q9P086), MED17


(Q9NVC6), MED18 (Q9BUE0), MED20 (Q9H944), MED23 (Q9ULK4), MED24 (O75448),


MED28 (Q9H204), MED31 (Q9Y3C7), MEMO1 (Q9Y316), MEN1 (O00255), MERIT40


(Q9NWV8), METAP1 (P53582), METAP2 (P50579), METT10D (Q86W50), METTL1 (Q9UBP6),


METTL11A (Q9BV86), METTL13 (Q8N6R0), METTL2B (Q6P1Q9), METTL5 (Q9NRN9),


METTL9 (Q9H1A3), MFAP2 (P55001), MFAP4 (P55083), MFGE8 (Q08431), MFI2 (P08582),


MGEA5 (O60502), MICA (Q29983), MICAL1 (Q8TDZ2), MIF (P14174), MINA (Q8IUF8), MIOS


(Q9NXC5), MKI67IP (Q9BYG3), MLEC (Q14165), MLLT4 (P55196), MLST8 (Q9BVC4), MLTK


(Q9NYL2), MMP14 (P50281), MMP2 (P08253), MMS19 (Q96T76), MOB2 (Q70IA6), MOBKL1B


(Q9H8S9), MOBKL2A (Q96BX8), MOBKL3 (Q9Y3A3), MOCS2 (O96033), MOGS (Q13724),


MON2 (Q7Z3U7), MORC2 (Q9Y6X9), MOV10 (Q9HCE1), MOXD1 (Q6UVY6), MPG (P29372),


MPI (P34949), MPP6 (Q9NZW5), MPRIP (Q6WCQ1), MPST (P25325), MPZL1 (O95297),


MRC2 (Q9UBG0), MRE11A (P49959), MRI1 (Q9BV20), MRPS27 (Q92552), MRPS28


(Q9Y2Q9), MRPS33 (Q9Y291), MRPS34 (P82930), MRPS6 (P82932), MRTO4 (Q9UKD2),


MSH2 (P43246), MSH3 (P20585), MSH6 (P52701), MSN (P26038), MSTO1 (Q9BUK6), MTA1


(Q13330), MTA2 (O94776), MTAP (Q13126), MTHFD1 (P11586), MTHFS (P49914), MTM1


(Q13496), MTMR1 (Q13613), MTMR2 (Q13614), MTMR6 (Q9Y217), MTMR9 (Q96QG7),


MTOR (P42345), MTPN (P58546), MTR (Q99707), MTRR (Q9UBK8), MVD (P53602), MVK


(Q03426), MVP (Q14764), MX1 (P20591), MYADM (Q96S97), MYBBP1A (Q9BQG0), MYCBP


(Q99417), MYD88 (Q99836), MYH10 (P35580), MYH14 (Q7Z406), MYH9 (P35579), MYL12B


(O14950), MYL6 (P60660), MYO18A (Q92614), MYO1B (O43795), MYO1C (O00159), MYO1E


(Q12965), MYO5A (Q9Y4I1), MYO6 (Q9UM54), MYOF (Q9NZM1), NAA10 (P41227), NAA15


(Q9BXJ9), NAA16 (Q6N069), NAA25 (Q14CX7), NAA38 (O95777), NAA50 (Q9GZZ1), NACA


(Q13765), NAE1 (Q13564), NAGK (Q9UJ70), NAGLU (P54802), NAMPT (P43490), NANS


(Q9NR45), NAP1L1 (P55209), NAP1L4 (Q99733), NAPA (P54920), NAPG (Q99747), NAPRT1


(Q6XQN6), NARFL (Q9H6Q4), NARS (O43776), NASP (P49321), NAT10 (Q9H0A0), NAT9


(Q9BTE0), NCAM1 (P13591), NCAN (O14594), NCAPD2 (Q15021), NCAPG (Q9BPX3),


NCBP1 (Q09161), NCCRP1 (Q6ZVX7), NCDN (Q9UBB6), NCKAP1 (Q9Y2A7), NCKIPSD


(Q9NZQ3), NCL (P19338), NCLN (Q969V3), NCS1 (P62166), NCSTN (Q92542), NDOR1


(Q9UHB4), NDRG3 (Q9UGV2), NDRG4 (Q9ULP0), NDUFA2 (O43678), NDUFA7 (O95182),


NDUFAB1 (O14561), NDUFB4 (O95168), NDUFC2 (O95298), NDUFS5 (O43920), NDUFS6


(O75380), NEDD8 (Q15843), NEFL (P07196), NEFM (P07197), NEK6 (Q9HC98), NEK9


(Q8TD19), NES (P48681), NF1 (P21359), NF2 (P35240), NFIX (Q14938), NHLRC2 (Q8NBF2),


NHP2L1 (P55769), NID1 (P14543), NIP7 (Q9Y221), NIPSNAP1 (Q9BPW8), NIT1 (Q86X76),


NIT2 (Q9NQR4), NKRF (O15226), NLE1 (Q9NVX2), NLGN4X (Q8N0W4), NLN (Q9BYT8),


NMD3 (Q96D46), NME2 (P22392), NME3 (Q13232), NME7 (Q9Y5B8), NMT1 (P30419), NNMT


(P40261), NOB1 (Q9ULX3), NOC2L (Q9Y3T9), NOC3L (Q8WTT2), NOC4L (Q9BVI4), NOG


(Q13253), NOL11 (Q9H8H0), NOL6 (Q9H6R4), NOL9 (Q5SY16), NOMO2 (Q5JPE7), NONO


(Q15233), NOP10 (Q9NPE3), NOP16 (Q9Y3C1), NOP2 (P46087), NOP56 (O00567), NOP58


(Q9Y2X3), NOS1AP (O75052), NOSIP (Q9Y314), NOTCH2 (Q04721), NOVA2 (Q9UNW9),


NPC1 (O15118), NPC2 (P61916), NPEPPS (P55786), NPLOC4 (Q8TAT6), NPM1 (P06748),


NPTN (Q9Y639), NPW (Q8N729), NQO1 (P15559), NQO2 (P16083), NRAS (P01111), NRBP1


(Q9UHY1), NRD1 (O43847), NRP1 (O14786), NRP2 (O60462), NSDHL (Q15738), NSF


(P46459), NSUN2 (Q08J23), NSUN5 (Q96P11), NSUN6 (Q8TEA1), NT5C (Q8TCD5), NT5C2


(P49902), NT5C3L (Q969T7), NT5E (P21589), NTN1 (O95631), NUBP1 (P53384), NUBP2


(Q9Y5Y2), NUCB1 (Q02818), NUCKS1 (Q9H1E3), NUDC (Q9Y266), NUDCD1 (Q96RS6),


NUDCD2 (Q8WVJ2), NUDT1 (P36639), NUDT10 (Q8NFP7), NUDT16 (Q96DE0), NUDT16L1


(Q9BRJ7), NUDT21 (O43809), NUDT4 (Q9NZJ9), NUDT5 (Q9UKK9), NUMA1 (Q14980),


NUP188 (Q5SRE5), NUP210 (Q8TEM1), NUP37 (Q8NFH4), NUP43 (Q8NFH3), NUP54


(Q7Z3B4), NUP62 (P37198), NUP85 (Q9BW27), NUP88 (Q99567), NUP93 (Q8N1F7), NUTF2


(P61970), NXF1 (Q9UBU9), NXN (Q6DKJ4), NXT1 (Q9UKK6), OAT (P04181), OBSL1


(O75147), OCRL (Q01968), ODR4 (Q5SWX8), ODZ2 (Q9NT68), ODZ3 (Q9P273), OGFOD1


(Q8N543), OGT (O15294), OLA1 (Q9NTK5), OLFML3 (Q9NRN5), OPA1 (O60313), ORC3


(Q9UBD5), OSBP (P22059), OSBPL6 (Q9BZF3), OSGEP (Q9NPF4), OTUB1 (Q96FW1),


OVCA2 (Q8WZ82), OXCT1 (P55809), OXSR1 (O95747), P4HA1 (P13674), P4HB (P07237),


PA2G4 (Q9UQ80), PAAF1 (Q9BRP4), PABPC1 (P11940), PABPC4 (Q13310), PABPN1


(Q86U42), PACSIN2 (Q9UNF0), PACSIN3 (Q9UKS6), PAF1 (Q8N7H5), PAFAH1B1 (P43034),


PAFAH1B2 (P68402), PAFAH1B3 (Q15102), PAICS (P22234), PAIP1 (Q9H074), PAK1IP1


(Q9NWT1), PAK2 (Q13177), PALD (Q9ULE6), PALLD (Q8WX93), PANK4 (Q9NVE7), PAPOLA


(P51003), PAPSS1 (O43252), PARK7 (Q99497), PARN (O95453), PARP1 (P09874), PARP4


(Q9UKK3), PARVA (Q9NVD7), PBLD (P30039), PCBD1 (P61457), PCBP1 (Q15365), PCBP2


(Q15366), PCDHB2 (Q9Y5E7), PCDHGC3 (Q9UN70), PCID2 (Q5JVF3), PCMT1 (P22061),


PCNA (P12004), PCOLCE2 (Q9UKZ9), PCYOX1 (Q9UHG3), PCYOX1L (Q8NBM8), PCYT2


(Q99447), PDCD10 (Q9BUL8), PDCD11 (Q14690), PDCD4 (Q53EL6), PDCD5 (O14737),


PDCD6 (O75340), PDCD6IP (Q8WUM4), PDCL3 (Q9H2J4), PDDC1 (Q8NB37), PDE12


(Q6L8Q7), PDGFRA (P16234), PDIA3 (P30101), PDIA4 (P13667), PDIA5 (Q14554), PDIA6


(Q15084), PDLIM1 (O00151), PDLIM4 (P50479), PDLIM5 (Q96HC4), PDLIM7 (Q9NR12),


PDRO (Q6IAA8), PDS5A (Q29RF7), PDS5B (Q9NTI5), PDXK (O00764), PDXP (Q96GD0),


PEA15 (Q15121), PEBP1 (P30086), PECI (O75521), PEF1 (Q9UBV8), PELO (Q9BRX2),


PELP1 (Q8IZL8), PEPD (P12955), PES1 (O00541), PFAS (O15067), PFDN1 (O60925), PFDN2


(Q9UHV9), PFDN4 (Q9NQP4), PFDN5 (Q99471), PFDN6 (O15212), PFKL (P17858), PFKM


(P08237), PFKP (Q01813), PFN1 (P07737), PFN2 (P35080), PGAM1 (P18669), PGAM5


(Q96HS1), PGD (P52209), PGGT1B (P53609), PGK1 (P00558), PGLS (O95336), PGLYRP2


(Q96PD5), PGM1 (P36871), PGM2L1 (Q6PCE3), PGM3 (O95394), PGP (A6NDG6), PGRMC1


(O00264), PGRMC2 (O15173), PHB (P35232), PHB2 (Q99623), PHF5A (Q7RTV0), PHF6


(Q8IWS0), PHGDH (O43175), PHKB (Q93100), PHLDA1 (Q8WV24), PHLDA3 (Q9Y5J5),


PHLDB1 (Q86UU1), PHPT1 (Q9NRX4), PI15 (O43692), PI4KA (P42356), PICALM (Q13492),


PIGT (Q969N2), PIK3CA (P42336), PIK3R4 (Q99570), PIN1 (Q13526), PIP4K2A (P48426),


PIP4K2B (P78356), PIP4K2C (Q8TBX8), PIPOX (Q9P0Z9), PIPSL (A2A3N6), PITPNB


