KR101399056B1 - Pharmaceutical Compositions For Preventing or Treating Neuronal Diseases Comprising Stem Cell-derived Microvesicles as Active Ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neural disease comprising stem cell-derived micro-vesicle as an active ingredient. According to the present invention, the composition of the present invention provides a cell therapeutic agent free from ethical problems and immunogenicity problems, thereby providing a safer and more effective therapeutic effect as compared with the conventional method using stem cells directly.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating neurological diseases, which comprises stem cell-derived micro-endoplasmic reticulum as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating neurological diseases comprising stem cell-derived micro-vesicle as an active ingredient.

Mesenchymal Stem Cells (MSCs) have a heterogeneous population of stem cells with the ability to differentiate into other embryonic lines such as self-renewing and mesenchymal lineage and endoderm and ectoderm. ^1-4 Moreover, mesenchymal stem cells have effects for immunoregulatory function or anti-inflammatory and trophic effects. ^{Compared to} embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other tissue-specific stem cells, mesenchymal stem cells have excellent availability, low ethical issues and immunity It is an ideal candidate for the clinical application of regenerative medicine because it has originality. Mesenchymal stem cells can be isolated from the brain, liver, lung, fetal blood, cord blood, kidney, adipose tissue and placenta. ⁷ This broad distribution of whole bodies suggests that mesenchymal stem cells play an important role in maintaining tissue homeostasis. ⁸ Numerous studies have demonstrated the ability of mesenchymal stem cells to excrete in vitro into Lineage cells such as bone, cartilage, tendons and fats. ² This shows the potential for therapeutic agents in preclinical and clinical studies of bone and cartilage regeneration. ^In addition, clinical studies of mesenchymal stem cell-based cell therapy have shown that allergic bone marrow transplantation or hematopoietic stem cell transplantation, myocardial stiffness, systemic lupus erythematosus, cirrhosis, diabetes, acute kidney damage and various degenerative neurological diseases, And Graft-versus-host disease (GvHD) in a wide spectrum of human diseases. ^10-16

However, the fundamental mechanism of regeneration of mesenchymal stem cells is not known. In addition, the therapeutic effect observed in the absence of mesenchymal stem cell transplantation such as other connective tissues and organs other than the target tissue is also a suspicious effect of mesenchymal stem cell-based cell therapy.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a cell therapy agent for prevention or treatment of neurological diseases and a cell therapy agent having no ethical problems and no immunogenicity. As a result, the inventors of the present invention have completed the present invention by confirming that neural diseases can be effectively prevented or treated while not showing the ethical problems and immunogenicity of the conventional cell therapy agents when the stem cell-derived microemboli is used.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating neurological diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating neural disease comprising stem cell-derived micro-vesicle as an active ingredient.

The present inventors have sought to develop a cell therapy agent for prevention or treatment of neurological diseases and a cell therapy agent having no ethical problems and no immunogenicity. As a result, it has been found that the use of stem cell-derived micro-vesicles can effectively prevent or treat neurological diseases without showing ethical problems and immunogenicity against conventional cell therapy agents.

As used herein, the term " stem cell-derived microemboli " is a cell membrane particle derived from a stem cell. The micro-vesicle has the same meaning as the exosome, the circulating micro-vesicle or the micro-vesicle in the art, and refers to a fragment having a plasma membrane of 50 nm to 1000 nm removed from the cell. The micro-vesicles mediate the transport of mRNA, miRNA and protein between cells and play an important role in intracellular interactions. Microemboli expresses inhomogeneous population depending on the derived cells, the number of cells, the size of the cells and the composition of the antigen. The term " stem cell " includes any undifferentiated or partially undifferentiated cells capable of proliferation (self-renewal) and differentiation (plasticity). These stem cells replace mature cells of specific cell lineage cells that are cells at the endpoint stage during development. In addition, the stem cells include stem cells having unlimited self-renewing ability and pre-differentiating ability plasticity, and progenitor cells having multipotential or monodisperse ability plasticity.

Preferably, the stem cell is a mesenchymal stem cell, and the mesenchymal stem cell is a mesenchymal stem cell derived from brain, liver, lung, cord blood, fetal blood, kidney, adipose tissue, placenta or bone marrow.

According to a preferred embodiment, the stem cell-derived microvesicles of the present invention are mesenchymal stem cell-derived microspores, more preferably mesenchymal stem cell-derived microspores isolated from human bone marrow.