(P48739), PKM2 (P14618), PKP1 (Q13835), PLAA (Q9Y263), PLCB3 (Q01970), PLCD1


(P51178), PLCD3 (Q8N3E9), PLCG1 (P19174), PLCG2 (P16885), PLD3 (Q8IV08), PLEC


(Q15149), PLIN2 (Q99541), PLIN3 (O60664), PLK1 (P53350), PLOD1 (Q02809), PLOD2


(O00469), PLOD3 (O60568), PLRG1 (O43660), PLS1 (Q14651), PLS3 (P13797), PLSCR3


(Q9NRY6), PLTP (P55058), PLXNA1 (Q9UIW2), PLXNB2 (O15031), PLXND1 (Q9Y4D7),


PMM2 (O15305), PMPCA (Q10713), PMPCB (O75439), PMVK (Q15126), PNMA2 (Q9UL42),


PNN (Q9H307), PNO1 (Q9NRX1), PNP (P00491), PNPLA2 (Q96AD5), PODXL (O00592),


POLD1 (P28340), POLD2 (P49005), POLE3 (Q9NRF9), POLR1A (O95602), POLR1B


(Q9H9Y6), POLR1C (O15160), POLR1D (Q9Y2S0), POLR2A (P24928), POLR2B (P30876),


POLR2C (P19387), POLR2E (P19388), POLR2G (P62487), POLR2H (P52434), POLR2J


(P52435), POLR2K (P53803), POLR3A (O14802), POLR3B (Q9NW08), POLR3C (Q9BUI4),


POP1 (Q99575), POP4 (O95707), POP7 (O75817), POR (P16435), PPA1 (Q15181), PPA2


(Q9H2U2), PPAN (Q9NQ55), PPAP2A (O14494), PPAT (Q06203), PPCS (Q9HAB8), PPFIBP1


(Q86W92), PPIA (P62937), PPIB (P23284), PPIC (P45877), PPID (Q08752), PPIF (P30405),


PPIH (O43447), PPIL1 (Q9Y3C6), PPM1F (P49593), PPM1G (O15355), PPME1 (Q9Y570),


PPP1CA (P62136), PPP1CB (P62140), PPP1CC (P36873), PPP1R14B (Q96C90), PPP1R7


(Q15435), PPP1R8 (Q12972), PPP2CA (P67775), PPP2CB (P62714), PPP2R1A (P30153),


PPP2R2A (P63151), PPP2R2D (Q66LE6), PPP2R4 (Q15257), PPP2R5D (Q14738), PPP2R5E


(Q16537), PPP3CA (Q08209), PPP4C (P60510), PPP4R1 (Q8TF05), PPP5C (P53041), PPP6C


(O00743), PPP6R3 (Q5H9R7), PPPDE2 (Q6ICB0), PPT1 (P50897), PPWD1 (Q96BP3), PRCP


(P42785), PRDX1 (Q06830), PRDX2 (P32119), PRDX3 (P30048), PRDX4 (Q13162), PRDX6


(P30041), PREP (P48147), PREPL (Q4J6C6), PRIM1 (P49642), PRIM2 (P49643), PRKAA1


(Q13131), PRKACA (P17612), PRKACB (P22694), PRKAG1 (P54619), PRKAR1A (P10644),


PRKAR2A (P13861), PRKCA (P17252), PRKCI (P41743), PRKCSH (P14314), PRKDC


(P78527), PRKRA (O75569), PRMT1 (Q99873), PRMT10 (Q6P2P2), PRMT3 (O60678),


PRMT5 (O14744), PRMT7 (Q9NVM4), PROSC (O94903), PRPF19 (Q9UMS4), PRPF3


(O43395), PRPF31 (Q8WWY3), PRPF4 (O43172), PRPF40A (O75400), PRPF4B (Q13523),


PRPF6 (O94906), PRPF8 (Q6P2Q9), PRPS1 (P60891), PRPS2 (P11908), PRPSAP2


(O60256), PRRC1 (Q96M27), PRSS23 (O95084), PRTFDC1 (Q9NRG1), PSAP (P07602),


PSAT1 (Q9Y617), PSD3 (Q9NYI0), PSENEN (Q9NZ42), PSIP1 (O75475), PSMA1 (P25786),


PSMA2 (P25787), PSMA3 (P25788), PSMA4 (P25789), PSMA5 (P28066), PSMA6 (P60900),


PSMA7 (O14818), PSMB1 (P20618), PSMB2 (P49721), PSMB3 (P49720), PSMB4 (P28070),


PSMB5 (P28074), PSMB6 (P28072), PSMB7 (Q99436), PSMC1 (P62191), PSMC2 (P35998),


PSMC3 (P17980), PSMC4 (P43686), PSMC5 (P62195), PSMC6 (P62333), PSMD1 (Q99460),


PSMD10 (O75832), PSMD11 (O00231), PSMD12 (O00232), PSMD13 (Q9UNM6), PSMD14


(O00487), PSMD2 (Q13200), PSMD3 (O43242), PSMD4 (P55036), PSMD5 (Q16401), PSMD6


(Q15008), PSMD7 (P51665), PSMD8 (P48556), PSMD9 (O00233), PSME1 (Q06323), PSME2


(Q9UL46), PSME3 (P61289), PSME4 (Q14997), PSMG1 (O95456), PSMG2 (Q969U7), PSPC1


(Q8WXF1), PSPH (P78330), PTBP1 (P26599), PTGES2 (Q9H7Z7), PTGES3 (Q15185),


PTGFRN (Q9P2B2), PTGR1 (Q14914), PTHLH (P12272), PTK2 (Q05397), PTK7 (Q13308),


PTMA (P06454), PTN (P21246), PTP4A1 (Q93096), PTPN1 (P18031), PTPN11 (Q06124),


PTPN23 (Q9H3S7), PTPRA (P18433), PTPRE (P23469), PTPRG (P23470), PTPRJ (Q12913),


PTPRZ1 (P23471), PUF60 (Q9UHX1), PURA (Q00577), PURB (Q96QR8), PUS1 (Q9Y606),


PUS7 (Q96PZ0), PVR (P15151), PVRL2 (Q92692), PWP1 (Q13610), PWP2 (Q15269), PXDN


(Q92626), PXK (Q7Z7A4), PXN (P49023), PYCR1 (P32322), PYCRL (Q53H96), PYGB


(P11216), PYGL (P06737), QARS (P47897), QDPR (P09417), QKI (Q96PU8), QTRT1


(Q9BXR0), RAB10 (P61026), RAB11A (P62491), RAB11FIP1 (Q6WKZ4), RAB12 (Q6IQ22),


RAB13 (P51153), RAB14 (P61106), RAB18 (Q9NP72), RAB1A (P62820), RAB1B (Q9H0U4),


RAB21 (Q9UL25), RAB22A (Q9UL26), RAB23 (Q9ULC3), RAB27A (P51159), RAB2A


(P61019), RAB2B (Q8WUD1), RAB32 (Q13637), RAB34 (Q9BZG1), RAB35 (Q15286), RAB3A


(P20336), RAB3GAP1 (Q15042), RAB3GAP2 (Q9H2M9), RAB4A (P20338), RAB5A (P20339),


RAB5B (P61020), RAB5C (P51148), RAB6A (P20340), RAB7A (P51149), RAB8A (P61006),


RAB8B (Q92930), RABAC1 (Q9UI14), RABGAP1 (Q9Y3P9), RABGGTA (Q92696), RABGGTB


(P53611), RABL2A (Q9UBK7), RABL3 (Q5HYI8), RAC1 (P63000), RAC3 (P60763), RAD23B


(P54727), RAD50 (Q92878), RAE1 (P78406), RAF1 (P04049), RALA (P11233), RALB


(P11234), RALY (Q9UKM9), RAN (P62826), RANBP1 (P43487), RANBP2 (P49792),


RANGAP1 (P46060), RAP1A (P62834), RAP1B (P61224), RAP1GDS1 (P52306), RAP2B


(P61225), RAPH1 (Q70E73), RARS (P54136), RASA1 (P20936), RASA3 (Q14644), RBBP4


(Q09028), RBBP5 (Q15291), RBBP7 (Q16576), RBM12 (Q9NTZ6), RBM14 (Q96PK6), RBM15


(Q96T37), RBM22 (Q9NW64), RBM25 (P49756), RBM26 (Q5T8P6), RBM28 (Q9NW13),


RBM39 (Q14498), RBM4 (Q9BWF3), RBM8A (Q9Y5S9), RBMX (P38159), RBP1 (P09455),


RBPJ (Q06330), RBX1 (P62877), RCC1 (P18754), RCC2 (Q9P258), RCL (O43598), RCL1


(Q9Y2P8), RCN1 (Q15293), RDH11 (Q8TC12), RDH13 (Q8NBN7), RDX (P35241), RECQL


(P46063), RELA (Q04206), REPS1 (Q96D71), RETSAT (Q6NUM9), RFC2 (P35250), RFC3


(P40938), RFC4 (P35249), RFC5 (P40937), RFFL (Q8WZ73), RFTN1 (Q14699), RHEB


(Q15382), RHOA (P61586), RHOB (P62745), RHOC (P08134), RHOF (Q9HBH0), RHOG


(P84095), RHOT2 (Q8IXI1), RIC8A (Q9NPQ8), RNASEH2C (Q8TDP1), RNF114 (Q9Y508),


RNF20 (Q5VTR2), RNF213 (Q63HN8), RNF7 (Q9UBF6), RNGTT (O60942), RNH1 (P13489),


RNMT (O43148), RNPEP (Q9H4A4), ROBLD3 (Q9Y2Q5), ROCK1 (Q13464), ROCK2


(O75116), RP2 (O75695), RPA1 (P27694), RPA2 (P15927), RPA3 (P35244), RPE (Q96AT9),


RPF2 (Q9H7B2), RPIA (P49247), RPL10 (P27635), RPL10A (P62906), RPL11 (P62913),


RPL12 (P30050), RPL13 (P26373), RPL13A (P40429), RPL14 (P50914), RPL15 (P61313),


RPL17 (P18621), RPL18 (Q07020), RPL18A (Q02543), RPL19 (P84098), RPL21 (P46778),


RPL22 (P35268), RPL22L1 (Q6P5R6), RPL23 (P62829), RPL23A (P62750), RPL24 (P83731),


RPL26 (P61254), RPL26L1 (Q9UNX3), RPL27 (P61353), RPL27A (P46776), RPL28 (P46779),


RPL29 (P47914), RPL3 (P39023), RPL30 (P62888), RPL31 (P62899), RPL32 (P62910),


RPL34 (P49207), RPL35 (P42766), RPL35A (P18077), RPL36 (Q9Y3U8), RPL36A (P83881),


RPL36AL (Q969Q0), RPL37 (P61927), RPL37A (P61513), RPL38 (P63173), RPL4 (P36578),