One of the main features of the present invention is to analyze the proteome of the stem cell-derived microemboli to investigate the preventive or therapeutic effect of neurological diseases. The present inventors have identified 730 micro-vesicle proteins through LC-MS / MS (Liquid Chromatography-Mass Spectrometry) analysis of stem cell-derived microvesicles.

According to the results of the analysis of the present invention, preferably, said microemboli has (a) an immunological characteristic of positive for CD13, CD29, CD44, CD73 and CD105 surface antigens; And (b) variable immunological properties for CD10 and CD90.

According to a preferred embodiment, the proteome of the stem cell-derived micro-endoplasmic reticulum of the present invention comprises molecules of the signal transduction pathway involved in self-renewal and differentiation of stem cells. Briefly, first, microfibrillar proteomes contain 13 proteins involved in the self-renewal of mesenchymal stem cells based on the KEGG pathway pathway and the GOBP signaling pathway, the growth factor receptor and the two signaling pathways involved in the Wnt signaling pathway do. Second, it contains 38 proteins involved in the differentiation of mesenchymal stem cells, the five signaling pathways involved in TGFβ, MAPK, PPAR and BMP signaling pathways. The Wnt signaling pathway is reported to be involved in the self-renewal and differentiation of mesenchymal stem cells. These results suggest that the mesenchymal stem cell-derived microfilament is characterized by mesenchymal stem cells and that these microfilaments may affect self-regeneration and differentiation through proteins involved in the signal transduction pathway.

Preferably, the micro-vesicle is at least one mesenchymal stem cell selected from the group consisting of Plasmin-Derived Growth Factor Receptor (PDGFRB), Epidermal Growth Factor Receptor (EGFR) and Plasminogen Activator (PLAUR) Self-regenerating surface receptors.

The PEGFRB encodes a cell surface tyrosine kinase receptor for the PDGF (Platelet-Derived Growth Factor) family. These growth factors are mitogens for cells of mesenchymal origin. The EGFR is a cell-surface receptor for the EGF (Epidermal Growth Factor) family of extracellular protein ligands. EGFR is one of the ErbB families such as EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). The PLAUR is a multi-domain glycoprotein bound to a cell membrane.

Preferably, the micro-vesicle is selected from the group consisting of RRAS (Ras-related protein R-Ras) / NRAS (Neuroblastoma RAS viral oncogene homolog), MAPK1 (Mitogen- activated protein kinase 1), GNA13 (Guanine Nucleotide-binding protein subunit Alpha-13) From the group consisting of CD22 (guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit Gamma-12), CDC42 (Cell Division Control protein 42 homolog) and VAV2 (Guanine nucleotide exchange factor VAV2) Have at least one signaling molecule selected.

The RRAS / NARS, MAPK1 and VAV2 are involved in the RAS-MAPK (Ras / Mitogen-Activated Protein Kinase) signaling pathway, GNA13 is involved in RHO (Rhodopsin) signaling pathway and GNG12 and CDC42 are involved in the CDC42 signaling pathway I am involved.

Preferably, the microvesicles are selected from the group consisting of FN1 (Fibronectin), EZR (Ezrin), IQGAP1 (IQ motif containing GTPase Activating Protein 1), CD47, Integrin and LGALS1 (Galectin-1) / LGALS3 At least one cell attachment molecule selected.

The FN1 is an extracellular matrix polymeric glycoprotein that binds to a membrane-spanning receptor protein called integrin. FN1 binds to extracellular matrix elements such as collagen, fibrin and heparan sulfate proteoglycans (e.g., cindecane). FN1 consists of a dimer consisting of the same monomers linked by disulfide bonds. The EZR is a protein that encodes a human EZR gene known as cytovillin or borin-2. The cytoplasmic membrane protein encoded by the EZR gene functions as a protein-tyrosine kinase substrate in the microvilli. One type of ERM protein family, the EZR protein acts as a mediator between the plasma membrane and the actin cytoskeleton. The IQGAP1 is a protein that is encoded by the IQGAP1 gene and is ubiquitously expressed. IQGAP1 is a scaffold protein that regulates a variety of cellular processes ranging from the organization of the actin fascia skeleton to the attachment of cells that regulate transcription and cell cycle. The CD47 is a membrane protein associated with an increase in intracellular calcium concentration upon cell attachment to the extracellular matrix. CD47 is also a receptor for the C-terminal cell binding domain of thrombospondin and plays an important role in membrane transport and signal transduction. Integrins are receptors mediating adhesion between cells and their surrounding tissues, other cells, or extracellular matrix. The integrins regulate cell shape, motility and cell cycle by cell signal transduction and signal transduction. The LGALS1 and LGALS3 are beta-galactoside-binding proteins mediating cell-cell and cell-substrate interactions. LGALS1 acts as an autocrine negative growth factor that regulates cell proliferation.

Preferably, said microemboli has at least one mesenchymal stem cell-associated antigen selected from the group consisting of CD9, CD63, CD81, CD109, CD151, CD248 and CD276.

The CD9, CD63, CD81 and CD151 are one of the transmembrane 4 superfamilies known as the tetraspanin family. A member of this family is a cell-surface protein with a hydrophobic domain. The cell-surface proteins carry signals associated with cell development, activation, growth and migration. The CD109 is a GPI-linked cell surface antigen expressed by CD34 + acute myeloid leukemia cell line, T-cell line, activated T lymphocyte, endothelial cell, and activated platelet. CD248 is one of the XIV family of C-type lectin transmembrane receptors which plays an important role in cell-cell adhesion process and host defense. The XIV group consists of two different members, CD93 and thrombomodulin. CD248 is involved in angiogenesis in embryonic, uterine, and tumorigenesis and growth.

The composition comprising the stem cell-derived microvesicle of the present invention as an active ingredient is a pharmaceutical composition for preventing or treating neurological diseases.

Preferably, said neurological disease is a neurological disease selected from the group consisting of neural injury, neurodegenerative disease, and ischemic or reperfusion disease, more preferably said nerve injury is a central nervous system injury, The neurodegenerative disease is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis, and the ischemic or reperfusion-induced disease is ischemic stroke.

According to a preferred embodiment of the present invention, when injecting mesenchymal stem cell-derived micro-endoplasmic reticulum into an animal model that induced stroke, the function of the brain is restored and the infarct area is reduced.

The composition of the present invention comprises (a) a pharmaceutically effective amount of the above-described stem cell-derived microvesicles; And (b) a pharmaceutically acceptable carrier.

As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve efficacy or activity of the stem cell-derived microemboli as described above.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered parenterally, for example, by subcutaneous injection, intramuscular injection, percutaneous injection, intramuscular injection, intrathecal injection, intravenous injection, intracerebral injection, It is administered by injection.

Preferably, the pharmaceutical composition of the present invention can be injected in single or repeated administration, more preferably repeated administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of this invention are 0.001-1000 mg per day on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating neurological diseases comprising stem cell-derived micro-vesicle as an active ingredient.

(b) The composition of the present invention provides a cell therapy agent free from ethical problems and immunogenicity problems.

(c) The composition of the present invention provides a cost-effective effect by using the conventional method of directly using stem cells and mass production and manufacturing.