RPL5 (P46777), RPL6 (Q02878), RPL7 (P18124), RPL7A (P62424), RPL7L1 (Q6DKI1), RPL8


(P62917), RPL9 (P32969), RPLP0 (P05388), RPLP1 (P05386), RPLP2 (P05387), RPN1


(P04843), RPN2 (P04844), RPP30 (P78346), RPP38 (P78345), RPRD1A (Q96P16), RPRD1B


(Q9NQG5), RPS10 (P46783), RPS11 (P62280), RPS12 (P25398), RPS13 (P62277), RPS14


(P62263), RPS15 (P62841), RPS15A (P62244), RPS16 (P62249), RPS17 (P08708), RPS18


(P62269), RPS19 (P39019), RPS2 (P15880), RPS20 (P60866), RPS21 (P63220), RPS23


(P62266), RPS24 (P62847), RPS25 (P62851), RPS26 (P62854), RPS27 (P42677), RPS27A


(P62979), RPS27L (Q71UM5), RPS28 (P62857), RPS29 (P62273), RPS3 (P23396), RPS3A


(P61247), RPS4X (P62701), RPS4Y1 (P22090), RPS5 (P46782), RPS6 (P62753), RPS6KA1


(Q15418), RPS6KA3 (P51812), RPS7 (P62081), RPS8 (P62241), RPS9 (P46781), RPSA


(P08865), RQCD1 (Q92600), RRAGC (Q9HB90), RRAS2 (P62070), RRBP1 (Q9P2E9), RRM1


(P23921), RRM2 (P31350), RRM2B (Q7LG56), RRP1 (P56182), RRP12 (Q5JTH9), RRP1B


(Q14684), RRP7A (Q9Y3A4), RRP9 (O43818), RRS1 (Q15050), RSL1D1 (O76021), RSL24D1


(Q9UHA3), RSPRY1 (Q96DX4), RSU1 (Q15404), RTCD1 (O00442), RTKN (Q9BST9), RTN3


(O95197), RTN4 (Q9NQC3), RUVBL1 (Q9Y265), RUVBL2 (Q9Y230), RWDD2B (P57060),


S100A10 (P60903), S100A11 (P31949), S100A13 (Q99584), S100A16 (Q96FQ6), S100A2


(P29034), S100A4 (P26447), S100A6 (P06703), S100A7 (P31151), S100A8 (P05109), S100A9


(P06702), SAAL1 (Q96ER3), SACS (Q9NZJ4), SAE1 (Q9UBE0), SAMHD1 (Q9Y3Z3), SAP18


(O00422), SAR1A (Q9NR31), SARM1 (Q6SZW1), SARNP (P82979), SARS (P49591), SARS2


(Q9NP81), SART3 (Q15020), SBDS (Q9Y3A5), SBF1 (O95248), SCARB1 (Q8WTV0), SCARB2


(Q14108), SCCPDH (Q8NBX0), SCFD1 (Q8WVM8), SCFD2 (Q8WU76), SCP2 (P22307),


SCPEP1 (Q9HB40), SCRG1 (O75711), SCRIB (Q14160), SCRN1 (Q12765), SCRN2


(Q96FV2), SCYL1 (Q96KG9), SDC2 (P34741), SDC4 (P31431), SDCBP (O00560), SDCCAG1


(O60524), SDCCAG3 (Q96C92), SDHA (P31040), SDHB (P21912), SDK1 (Q7Z5N4), SDSL


(Q96GA7), SEC13 (P55735), SEC14L2 (O76054), SEC22B (O75396), SEC23A (Q15436),


SEC23B (Q15437), SEC23IP (Q9Y6Y8), SEC24A (O95486), SEC24B (O95487), SEC24C


(P53992), SEC24D (O94855), SEC31A (O94979), SEC61B (P60468), SEC61G (P60059),


SEH1L (Q96EE3), SELH (Q8IZQ5), SELO (Q9BVL4), SEMA3A (Q14563), SENP3 (Q9H4L4),


SEPSECS (Q9HD40), 40422 (Q9P0V9), 40787 (Q9NVA2), 37500 (Q15019), 38596 (Q99719),


39326 (Q16181), 40057 (Q9UHD8), SERBP1 (Q8NC51), SERPINB12 (Q96P63), SERPINB3


(P29508), SERPINB6 (P35237), SERPINH1 (P50454), SESN2 (P58004), SET (Q01105),


SETD3 (Q86TU7), SF3A1 (Q15459), SF3A2 (Q15428), SF3A3 (Q12874), SF3B1 (O75533),


SF3B14 (Q9Y3B4), SF3B2 (Q13435), SF3B3 (Q15393), SF3B4 (Q15427), SF3B5 (Q9BWJ5),


SFN (P31947), SFPQ (P23246), SFRP4 (Q6FHJ7), SFXN3 (Q9BWM7), SGTA (O43765),


SH3BGRL3 (Q9H299), SH3BP4 (Q9P0V3), SH3GL1 (Q99961), SH3GLB1 (Q9Y371), SHC1


(P29353), SHMT1 (P34896), SHMT2 (P34897), SHOC2 (Q9UQ13), SHPK (Q9UHJ6), SIRT5


(Q9NXA8), SKIV2L (Q15477), SKIV2L2 (P42285), SKP1 (P63208), SLC12A2 (P55011),


SLC12A4 (Q9UP95), SLC16A1 (P53985), SLC1A3 (P43003), SLC1A5 (Q15758), SLC25A10


(Q9UBX3), SLC25A11 (Q02978), SLC25A13 (Q9UJS0), SLC25A22 (Q9H936), SLC25A3


(Q00325), SLC25A5 (P05141), SLC25A6 (P12236), SLC26A2 (P50443), SLC29A1 (Q99808),


SLC29A2 (Q14542), SLC2A1 (P11166), SLC30A1 (Q9Y6M5), SLC38A1 (Q9H2H9), SLC3A2


(P08195), SLC44A2 (Q8IWA5), SLC4A2 (P04920), SLC4A7 (Q9Y6M7), SLC5A3 (P53794),


SLC5A6 (Q9Y289), SLC6A8 (P48029), SLC7A1 (P30825), SLC7A5 (Q01650), SLC9A3R1


(O14745), SLC9A3R2 (Q15599), SLIRP (Q9GZT3), SLK (Q9H2G2), SMAD1 (Q15797), SMAD2


(Q15796), SMARCA4 (P51532), SMARCA5 (O60264), SMARCB1 (Q12824), SMARCC1


(Q92922), SMARCC2 (Q8TAQ2), SMARCD2 (Q92925), SMC1A (Q14683), SMC2 (O95347),


SMC3 (Q9UQE7), SMC4 (Q9NTJ3), SMC5 (Q8IY18), SMCHD1 (A6NHR9), SMEK1 (Q6IN85),


SMG1 (Q96Q15), SMN1 (Q16637), SMS (P52788), SMU1 (Q2TAY7), SMYD3 (Q9H7B4),


SMYD5 (Q6GMV2), SNAP23 (O00161), SND1 (Q7KZF4), SNF8 (Q96H20), SNRNP200


(O75643), SNRNP40 (Q96DI7), SNRNP70 (P08621), SNRPA1 (P09661), SNRPB (P14678),


SNRPB2 (P08579), SNRPD1 (P62314), SNRPD2 (P62316), SNRPD3 (P62318), SNRPE


(P62304), SNRPF (P62306), SNRPG (P62308), SNTB1 (Q13884), SNTB2 (Q13425), SNX1


(Q13596), SNX12 (Q9UMY4), SNX17 (Q15036), SNX18 (Q96RF0), SNX2 (O60749), SNX27


(Q96L92), SNX3 (O60493), SNX5 (Q9Y5X3), SNX6 (Q9UNH7), SNX9 (Q9Y5X1), SOD1


(P00441), SOD2 (P04179), SORD (Q00796), SORT1 (Q99523), SPATS2L (Q9NUQ6), SPC24


(Q8NBT2), SPCS2 (Q15005), SPCS3 (P61009), SPG21 (Q9NZD8), SPIN1 (Q9Y657), SPR


(P35270), SPRR1B (P22528), SPRR2E (P22531), SPTAN1 (Q13813), SPTBN1 (Q01082),


SPTBN2 (O15020), SR140 (O15042), SRBD1 (Q8N5C6), SRCRL (A1L4H1), SRGAP2


(O75044), SRI (P30626), SRM (P19623), SRP14 (P37108), SRP19 (P09132), SRP54


(P61011), SRP68 (Q9UHB9), SRP72 (O76094), SRP9 (P49458), SRPK1 (Q96SB4), SRPR


(P08240), SRPRB (Q9Y5M8), SRPX (P78539), SRPX2 (O60687), SRR (Q9GZT4), SRRM1


(Q8IYB3), SRRM2 (Q9UQ35), SRRT (Q9BXP5), SRSF1 (Q07955), SRSF10 (O75494),


SRSF11 (Q05519), SRSF2 (Q01130), SRSF3 (P84103), SRSF5 (Q13243), SRSF6 (Q13247),


SRSF7 (Q16629), SRSF9 (Q13242), SRXN1 (Q9BYN0), SSB (P05455), SSBP1 (Q04837),


SSR1 (P43307), SSR3 (Q9UNL2), SSRP1 (Q08945), SSSCA1 (O60232), SSU72 (Q9NP77),


ST13 (P50502), STAG1 (Q8WVM7), STAM (Q92783), STAMBP (O95630), STAT1 (P42224),


STAT2 (P52630), STAT3 (P40763), STAU1 (O95793), STIP1 (P31948), STK10 (O94804),


STK24 (Q9Y6E0), STK25 (O00506), STK38 (Q15208), STK38L (Q9Y2H1), STOM (P27105),


STOML2 (Q9UJZ1), STON2 (Q8WXE9), STRAP (Q9Y3F4), STT3A (P46977), STUB1


(Q9UNE7), STX12 (Q86Y82), STX4 (Q12846), STX5 (Q13190), STXBP1 (P61764), STXBP3


(O00186), STYX (Q8WUJ0), SUB1 (P53999), SUCLA2 (Q9P2R7), SUCLG2 (Q96I99), SUGT1


(Q9Y2Z0), SULF2 (Q8IWU5), SUMO1 (P63165), SUPT16H (Q9Y5B9), SUPT4H1 (P63272),


SUPT5H (O00267), SUPT6H (Q7KZ85), SUSD5 (O60279), SVEP1 (Q4LDE5), SVIL (O95425),


SWAP70 (Q9UH65), SYMPK (Q92797), SYNCRIP (O60506), SYNGR2 (O43760), SYNJ2BP


(P57105), SYNM (O15061), SYPL1 (Q16563), TAB1 (Q15750), TAF9 (Q9Y3D8), TAGLN


(Q01995), TAGLN2 (P37802), TALDO1 (P37837), TAOK1 (Q7L7X3), TARDBP (Q13148),


TARS (P26639), TATDN1 (Q6P1N9), TAX1BP3 (O14907), TBC1D13 (Q9NVG8), TBC1D15