FIG. 1 shows the results of purification and characterization of human myeloid mesenchymal stem cell-derived micro-endoplasmic reticulum. 1A shows a fusiform shape of mesenchymal stem cells (x100). Fig. 1B shows a typical mesenchymal stem cell morphology by immunohistochemistry of human bone marrow-derived mesenchymal stem cells. Histograms of mesenchymal stem cells stained with anti-CD29, anti-CD44, anti-CD73, anti-CD90, anti-CD105, anti-CD31, anti-CD34, anti-CD45 and CD106 overlap with the isotype control. Figure 1c shows multilineage differentiation of mesenchymal stem cells. Adipocyte differentiation was analyzed by staining lipid vacuoles with Oil Red O (top, x40 magnification); Chondrocytic differentiation was confirmed by positive staining of proteoglycan using saffranin O (medium, × 200 magnification); Bone cell differentiation was confirmed by mineralization of the substrate by von Kossa staining (below, × 40 magnification). Figure 1d shows an electron microscope image of a tablet micro-vesicle having various sizes ranging from 50 to 200 nm. The sample was not contaminated by cell debris or protein aggregates, and the scale bar exhibits 200 or 500 nm. Figure 1e shows galectin-1, HSP90, beta -actin and CD63 in immunoblots in whole-cell lysates (mesenchymal stem cells) and microemboli of purified mesenchymal stem cells.
Fig. 2 shows the protein identified from the mesenchymal stem cell-derived micro-endoplasmic reticulum. Figure 2a shows the total number of spectra, authenticated peptides and identified proteins to mesenchymal stem cell-derived microvesicles. Figure 2b identifies a total of 730 proteins with three independent LC-MS / MS analyzes. FIG. 2C shows that the mesenchymal stem cell-derived microfibrillar proteome contains a surface marker protein of mesenchymal stem cells and is involved in self-renewal and differentiation of mesenchymal stem cells.
Figure 3 shows the functional analysis of the mesenchymal stem cell-derived microembologue proteome. The 730 mesenchymal stem cell-derived micro-vesicle proteins identified using DAVID software were analyzed by GOBPs (Gene Ontology Biological Processes, FIG. 3a), GOMFs (Gene Ontology Molecular Functions (FIG. 3b), and GOCCs (Gene Ontology Cellular Components, FIG. 3c) (p < 0.05).
FIG. 4 shows the results of integrating mesenchymal stem cell-derived micro-expanded proteome and mesenchymal stem cell-conditioned medium proteome and differentiated cells, dedifferentiated cells and human mesenchymal stem cell mRNA data. FIG. 4A shows the differentiation of differentiated and differentiated cells of human mesenchymal stem cells. The arrows show changes in the degree of expression of self-regenerating (green) and differentiating (red) related genes. FIG. 4B is a graph showing the relationship between mesenchymal stem cell-derived microembodid protein and self-renewal and differentiation-related genes. FIG. 4c compares mesenchymal stem cell-derived microembodermes, mesenchymal stem cell-conditioned medium proteomes, self-renewal and differentiation-related genes.
Figure 5 shows a network model of candidate molecules and their associated pathways. The nodes exhibit microembolite proteins (orange) and their interactions (gray) depending on the color. The color of the edge represents protein-protein interaction (gray), activation (arrow), inhibition (inhibitory label), and direct (solid) and indirect (dotted) interactions. Activation and suppression information was obtained from the &quot; control of actin cytoskeleton &quot; and &quot; focus attachment sites &quot; of the KEGG pathway database. The nodes were arranged based on these two KEGG pathways and were placed in close proximity to the same GOBPs. The blue boundary represents the selected candidate molecule.
FIG. 6 shows that mesenchymal stem cells and mesenchymal stem cell-derived micro-endothelial cells promote recovery after ischemic infarction by permanent mid cerebral artery occlusion (pMCAo). Behavioral testing of the elevated body swing test, the Beam balance test and the modified Neurological Severity Score (mNSS) was performed before and after the pMCAo of the assessment phase during the conditional phase. FIG. 6A shows the results of the increased body swing test, FIG. 6B shows the results of the mean band test, and FIG. 6C shows the results of the modified nerve behavior index. After 48 hours of focal anemia, 5 × 10 ⁵ cells (mesenchymal stem cells and mesenchymal stem cell-derived microfilaments, n = 8) or PBS (Phosphate Buffered Saline, n = 5) were injected into rats as a control group. Significant differences were found in each test at 7 days post-transplantation or at 14 days post-pMCAo. Data are presented as median &lt; RTI ID = 0.0 &gt;
7 shows the result of cerebral infarction analysis of the focal anemic brain. (Ratio of ipsilateral ipsilateral to contralateral) in the mesenchymal stem cell-treated and mesenchymal stem cell-derived microfibrillar-treated groups was significantly reduced (* p &lt; 0.05, ** p &lt; 0.01 ).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

Reagents and Chemicals

Dulbecco's modified Eagle's medium-low glucose and sucrose, gelatin, von Kossa stain, oil red O, sapranin O and HEPES were purchased from Sigma Chemical Company. Insulin-Transferrin-Selenium (ITS) was purchased from Lonza. FBS (Fetal Bovine Serum), penicillin / streptomycin, 2-mercaptoethanol (× 1000), trypsin / EDTA (0.05%) and trypan blue (0.4%) were purchased from Invitrogen. TGF-β3 was purchased from Pepro Tech. Bradford dye was purchased from BioRad, and bFGF (basic Fibroblast Growth Factor) was purchased from CHA Biomed. OptiPrep was purchased from Axis-Shells.