(Q8TC07), TBC1D23 (Q9NUY8), TBC1D24 (Q9ULP9), TBC1D4 (O60343), TBC1D9B


(Q66K14), TBCA (O75347), TBCB (Q99426), TBCC (Q15814), TBCD (Q9BTW9), TBCE


(Q15813), TBK1 (Q9UHD2), TBL1XR1 (Q9BZK7), TBL2 (Q9Y4P3), TBL3 (Q12788), TBPL1


(P62380), TCEA1 (P23193), TCEB1 (Q15369), TCEB2 (Q15370), TCERG1 (O14776), TCF25


(Q9BQ70), TCP1 (P17987), TELO2 (Q9Y4R8), TEX10 (Q9NXF1), TEX15 (Q9BXT5), TF


(P02787), TFCP2 (Q12800), TFG (Q92734), TFRC (P02786), TGFB1 (P01137), TGFB2


(P61812), TGFBI (Q15582), TGFBRAP1 (Q8WUH2), TGM1 (P22735), TGM3 (Q08188), TH1L


(Q8IXH7), THBS1 (P07996), THBS3 (P49746), THG1L (Q9NWX6), THOC2 (Q8NI27), THOC3


(Q96J01), THOC5 (Q13769), THOC6 (Q86W42), THOC7 (Q6I9Y2), THOP1 (P52888), THTPA


(Q9BU02), THUMPD1 (Q9NXG2), THUMPD3 (Q9BV44), THY1 (P04216), THYN1 (Q9P016),


TIA1 (P31483), TIAL1 (Q01085), TIGAR (Q9NQ88), TIMM13 (Q9Y5L4), TIMM44 (O43615),


TIMM50 (Q3ZCQ8), TIMM8A (O60220), TIMM8B (Q9Y5J9), TIMM9 (Q9Y5J7), TIMP2


(P16035), TIPRL (O75663), TJP1 (Q07157), TKT (P29401), TLN1 (Q9Y490), TLN2 (Q9Y4G6),


TM9SF3 (Q9HD45), TMED10 (P49755), TMED2 (Q15363), TMED5 (Q9Y3A6), TMED7


(Q9Y3B3), TMED9 (Q9BVK6), TMEFF2 (Q9UIK5), TMEM132A (Q24JP5), TMEM2 (Q9UHN6),


TMEM30A (Q9NV96), TMEM33 (P57088), TMOD3 (Q9NYL9), TMPO (P42166), TMX1


(Q9H3N1), TNC (P24821), TNKS1BP1 (Q9C0C2), TNPO1 (Q92973), TNPO2 (O14787),


TNPO3 (Q9Y5L0), TOM1L2 (Q6ZVM7), TOMM20 (Q15388), TOMM34 (Q15785), TOMM5


(Q8N4H5), TOMM70A (O94826), TOP1 (P11387), TOP2A (P11388), TOP2B (Q02880), TP53I3


(Q53FA7), TP53RK (Q96S44), TPBG (Q13641), TPD52 (P55327), TPI1 (P60174), TPM1


(P09493), TPM2 (P07951), TPM3 (P06753), TPM3L (A6NL28), TPM4 (P67936), TPP2


(P29144), TPT1 (P13693), TRA2A (Q13595), TRA2B (P62995), TRAF2 (Q12933), TRAP1


(Q12931), TRAPPC1 (Q9Y5R8), TRAPPC2L (Q9UL33), TRAPPC3 (O43617), TRAPPC4


(Q9Y296), TRAPPC5 (Q8IUR0), TRIM16 (O95361), TRIM22 (Q8IYM9), TRIM25 (Q14258),


TRIM26 (Q12899), TRIM28 (Q13263), TRIM47 (Q96LD4), TRIM5 (Q9C035), TRIO (O75962),


TRIP13 (Q15645), TRIP6 (Q15654), TRMT1 (Q9NXH9), TRMT112 (Q9UI30), TRMT5


(Q32P41), TRMT6 (Q9UJA5), TRMT61A (Q96FX7), TRNT1 (Q96Q11), TROVE2 (P10155),


TRRAP (Q9Y4A5), TSG101 (Q99816), TSKU (Q8WUA8), TSN (Q15631), TSPAN14 (Q8NG11),


TSPAN6 (O43657), TSR1 (Q2NL82), TSSC1 (Q53HC9), TSTA3 (Q13630), TTC1 (Q99614),


TTC15 (Q8WVT3), TTC27 (Q6P3X3), TTC37 (Q6PGP7), TTC38 (Q5R3I4), TTC7B (Q86TV6),


TTC9C (Q8N5M4), TTL (Q8NG68), TTLL12 (Q14166), TTN (Q8WZ42), TTYH1 (Q9H313),


TTYH3 (Q9C0H2), TUBA1B (P68363), TUBA4A (P68366), TUBB (P07437), TUBB2B


(Q9BVA1), TUBB2C (P68371), TUBB3 (Q13509), TUBB6 (Q9BUF5), TUBG1 (P23258),


TUBGCP2 (Q9BSJ2), TUBGCP3 (Q96CW5), TUFM (P49411), TWF1 (Q12792), TWF2


(Q6IBS0), TXN (P10599), TXNDC17 (Q9BRA2), TXNDC5 (Q8NBS9), TXNDC9 (O14530),


TXNL1 (O43396), TXNRD1 (Q16881), TYK2 (P29597), TYMS (P04818), U2AF1 (Q01081),


U2AF2 (P26368), UAP1 (Q16222), UBA1 (P22314), UBA2 (Q9UBT2), UBA3 (Q8TBC4), UBA52


(P62987), UBA6 (A0AVT1), UBE2D1 (P51668), UBE2D3 (P61077), UBE2E1 (P51965),


UBE2G2 (P60604), UBE2I (P63279), UBE2J2 (Q8N2K1), UBE2K (P61086), UBE2L3 (P68036),


UBE2M (P61081), UBE2N (P61088), UBE2O (Q9C0C9), UBE2S (Q16763), UBE2V1 (Q13404),


UBE2V2 (Q15819), UBE3A (Q05086), UBE3C (Q15386), UBE4A (Q14139), UBE4B (O95155),


UBFD1 (O14562), UBL3 (O95164), UBL4A (P11441), UBL5 (Q9BZL1), UBLCP1 (Q8WVY7),


UBP1 (Q9NZI7), UBQLN2 (Q9UHD9), UBR1 (Q8IWV7), UBR4 (Q5T4S7), UBTD1 (Q9HAC8),


UBXN1 (Q04323), UBXN6 (Q9BZV1), UCHL1 (P09936), UCHL3 (P15374), UCHL5 (Q9Y5K5),


UCK2 (Q9BZX2), UFC1 (Q9Y3C8), UFD1L (Q92890), UGDH (O60701), UGGT1 (Q9NYU2),


UGP2 (Q16851), ULK3 (Q6PHR2), UMPS (P11172), UNC119B (A6NIH7), UNC45A (Q9H3U1),


UPF1 (Q92900), UPP1 (Q16831), UQCRC1 (P31930), UQCRC2 (P22695), UQCRFS1


(P47985), URB1 (O60287), URB2 (Q14146), UROD (P06132), UROS (P10746), USO1


(O60763), USP10 (Q14694), USP11 (P51784), USP13 (Q92995), USP14 (P54578), USP15


(Q9Y4E8), USP24 (Q9UPU5), USP39 (Q53GS9), USP5 (P45974), USP7 (Q93009), USP9X


(Q93008), UTP15 (Q8TED0), UTP18 (Q9Y5J1), UTP20 (O75691), UTP6 (Q9NYH9), UTRN


(P46939), UXS1 (Q8NBZ7), UXT (Q9UBK9), VAC14 (Q08AM6), VAMP3 (Q15836), VAMP5


(O95183), VAPA (Q9P0L0), VAPB (O95292), VARS (P26640), VASP (P50552), VAT1


(Q99536), VAV2 (P52735), VBP1 (P61758), VCAN (P13611), VCL (P18206), VCP (P55072),


VDAC1 (P21796), VDAC2 (P45880), VDAC3 (Q9Y277), VIM (P08670), VPRBP (Q9Y4B6),


VPS11 (Q9H270), VPS13A (Q96RL7), VPS13C (Q709C8), VPS16 (Q9H269), VPS18


(Q9P253), VPS24 (Q9Y3E7), VPS25 (Q9BRG1), VPS26A (O75436), VPS26B (Q4G0F5),


VPS28 (Q9UK41), VPS29 (Q9UBQ0), VPS33A (Q96AX1), VPS33B (Q9H267), VPS35


(Q96QK1), VPS36 (Q86VN1), VPS37B (Q9H9H4), VPS39 (Q96JC1), VPS41 (P49754), VPS45


(Q9NRW7), VPS4A (Q9UN37), VPS4B (O75351), VPS53 (Q5VIR6), VPS8 (Q8N3P4), VRK1


(Q99986), VTA1 (Q9NP79), VWA1 (Q6PCB0), VWA5A (O00534), WARS (P23381), WASF2


(Q9Y6W5), WASL (O00401), WBSCR22 (O43709), WDFY1 (Q8IWB7), WDR1 (O75083),


WDR11 (Q9BZH6), WDR12 (Q9GZL7), WDR18 (Q9BV38), WDR26 (Q9H7D7), WDR3


(Q9UNX4), WDR36 (Q8NI36), WDR4 (P57081), WDR43 (Q15061), WDR45L (Q5MNZ6),


WDR48 (Q8TAF3), WDR5 (P61964), WDR54 (Q9H977), WDR6 (Q9NNW5), WDR61


(Q9GZS3), WDR73 (Q6P4I2), WDR74 (Q6RFH5), WDR75 (Q8IWA0), WDR77 (Q9BQA1),


WDR82 (Q6UXN9), WDR92 (Q96MX6), WHSC2 (Q9H3P2), WRNIP1 (Q96S55), XP32


(Q5T750), XPC (Q01831), XPNPEP1 (Q9NQW7), XPO1 (O14980), XPO4 (Q9C0E2), XPO5


(Q9HAV4), XPO6 (Q96QU8), XPO7 (Q9UIA9), XPOT (O43592), XRCC1 (P18887), XRCC5


(P13010), XRCC6 (P12956), XRN2 (Q9H0D6), YARS (P54577), YBX1 (P67809), YES1


(P07947), YKT6 (O15498), YRDC (Q86U90), YTHDC1 (Q96MU7), YTHDF2 (Q9Y5A9),


YWHAB (P31946), YWHAE (P62258), YWHAG (P61981), YWHAH (Q04917), YWHAQ


(P27348), YWHAZ (P63104), ZC3H15 (Q8WU90), ZC3HAV1 (Q7Z2W4), ZC3HAV1L


(Q96H79), ZCCHC3 (Q9NUD5), ZFAND1 (Q8TCF1), ZFR (Q96KR1), ZMAT2 (Q96NC0),


ZNF259 (O75312), ZNF326 (Q5BKZ1), ZNF330 (Q9Y3S2), ZNF622 (Q969S3), ZNF765


(Q7L2R6), ZNFX1 (Q9P2E3), ZW10 (O43264), ZWILCH (Q9H900), ZYG11B (Q9C0D3), ZYX


(Q15942).
















TABLE 21







100 most abundant proteins (name and SwissProt accession number)


in CTX0E03 microvesicles








Identified proteins
Accession number





Actin, cytoplasmic 2
P63261


Histone H4
P62805


Histone H2B
Q99879


Histone H3.2
Q71DI3


Histone H2B type 1
P23527


Glyceraldehyde-3-phosphate dehydrogenase
P04406


Histone H2A type 2-A
Q6FI13


Ubiquitin-40S ribosomal protein S27a
P62979


Annexin A2
P07355


Alpha-enolase
P06733


Pyruvate kinase isozymes M1/M2
P14618


60S ribosomal protein L6
Q02878


Histone H2B type 2-E
Q16778


Heat shock cognate 71 kDa protein
P11142


Actin, alpha cardiac muscle 1
P68032


Heat shock protein HSP 90-beta
P08238


Histone H2B type 1-J
P06899


Elongation factor 1-alpha 1
P68104


Tubulin beta-2C chain
P68371


60S ribosomal protein L18
Q07020


Tubulin beta chain
P07437


40S ribosomal protein S2
P15880


40S ribosomal protein S11
P62280


Histone H2B type 3-B
Q8N257


Tubulin alpha-1B chain
P68363


40S ribosomal protein S3
P23396


40S ribosomal protein S3a
P61247


Histone H2A type 1-D
P20671


Elongation factor 2
P13639


Heat shock protein HSP 90-alpha
P07900


GTP-binding nuclear protein Ran
P62826


60S ribosomal protein L4
P36578


40S ribosomal protein S9
P46781


Profilin-1
P07737


60S ribosomal protein L13a
P40429


Phosphoglycerate kinase 1
P00558


Fatty acid synthase
P49327


Annexin A1
P04083


Histone H2A.Z
P0C0S5


Vimentin
P08670


40S ribosomal protein S6
P62753


Moesin
P26038


Peptidyl-prolyl cis-trans isomerase A
P62937


60S ribosomal protein L26
P61254


60S ribosomal protein L3
P39023


40S ribosomal protein S8
P62241


60S ribosomal protein L28
P46779


Ezrin
P15311


40S ribosomal protein S4, X isoform
P62701


60S ribosomal protein L7a
P62424


60S ribosomal protein L13
P26373


60S ribosomal protein L7
P18124


40S ribosomal protein S23
P62266


60S ribosomal protein L5
P46777


Eukaryotic initiation factor 4A-I
P60842


40S ribosomal protein S24
P62847


Tubulin beta-2B chain
Q9BVA1


60S ribosomal protein L8
P62917


60S ribosomal protein L15
P61313


60S ribosomal protein L10
P27635


Peroxiredoxin-1
Q06830


Keratin, type I cytoskeletal 14
P02533


14-3-3 protein theta
P27348


40S ribosomal protein S18
P62269


Transketolase
P29401


60S ribosomal protein L24
P83731


Histone H1.5
P16401


Cofilin-1
P23528


Dihydropyrimidinase-related protein 3
Q14195


60S ribosomal protein L21
P46778


60S ribosomal protein L36
Q9Y3U8


Sodium/potassium-transporting ATPase subunit
P05023


alpha-1


40S ribosomal protein S16
P62249


T-complex protein 1 subunit gamma
P49368


Heterogeneous nuclear ribonucleoprotein A1
P09651


60S ribosomal protein L14
P50914


Heat shock 70 kDa protein 1A/1B
P08107


T-complex protein 1 subunit theta
P50990


60S ribosomal protein L30
P62888


Protein S100-A6
P06703


40S ribosomal protein SA
P08865


CD44 antigen
P16070


60S ribosomal protein L35a
P18077


Tubulin beta-3 chain
Q13509


T-complex protein 1 subunit delta
P50991


4F2 cell-surface antigen heavy chain
P08195


T-complex protein 1 subunit beta
P78371


Myosin-9
P35579


Adenosylhomocysteinase
P23526


Filamin-A
P21333


Fatty acid-binding protein, brain
O15540


Myristoylated alanine-rich C-kinase substrate
P29966


T-complex protein 1 subunit eta
Q99832


Fascin
Q16658


Fructose-bisphosphate aldolase A
P04075


60S ribosomal protein L27
P61353


60S ribosomal protein L17
P18621


Heterogeneous nuclear ribonucleoproteins A2/B1
P22626


60S ribosomal protein L10a
P62906


60S ribosomal protein L35
P42766









Discussion of Proteomic Data

CD63 (also known as MLA1 and TSPAN30), TSG101 (also known as ESCRT-I complex subunit TSG101), CD109 (also known as 150 kDa TGF-beta-1-binding protein) and thy-1 (also known as CD90) were detected in both exosomes and microvesicles.


Other tetraspanins were also detected: Tetraspanin-4, -5, -6, -9 and 14 were detected in the exosome fraction; tetraspanins-6 and -14 were detected in the microvesicles.


CD133 (also known as AC133, Prominin-1, PROM1, PROML1 and MSTP061) was detected in the exosomes but not the microvesicles.


CD53 (also known as MOX44 and TSPAN25), CD82 (also known as KAI1, SAR2, ST6 and TSPAN27), CD37 (also known as TSPAN26) and CD40 ligand (also known as CD40LG, CD40L and TNFSF5) were not detected in the exosomes or the microvesicles.


Nestin, GFAP and tubulin beta-3 chain (also known as TUBB3) were detected in both the exosome and microvesicle fractions, with tubulin beta-3 chain being particularly prominent within the top 100 proteins in both fractions. Sox2, DCX, GALC, GDNF and IDO were not detected.


Selectins and TNFRI (also known as TNF receptor 1, TNFRSF1A, TNFAR and TNFR1) were not detected.


Integrin alpha-2, -3, -4, -5, -6, -7, -V and integrin beta-1, -4 and -8 were detected in both exosome and microvesicle fractions. Integrin beta-3 and -5 were detected in the microvesicles only.


MHC Class I antigens (e.g. HLA_A1, HLA-A2 and HLA-B27) were detected in both the exosomes and microvesicles.


Cell-adhesion molecules (e.g. CADM1, CADM4, ICAM1, JAM3, L1CAM, NCAM) were detected in both the exosomes and microvesicles.


Cytoskeletal proteins (e.g. actin, vimentin, keratins, catenins, dystroglucan, neurofilament polypeptide, microtubule-associated protein, tubulin, desmoplaktin, plectin, plakophilin, septin, spectrin, talin, vinculin and zyxin) were detected in both the exosome and microvesicle fractions.


GTPases, clathrin, chaperones, heat-shock proteins (e.g. Hsp90, Hsp70), splicing factors, translation factors, annexins and growth factors (e.g. TGF-beta) were detected in both the exosomes and microvesicles.


Galectin-3, TIMP-1, thrombosponding-1, EGF receptor and CSK were detected in both the exosomes and microvesicles.



FIG. 18 compares the proteomic data from the exosomes and microvesicles. FIG. 18A illustrates the number of unique proteins within each micro particle population, isolated from week 2 Integra culture system. FIG. 18B compares the biological processes associated with the identified proteins within each micro particle population, isolated from week 2 Integra system. The proteins identified within exosomes and microvesicles are associated with very similar biological processes.


Proteins associated with biotin metabolism were only found in exosomes and proteins involved in tryptophan biosynthesis and taurine/alpha-linolenic acid metabolism were only identified in microvesicles.