Isolation and Characterization of Mesenchymal Stem Cells from Human Bone Marrow Samples

The experiment was conducted with approval of the Research Ethics Review Committee of Yonsei University Severance Hospital of Korea. Bone marrow cells were collected from the iliac crest of normal subjects. A non-filtered bone marrow harvest bag was used with the consent of the donor. Mononuclear cells (MNCs) from bone marrow samples were prepared by known methods. Briefly, mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation (GE, Sweden) and stained with low glucose DMEM, 10% FBS and penicillin-streptomycin, 2-mercapto Ethanol and FGF (4 ng / ml), and cultured in a gelatin-coated 75 cm 2 flask at 1 × 10 ⁶ cells / cm 2. After 3 days, the cultures were washed with PBS to remove unattached cells and a single layer of remaining adherent cells was cultured in 70-85% confluence in fresh medium. Cells were trypsinized, collected, and subcultured at 5000 cells / cm2. The medium was changed every 3 days, and the cell cultures were passaged with confluence of 70-85% and the 3-4th passage was used for the experiment.

Immunophenotypes of mesenchymal stem cells were examined in three passages. For the investigation, the following cell surface antigens were stained with anti-human antibodies or appropriate mouse isotype antibodies (all BD-Pharmingen San Jose, USA): CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD106. Cells were collected and analyzed with a flow cytometer (Beckman Coulter, USA) and analyzed using WinMDI.

3 &lt; / RTI &gt; cells were treated with a differentiation inducing medium to determine the ability of mesenchymal stem cells to differentiate into adipocytes, osteocytes and chondrocytes, according to a known method. Plate ³⁸ to 5 × 10 12-well to the mesenchymal stem cells to cartilage cells, and bone cell differentiation ⁴ cells / well seeded (seeding), and for adipocyte differentiation, were seeded 1 × 10 ⁵ cells / well. Chondrocytes were cultured by treatment with TGF-β3 (10 ng / ㎖) in monolayer of mesenchymal stem cells. The cultures were incubated for 2 weeks at 37 ° C in 95% air / 5% CO ₂ at 3-4 days intervals. Chondrocytosis was confirmed by saponin-O staining. Osteocytosis was confirmed by the accumulation of mineralized calcium phosphate through the main staining. The lipid saturation was confirmed by the oil red O staining of intracellular accumulation of lipid - rich vacuole. Cells cultured in basal culture medium were used as a control.

Isolation of microemboli

Microvesicles (MVs) were separated from conditioned media using centrifugation by modifying known methods. Free low glucose DMEM containing ²³ bFGF (4 ng / ml), 1 x ITS and 2-mercaptoethanol, followed by 10 minutes at 500 g for 24 hours, 15 minutes at 800 g for 24 hours The culture medium was collected by centrifugation twice. The supernatant was concentrated using a 100 kda membrane Minimate TFF capsule system (PALL Corporation, USA). For the microinfarnate of high quality, the concentrated supernatant was placed on 20 mM HEPES / 150 mM NaCl buffer (pH 7.2) containing 0.8 and 2.7 M sucrose cushion and subjected to ultra-high speed centrifugation at 100,000 g for 2 hours. After centrifugation, the interstices of 0.8 and 2.7 M sucrose cushions were collected by dilution in PBS. For further purification of the microvesicles, Opti-prep density gradient ultracentrifugation was performed. Briefly, sucrose-cushion-rich microvesicles were mixed with Opti prep (final concentration 30%) and pipetted to the bottom of a 12 ml tube. Next, 20 mM HEPES / 150 mM NaCl buffer (pH 7.2) containing 3 mL of 20% OptiPrep and 2.5 mL of 5% OptiPrep was prepared and the layered tube was ultracentrifuged for 3 hours at 200,000 g. 10 fractions of the same volume were collected from the bottom of the tube, diluted with 9 ml PBS and ultracentrifuged for 1 hour at 100000 g. Finally, the purified microvesicles were suspended in distilled water and the protein concentration of each fraction was determined by refractometer measurements and Bradford assay. All fractions were stored at -80 ° C until the experiment.

Transmission electron microscope

Tablet microvesicles were applied to a glow-out carbon-coated copper grid (EMS, USA). After the microvesicles were absorbed for 8 hours, PTA (Phosphor-Tungsten Acid) staining was performed for 10 minutes. The grid was washed with distilled water and dried. Transmission electron microscopy results were recorded at an acceleration voltage of 80 kV using JEM 1011 (Jeol, Japan).