FIG. 18C compares the CTX0E03 proteome to the Mesenchymal Stem Cell exosome proteome disclosed in Lai et al 2012, in which a total of 857 proteins were identified in exosomes released from mesenchymal stem cells.



FIG. 18D compares the biological processes associated with the identified proteins within the MSC derived exosomes (Lim 2012) with the neural stem cell derived exosomes of the invention. The three biological processes found to be associated with the MSC derived exosomes only are (in decreasing order of significance): Asthma; phenylalanine, tyrosine and tryptophan biosynthesis; and primary immunodeficiency. The thirty biological processes found to be associated only with the neural stem cell derived exosomes are shown in FIG. 19; the most significant biological function identified relates to RNA polymerase.


A further comparison of the 197 biological processes shared by both MSC derived exosomes and NSC derived exosomes shows that NSC exosomes contain notably more processes involved in RNA degradation, the Ribosome and spliceosomes, when compared to MSC exosomes.


The above comparison indicates a number of significant differences between NSC derived exosomes and MSC derived exosomes (as characterised by Lim et al 2012). The 4 most significant biological differences identified as present in NSC exosomes compared to being very low/absent in those identified by the Lim's group, all involve proteins associated with the production, packaging, function and degradation of genetic material, i.e RNA polymerase, RNA degradation, Ribosome and spliceosomes.


Example 14
Size Distribution of Microparticles

NanoSight analysis was undertaken to determine the particle size and concentration of microvesicles (“mv1” to “mv6”) and exosomes (“exo1” to “exo6”) isolated from CTX0E03 cells cultured in the Integra Celline system for 1, 2, 3, 4, 5 and 6 weeks. All results are based on 5 replicate measurements.


Particle size distribution was measured using Nanoparticle Tracking Analysis (NTA). NTA detects the movement of particles in solution and relates it to particle size. Mode and median particle size was calculated for all samples. Exosome samples were analysed using the most sensitive camera settings in order to capture the smallest vesicles. Microvesicle samples were analysed using less sensitive camera settings to prevent over exposure of the larger vesicles. As a result, some smaller vesicles were not detected in the samples. Although smaller vesicles were present in the MV samples, these represent a small percentage of the sample in terms of mass.


A proportion of Exo1 was labelled with a fluorescent membrane-specific dye (CellMask™) and a combination of NTA analysis with the CellMask™ labelling confirmed that the events detected by NTA correspond to membrane vesicles (data not shown).


The results are shown in Table 22 below, and in FIG. 17.


The exosomes show a drop in size at week six, from a mode of approximately 110 nm to approximately 70 nm, or from a median of approximately 130 nm to approximately 75 nm. The overall size range, from 70 nm to 150 nm, is consistent with the size of exosomes from other cell types, described in the art. The observed reduction in size of the exosomes to around 70 nm diameter after culturing the cells for 6 weeks correlates with the increased efficacy of exosomes isolated from CTX0E03 cells that have been cultured in a multi-compartment bioreactor for 6 weeks correlates, as reported in Example 8 and FIG. 6.


It is also noted that the concentration of microvesicles and exosomes decreases over the six week period of FIG. 17, broadly mirroring the improved efficacy observed over time.


The microvesicles are, as expected, larger, with a mode diameter of approximately 150 nm-200 nm, or a median diameter of approximately 180 nm-350 nm.









TABLE 22







Size distribution of CTX0E03 microvesicles and exosomes.















Concentration

Median


Sample
Count
Dilution
×1012/ml
Mode (nm)
(nm)















Exo1 (1)
5.204
10000
32.26
107
151


Exo1 (2)
1.734
10000
10.75
135
164


Exo1 (3)
6.55
10000
40.61
108
128


Exo2
14.33
10000
88.85
118
153


Exo3 (1)*
2.52
10000
15.62
89
115


Exo3 (2)
10.06
10000
62.37
115
146


Exo3 (3)
8.98
10000
55.68
128
147


Exo4 (1)
3.04
10000
18.85
111
136


Exo4 (2)
2.89
10000
17.92
110
120


Exo4 (3)
2.77
10000
17.17
116
134


Exo5 (1)
2.34
100
0.15
99
117


Exo5 (2)
2.02
100
0.13
102
124


Exo 5 (3)
2.08
100
0.13
116
127


Exo6 (1)
1.45
100
0.09
68
74


Exo6 (2)
1.19
100
0.07
69
75


MV1 (1)
9.314
200
1.15
183
212


MV1 (2)
10.76
200
1.33
161
214


MV1 (3)
10.738
200
1.33
173
198


MV2
5.89
1000
3.65
177
194


MV3 (1)*
5.68
2000
7.04
150
186


MV3 (2)
11.5
2000
14.26
221
351


MV3 (3)
9.57
2000
11.87
214
270


MV4 (1)
4.894
400
1.21
209
240


MV4 (2)
2.934
1000
1.82
195
212


MV4 (3)
2.55
1000
1.58
184
221


MV5 (1)
1.086
200
0.13
164
237


MV5 (2)
1.458
200
0.18
205
205


MV 5 (3)
1.3
200
0.16
219
210


MV6 (1)
0.346
200
0.04
171
186


MV6 (2)
0.37
200
0.05
168
212


Media
0.14
10
0.00
100
149





*large aggregates.






Example 15
Exosomes Produced by Human CD34+Progenitor Cells Derived from Cord Blood Conditionally Immortalized with a c-mycERTAM Lentivirus

Human CD34+ progenitor cells derived from cord blood were conditionally immortalized with a c-mycERTAM lentivirus. qRT-PCR confirmed the presence of c-mycERTAM mRNA in lentivirus infected human CD34+ progenitor derived from cord blood (as shown in FIG. 20A).


Western Blot analysis of exosomes from cell culture supernatants was then performed. The analysis of samples was done as specified below:

    • 1. Concentration of exosomes from two cell supernatant samples, 1 ml volume each, using exosome capture immunobeads;
    • 2. SDS-PAGE and WB analysis for expression of common exosome associated proteins (Alix and HSP70) including normalisation control and reference sample with known expression of analysed markers and known protein content.


Material Analysed:

Aliquots of supernatant from cell culture contained three 1.5 ml vials isolated from CD34 and conditionally immortalised CD34 c-mycERTAM cells, stored at −80° C. until processing.


Description of the Activity:

Exosomes are captured and concentrated from precleared supernatants by incubation with exosome capture immunobeads ON at 4° C.


After immunobeads recovery, remaining supernatant we as ultracentrifuged 2 hours at 120,000×g to precipitate residual exosomes (if any) and check efficiency of immunocapturing.


Total IP and UC pellets, corresponding each to 1 ml of original supernatant samples, were lysed and loaded on gel and analyzed by WB for Alix (a protein involved in the concentration and sorting of cargo proteins at the multivesicular body level, and incorporation into IL vesicles, ubiquitously expressed in all exosomes) and HSP70 (molecular chaperone facilitating the assembly of multi-protein complexes, participate in the translocation of polypeptides across cell membranes, expressed on exosomes from most epithelial like human cells).


Concentrated supernatants were too dense and thus not suitable for WB analysis.


Overall Results of the Analysis:
Control Sample:

We first applied the protocol to a control sample, supernatant derived from a human cell culture, showing that 1 mL of sample is sufficient to obtain high expression signal.


We harvested control sample supernatant from 72 hours old cell culture at 80% confluence, in a way that 1 ml of conditioned medium corresponded to 1.1×106 cells with a total yield of 8.3 μg total exosomes protein concentration/ml supernatant.


As a reference to estimate band's density we loaded three different concentrations of exosomes purified from the same human cell culture supernatant.


As expected immunoprecipitated sample (from 1 ml of supernatant) yields a signal comparable to 5-10 μg of purified exosomes. This result is shown in FIG. 20B.


Sample: Cell Culture Supernatant
CD34

CD34 cmycERTAM


Exosomes from sample supernatants were successfully concentrated using exosome capturing immunobeads (based on binding tetraspanins as common exosomal markers).


Samples loaded after Immunoprecipitation (IP) shows Alix expression corresponding to overall yield of less than 1 μg of exosomes while no clear differences can be appreciated between two samples: both CD34+ and CD34+ c-MycERTam cells produce exosomes. A different method should be used for accurate quantitative comparison. Samples appear negative for HSP70 expression (possibly due to tissue/cell type). These results are shown in FIG. 20C.


Used immunobeads have previously shown to precipitate total exosomes from cell supernatant as confirmed by absence of signal after ultracentrifugation (UC) of residual supernatants. The same was confirmed also for analyzed sample.


Embodiments of the invention comprise:

    • 1. A neural stem cell microparticle.
    • 2. The neural stem cell microparticle of embodiment 1, wherein the microparticle is an exosome, microvesicle, membrane particle, membrane vesicle, exosome-like vesicle, ectosome-like vesicle, ectosome or exovesicle.
    • 3. The neural stem cell microparticle of embodiments 1 or 2, wherein the microparticle is derived from a neural stem cell line.
    • 4. The neural stem cell microparticle of embodiment 3, wherein the neural stem cell line is conditionally-immortalised and/or grown in serum free medium.
    • 5. The neural stem cell microparticle of embodiment 4, wherein the neural stem cell line is
    • CTX0E03 having ECACC Accession No. 04091601, STR0C05 having ECACC Accession No. 04110301 and HPC0A07 having ECACC Accession No. 04092302.
    • 6. The neural stem cell microparticle of any preceding embodiment, wherein the microparticle has:
      • (a) a size of between 30 nm and 1000 nm, or between 30 and 200 nm, or between 30 and 100 nm, as determined by electron microscopy; or
      • (b) a density in sucrose of 1.1-1.2 g/ml.
    • 7. The neural stem cell microparticle of any preceding embodiment, comprising RNA.
    • 8. The neural stem cell microparticle of embodiment 7, wherein the RNA is mRNA and/or miRNA.
    • 9. The neural stem cell microparticle of embodiment 8, wherein the microparticle comprises one, two, three or four of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532.
    • 10. The neural stem cell microparticle of any preceding embodiment, comprising one or more of:
      • (a) a lipid selected from ceramide, cholesterol, sphingomyelin, phosphatidylserine, phosphatidylinositol, and/or phosphatidylcholine;
      • (b) miRNA, optionally selected from hsa-let-7 g, hsa-miR-101, hsa-miR-10a, hsa-miR-10b, hsa-miR-126, hsa-miR-128, hsa-miR-129-5p, hsa-miR-130a, hsa-miR-134, hsa-miR-137, hsa-miR-155, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-17, hsa-miR-182, hsa-miR-183, hsa-miR-185, hsa-miR-18b, hsa-miR-192, hsa-miR-194, hsa-miR-195, hsa-miR-20a, hsa-miR-20b, hsa-miR-210, hsa-miR-218, hsa-miR-301a, hsa-miR-302a, hsa-miR-302c, hsa-miR-345, hsa-miR-375, hsa-miR-378, hsa-miR-7, hsa-miR-9, hsa-miR-93, hsa-miR-96, and hsa-miR-99a;
      • (c) a tetraspanin, optionally selected from CD63, CD81, CD9, CD53, CD82 and/or CD37;
      • (d) TSG101, Alix, CD109 and/or thy-1; and/or
      • (e) CD133.
    • 11. The neural stem cell microparticle of any preceding embodiment, comprising at least 10 of the proteins present in Table 19 or Table 21.
    • 12. The neural stem cell microparticle of any preceding embodiment, comprising at least one biological activity of a neural stem cell or a neural stem cell-conditioned medium.
    • 13. The neural stem cell microparticle of embodiment 12, wherein the at least one biological activity is regenerative activity.
    • 14. The neural stem cell microparticle of any preceding embodiment, for use in therapy.
    • 15. The neural stem cell microparticle of embodiment 14, wherein the therapy is regenerative therapy.
    • 16. The neural stem cell microparticle of embodiments 14 or 15, wherein the therapy is for a
      • (i) Neurological disorder, disease or deficit, such as Parkinson's, Alzheimer's, Stroke, or ALS;
      • (ii) Lysosomal storage disorder;
      • (iii) Cardiovascular disorder, such as Myocardial Infarction, congestive heart failure, Peripheral Arterial Disease, diabetic ulcers, wound healing;
      • (iv) Diseases of the lung, including Idiopathic Pulmonary Fibrosis, Respiratory Distress Syndrome, Chronic Obstructive Pulmonary Disease, Idiopathic Pulmonary Hypertension, Cystic Fibrosis and Asthma
      • (v) Metabolic or inflammatory disorder, such as Diabetes (I or II), rheumatoid arthritis, osteoarthritis, lupus, Crohn's disease, Irritable Bowel Disease, or Graft versus Host Disease;
      • (vi) Psychiatric disorder, such as: Depression, Bipolar, Schizophrenia or an Autistic syndrome disorder such as Autism, Asperger's syndrome or Rett Syndrome;
      • (vii) Blindness-causing disease of the retina, such as Age-related macular degeneration, Stargardt disease, diabetic retinopathy, or retinitis pigmentosa; and
      • (viii) Demyelinating disease, such as multiple sclerosis, central pontine myelinolysis, tabes dorsalis, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukodystrophies, Guillain-Barre syndrome, Anti-MAG peripheral neuropathy and Charcot-Marie-Tooth disease.
    • 17. The neural stem cell microparticle of embodiments 14-16, wherein the therapy improves functional and/or cognitive recovery.
    • 18. The neural stem cell microparticle of embodiments 14-17, wherein the therapy is of stroke, peripheral arterial disease or blindness-causing diseases of the retina.
    • 19. The neural stem cell microparticle of embodiments 14 to 17, wherein:
      • (i) the microparticle is an exosome and therapy is of a disease or condition requiring tissue replacement, regeneration or repair; or
      • (ii) the microparticle is a microvesicle and the therapy is of a disease requiring angiogenesis or a neurological disease, disorder or deficit.
    • 20. Use of a neural stem cell microparticle according to any preceding embodiment, in the manufacture of a medicament for the treatment of a disease.
    • 21. A method of producing a neural stem cell microparticle as defined in embodiments 1-13, comprising isolating a microparticle from a neural stem cell-conditioned medium.
    • 22. A method of producing a stem cell microparticle, comprising isolating a microparticle from a stem cell-conditioned medium wherein:
      • (i) the stem cell-conditioned medium comprises one or more components which induce the release of microparticles by the stem cells into the medium;
      • (ii) the stem cells were cultured under hypoxic conditions;
      • (iii) the stem cells were co-cultured with a different cell type;
      • (iv) the stem cells were cultured in a multi-compartment bioreactor; and/or
      • (v) the stem cells were partially-differentiated.
    • 23. A method according to embodiment 22, wherein the stem cell is a neural stem cell, optionally as defined in any of embodiments 3 to 5.
    • 24. A method according to embodiment 22(i) or embodiment 23 when dependent upon embodiment 22(i), wherein the one or more components are selected from: transforming growth factor-beta (TGF-β), interferon-gamma (INF-γ) and tumour necrosis factor-alpha (TNF-α).
    • 25. A method according to embodiment 22(iii), embodiment 23 or 24 when dependent upon embodiment 22(iii), wherein the different cell type is an endothelial cell.
    • 26. A microparticle obtainable by the method of any of embodiments 22-25.
    • 27. A composition comprising a microparticle according to any of embodiments 1-13 or 26 and a pharmaceutically acceptable excipient, carrier or diluent.
    • 28. A kit for use in a method for producing the microparticle of any of embodiments 1-13 or 26 comprising: (a) a medium; (b) a neural stem cell; (c) optionally the one or more components of embodiments 22 to 24; (d) optionally the microparticle of any of embodiments 1-13 or 26 suitable for use as a control; (e) optionally a detection agent suitable for specific detection of the produced microparticles; and (f) instructions for producing the microparticle of any of embodiments 1-13 or 26 using the kit.
    • 29. A method of screening for an agent that alters the rate of production of a microparticle by a stem cell, comprising contacting a stem cell with a candidate agent and observing whether the rate production of microparticles by the contacted stem cell increases or decreases compared to a control.
    • 30. A method of producing a stem cell microparticle, comprising:
      • i. culturing the stem cells in an environment that allows stem cell differentiation; and
      • ii. collecting the microparticles that are produced by the cells.
    • 31. The method of embodiment 30, wherein an environment that allows stem cell differentiation is culture in a multi-compartment bioreactor, for example wherein the bioreactor contains at least two compartments separated by one or more membranes or barriers that separate the compartment containing the cells from one or more compartments containing gas and/or culture medium.
    • 32. The method of embodiment 31, wherein the culture is for more than seven days.
    • 33. The method of any one of embodiments 30-32, comprising isolating a microparticle from a stem cell-conditioned medium.
    • 34. The method of embodiment 33, wherein the stem cell-conditioned medium comprises one or more additive components or agents which stimulate the release of microparticles by the stem cells into the medium.
    • 35. The method of embodiment 34, wherein the one or more components are selected from transforming growth factor-beta (TGF-β), interferon-gamma (INF-γ) and/or tumour necrosis factor-alpha (TNF-α).
    • 36. The method of any one of embodiments 33-35, wherein the stem cells were cultured under hypoxic conditions.
    • 37. The method of any one of embodiments 33-36, wherein the stem cells were co-cultured with a different cell type.
    • 38. The method of embodiment 37, wherein the different cell type is an endothelial cell.
    • 39. The method of any one of embodiments 30 to 38, wherein the stem cell is a neural stem cell.
    • 40. The method of embodiment 39, wherein the stem cell is a neural stem cell line.
    • 41. The method of embodiment 40, wherein the neural stem cell line is conditionally-immortalised.
    • 42. The method of embodiments 40 or 41, wherein the neural stem cell line is CTX0E03 having ECACC Accession No. 04091601, STR0C05 having ECACC Accession No. 04110301, or HPC0A07 having ECACC Accession No. 04092302.
    • 43. The method of any one of embodiments 40-42, wherein the neural stem cell line is grown in serum-free medium.
    • 44. The method of any one of embodiments 39-43, wherein the neural stem cell expresses one or more of the markers: Nestin, Sox2, GFAP, 13111 tubulin, DCX, GALC, TUBB3, GDNF and 100.
    • 45. The method of any one of embodiments 39-44, wherein the microparticle is an exosome and the exosome expresses one or more of: DCX, GFAP, GALC, TUBB3, GDNF and IDO.
    • 46. The method of any one of embodiments 30 to 45, wherein at least two, three, four, five, six or seven miRNAs are up or down regulated in the microparticle compared to in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation.
    • 47. The method of any one of embodiments 30 to 46, wherein the microparticle is an exosome and the exosome expresses one, two, three, four, five, six, seven, eight, nine, ten or more or more of the following miRNAs at a higher level than is expressed in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation: hsa-miR-146b-5p; hsa-let-7c; hsa-miR-99a; hsa-miR-132; hsa-miR-378; hsa-miR-181a; hsa-let-7b; hsa-miR-100; hsa-let-7e; hsa-miR-23b; hsa-miR-185; hsa-let-7i; hsa-let-7a; hsa-let-7d; hsa-let-7 g; hsa-miR-222; hsa-let-7f; hsa-miR-218; hsa-miR-24; hsa-miR-9; hsa-miR-126; hsa-miR-134; hsa-miR-128; and hsa-miR-155.
    • 48. The method of any one of embodiments 30 to 47, wherein the microparticle is an exosome and the exosome expresses one, two, three, four, five, six, seven, eight, nine, ten or more of the following miRNAs at a lower level than is expressed in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation: hsa-miR-22; hsa-miR-26a; hsa-miR-210; hsa-miR-92a; hsa-miR-93; hsa-miR-424; hsa-miR-195; hsa-miR-127-5p; hsa-miR-21; hsa-miR-103a; hsa-miR-16; hsa-miR-125a-5p; hsa-miR-10a; hsa-miR-10b; hsa-miR-345; hsa-miR-130a; hsa-miR-15b; hsa-miR-20b; hsa-miR-20a; hsa-miR-17; hsa-miR-7; hsa-miR-106b; hsa-miR-101; hsa-miR-302a; hsa-miR-301a; hsa-miR-183; hsa-miR-219-5p; hsa-miR-18a; hsa-miR-15a; hsa-miR-182; hsa-miR-33a; hsa-miR-96; and hsa-miR-18b.
    • 49. The method of any of embodiments 30 to 48, wherein the microparticle comprises one, two, three or four of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532.
    • 50. The method of any one of embodiments 30 to 49, further comprising loading the isolated or purified microparticle with one or more exogenous nucleic acids, lipids, proteins, drugs or prodrugs.
    • 51. The method of embodiment 50, wherein the exogenous nucleic acid is siRNA capable of silencing one or more pathological genes.
    • 52. A microparticle obtainable by the method of any one of embodiments 30-51.
    • 53. A microparticle which is an exosome and the exosome expresses one or more of: DCX, GFAP, GALC, TUBB3, GDNF and IDO.
    • 54. The microparticle of any one of embodiments 52-53, wherein the exosome expresses one, two, three, four, five, six, seven, eight, nine, ten or more or more of the following miRNAs at a higher level than is expressed in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation: hsa-miR-146b-5p; hsa-let-7c; hsa-miR-99a; hsa-miR-132; hsa-miR-378; hsa-miR-181a; hsa-let-7b; hsa-miR-100; hsa-let-7e; hsa-miR-23b; hsa-miR-185; hsa-let-7i; hsa-let-7a; hsa-let-7d; hsa-let-7 g; hsa-miR-222; hsa-let-7f; hsa-miR-218; hsa-miR-24; hsa-miR-9; hsa-miR-126; hsa-miR-134; hsa-miR-128; and hsa-miR-155.
    • 55. The microparticle of any one of embodiments 52-54, wherein the exosome expresses one, two, three, four, five, six, seven, eight, nine, ten or more of the following miRNAs at a lower level than is expressed in the corresponding stem cells cultured in standard T-175 flasks, as calculated by Fold Regulation: hsa-miR-22; hsa-miR-26a; hsa-miR-210; hsa-miR-92a; hsa-miR-93; hsa-miR-424; hsa-miR-195; hsa-miR-127-5p; hsa-miR-21; hsa-miR-103a; hsa-miR-16; hsa-miR-125a-5p; hsa-miR-10a; hsa-miR-10b; hsa-miR-345; hsa-miR-130a; hsa-miR-15b; hsa-miR-20b; hsa-miR-20a; hsa-miR-17; hsa-miR-7; hsa-miR-106b; hsa-miR-101; hsa-miR-302a; hsa-miR-301a; hsa-miR-183; hsa-miR-219-5p; hsa-miR-18a; hsa-miR-15a; hsa-miR-182; hsa-miR-33a; hsa-miR-96; and hsa-miR-18b.
    • 56. The microparticle any one of embodiments 52-55, wherein the microparticle comprises one, two, three or four of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532.
    • 57. The microparticle of any one of embodiments 52-56, for use in therapy.
    • 58. The microparticle or embodiment 57, wherein the therapy is for a:
      • i. Neurological disorder, disease or deficit, such as Parkinson's, Alzheimer's, Stroke, or ALS;
      • ii. Lysosomal storage disorder;
      • iii. Cardiovascular disorder, such as Myocardial Infarction, congestive heart failure, Peripheral Arterial Disease, diabetic ulcers, wound healing;
      • iv. Diseases of the lung, including Idiopathic Pulmonary Fibrosis, Respiratory Distress Syndrome, Chronic Obstructive Pulmonary Disease, Idiopathic Pulmonary Hypertension, Cystic Fibrosis and Asthma;
      • v. Metabolic or inflammatory disorder, such as Diabetes (I or II), rheumatoid arthritis, osteoarthritis, lupus, Crohn's disease, Irritable Bowel Disease, or Graft versus Host Disease;
      • vi. Psychiatric disorder, such as: Depression, Bipolar, Schizophrenia or an Autistic syndrome disorder such as Autism, Asperger's syndrome or Rett Syndrome;
      • vii. Blindness-causing disease of the retina, such as Age-related macular degeneration, Stargardt disease, diabetic retinopathy, or retinitis pigmentosa; or
      • viii. Demyelinating disease, such as multiple sclerosis, cerebral palsy, central pontine myelinolysis, tabes dorsalis, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukodystrophies, Guillain-Barre syndrome, Anti-MAG peripheral neuropathy and Charcot-Marie-Tooth disease.
    • 59. A composition comprising the microparticle of any one of embodiments 52-56 and a stem cell, optionally wherein the stem cell is the stem cell from which the microparticle is derived, for example wherein the stem cell is CTX0E03 having ECACC Accession No. 04091601.
    • 60. The composition of embodiment 59, for use in therapy.
    • 61. The composition of embodiment 60, wherein the stem cell and the microparticle are administered:
      • i. together in a single pharmaceutical composition;
      • ii. contemporaneously or simultaneously but separately; or
      • iii. separately and sequentially, for example wherein the duration between the administration of the cell and microparticle is one hour, one day, one week, two weeks or more.
    • 62. The composition of any one of embodiments 60-61, wherein the therapy induces tolerance, typically immunotolerance, in a host that is to receive the stem cells from which the microparticle is derived; or wherein tolerance to the stem cells is increased by administering stem cells together with the microparticles.


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Claims
  • 1. A conditionally-immortalised cell that produces microparticles.
  • 2. The cell according to claim 1, wherein the conditionally-immortalised cell is: a mesenchymal stem cell, optionally selected from a bone marrow derived stem cell, an endometrial regenerative cell, a mesenchymal progenitor cell, an adipose derived stem cell or a multipotent adult progenitor cell;a neural stem cell, optionally selected from a neurosphere initiating stem cell, or an oligodendrocyte precursor cell;a haematopoietic stem cell, optionally a CD34+ cell and/or isolated from umbilical cord blood, or optionally a CD34+/CXCR4+ cell;a non-haematopoietic umbilical cord blood stem cell;a very small embryonic like stem cell (VSEL);an induced pluripotent stem (iPS) cell;a fibroblast; ora dendritic cell.
  • 3. The cell according to claim 1 or claim 2, wherein the cell comprises c-mycER.
  • 4. The cell of claim 1, wherein the microparticle is an exosome, microvesicle, membrane particle, membrane vesicle, exosome-like vesicle, ectosome-like vesicle, ectosome or exovesicle.
  • 5. The cell according to claim 1, wherein the cell is from a stem cell line, optionally a neural stem cell line or a non-neural stem cell line.
  • 6. The cell of claim 5, wherein the stem cell line is grown in serum free medium.
  • 7. The cell of claim 6, wherein the stem cell line is a neural stem cell line, optionally CTX0E03 having ECACC Accession No. 04091601, STR0C05 having ECACC Accession No. 04110301 and HPC0A07 having ECACC Accession No. 04092302.
  • 8. The cell of claim 1, wherein the microparticle has: (a) a size of between 30 nm and 1000 nm, or between 30 and 200 nm, or between 30 and 100 nm, as determined by electron microscopy; or(b) a density in sucrose of 1.1-1.2 g/ml.
  • 9. The cell of claim 1, wherein the microparticle comprises RNA.
  • 10. The cell of claim 9, wherein the RNA is mRNA and/or miRNA.
  • 11. The cell of claim 10, wherein the microparticle comprises one, two, three or four of hsa-miR-1246, hsa-miR-4492, hsa-miR-4488 and hsa-miR-4532.
  • 12. The cell of claim 1, wherein the microparticle comprises one or more of: (a) a lipid selected from ceramide, cholesterol, sphingomyelin, phosphatidylserine, phosphatidylinositol, and/or phosphatidylcholine;(b) miRNA, optionally selected from hsa-let-7 g, hsa-miR-101, hsa-miR-10a, hsa-miR-10b, hsa-miR-126, hsa-miR-128, hsa-miR-129-5p, hsa-miR-130a, hsa-miR-134, hsa-miR-137, hsa-miR-155, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-17, hsa-miR-182, hsa-miR-183, hsa-miR-185, hsa-miR-18b, hsa-miR-192, hsa-miR-194, hsa-miR-195, hsa-miR-20a, hsa-miR-20b, hsa-miR-210, hsa-miR-218, hsa-miR-301a, hsa-miR-302a, hsa-miR-302c, hsa-miR-345, hsa-miR-375, hsa-miR-378, hsa-miR-7, hsa-miR-9, hsa-miR-93, hsa-miR-96, and hsa-miR-99a;(c) a tetraspanin, optionally selected from CD63, CD81, CD9, CD53, CD82 and/or CD37;(d) TSG101, Alix, CD109 and/or thy-1; and/or(e) CD133.
  • 13. The cell of claim 1, wherein the microparticle comprises at least 10 of the proteins present in Table 19 or Table 21.
  • 14. The cell of claim 1, wherein the microparticle comprises at least one biological activity of a stem cell, a stem cell-conditioned medium, a neural stem cell or a neural stem cell-conditioned medium.
  • 15. The cell of claim 14, wherein the at least one biological activity is regenerative activity.
  • 16. A therapeutic method comprising administration of the cell of claim 1 to a patient.
  • 17. The method of claim 16, wherein the therapy is regenerative therapy.
  • 18. A method of producing a microparticle, comprising isolating a microparticle from a conditionally-immortalised cell-conditioned medium.
  • 19. A method of producing a microparticle according to claim 18, wherein: (i) the cell-conditioned medium comprises one or more components which induce the release of microparticles by the stem cells into the medium;(ii) the cells were cultured under hypoxic conditions;(iii) the cells were co-cultured with a different cell type;(iv) the cells were cultured in a multi-compartment bioreactor; and/or(v) the cells were stem cells that were partially-differentiated.
  • 20. A method according to claim 19(i), wherein the one or more components are selected from: transforming growth factor-beta (TGF-β), interferon-gamma (INF-γ) and tumour necrosis factor-alpha (TNF-α).
  • 21. A method according to claim 19(iii), or claim 20 when dependent upon claim 19(iii), wherein the different cell type is an endothelial cell.
  • 22. A microparticle obtainable by the method of claim 18 or 19.
  • 23. A composition comprising a microparticle according to claim 22 and a pharmaceutically acceptable excipient, carrier or diluent.
  • 24. A kit for use in a method for producing the microparticle of claim 22 comprising: (a) a medium; and (b) a conditionally-immortalised cell.
  • 25. A method of screening for an agent that alters the rate of production of a microparticle by a conditionally-immortalised cell, comprising contacting a conditionally immortalised cell with a candidate agent and observing whether the rate production of microparticles by the contacted stem cell increases or decreases compared to a control.
Priority Claims (2)
Number Date Country Kind
1302468.2 Feb 2013 GB national
1317888.4 Oct 2013 GB national
PCT Information
Filing Document Filing Date Country Kind
PCT/GB2014/050412 2/12/2014 WO 00