Western Blot

Whole cell lysates (10-20 ㎍) and microvesicles (0.5-2 ㎍) for each sample were loaded onto SDS polyacrylamide gels. Proteins were separated by electrophoresis and transferred to PVDF membrane at 250 mA for 90 minutes to block 5% defatted milk powder. The blocked membranes were reacted with anti-CD81 (BD-Pharmingen), CD63 and anti-actin (Santa Cruz, USA) and washed three times with TBST buffer for 10 minutes. The membrane was then reacted with HRT-conjugated anti-goto or anti-mouse IgG. Washed three times for 10 minutes with TBST buffer, and treated with a chemiluminescent substrate (Intron, Korea) and sensitized to an X-ray film.

SDS - PAGE  And phosphorus decomposition

Each isolated mesenchymal stem cell-derived microfilament was separated by SDS-PAGE with 4-12% gradient Novex bis-tris gel (Invitrogen) and the resulting protein bands were stained with gel code blue staining reagent (Pierce, USA) Respectively. The dyed gel was sliced into 10 bands of the same size and in-gel trypsin digestion was performed in a known manner. ³⁹

Nano- LC - ESI - MS / MS  analysis

(Linear Trap Quadrupole) mass spectrometer (Thermo Finnigan, USA) in which peptides prepared by in-gel digestion were combined with Excipient-Nano-Ultra Performance Liquid Chromatography (Eksigent Technologies, USA) Respectively. Briefly, trypsinized peptides were applied to a self-made analytical column (75 μm × 11 cm) packed with C18 regular 5 μm size resin.

A gradient of 45 minutes was performed on a 97% solution A (0.1% formic acid in distilled water) and a flow rate of 0.3 / / 60% solution B (0.1% formic acid in acetonitrile). The separated peptide ions were electrosprayed with a nano-ESI (Electro Spray Ionization) source. The electrospray voltage used in the MS / MS analysis is 1.9 kV and the standardized impact energy is 35%. All MS / MS spectra were segmented by total MS scans and the largest five spectra were obtained as result-dependent scans. The repetition factor for the dynamic exclusion was set to 1, the repetition period to 30 seconds, the dynamic exclusion period to 180 seconds, the exclusion mass width to 1.5 Da, and the dynamic exclusion list to 50.

Peptides  And protein identification

The S / MS spectra results were retrieved using the turbo-SEQUEST algorithm (Thermo Finnigan, USA) in the ipi.HUMAN.v3.76 database (89 378 entries). The following SEQUEST search parameters were used: allow for precursor and fragment ion masses of ± 2 Da and ± 1 Da, allow miscleavage, oxidize and fix the Met (+16 Da) by variable modulation Carbamidomethylation of Cys (+57 Da) by. After database searching, identified peptides and proteins were identified using Scarfold 2 (Proteome Software, USA). Of the peptides from the SEQUEST search, a set of peptides with a probability of peptide peptides of 0.95 or greater was selected. ⁴⁰ Also, a list of proteins having a ProteinProphet probability of 0.99 or more and having two or more unique peptides was obtained.

Gene Ontology Gene Ontology ; GO ) analysis

We used DAVID (Database for Annotation, Visualization and Integrated Discovery) to identify GO biological processes, molecular functions and cellular organs rich in proteins or gene sets. ⁴¹

Production of Stroke Animal Model Human bone marrow Liver  Stem cells or Mesenchymal stem cell - Analysis of therapeutic effect of derived micro-spermatids

The experimental animals were male mice of Sprague-Dawaur strain, weighing about 200 g purchased from Orient Bio, and water and pellet feed were allowed to freely eat. The temperature of the feeding room was 20-24 ℃, the humidity was 40-60% And day and night cycles were maintained for 12 hours each. All experimental animals were used in the laboratory after 2 weeks of adaptation and 250-300 g of rats used in the experiment.

The anesthesia of the experimental animals was maintained with proper isoflurane during the operation using 3% isoflurane (Hana Pharmaceutical Co., Korea), and the left middle cerebral artery occlusion was induced. To this end, the incision was made in the middle of the neck to expose the right common carotid artery, then to expose the internal carotid artery and external carotid artery, then to the right external carotid artery (nylon, 4-0) The pMCAo method was used to permanently block the blood flow by inserting it through the internal carotid artery into the origin of the middle cerebral artery (Circle of Willis). After that, the skin of the forehead was sutured and disinfected, and the animal that was awake from the anesthesia was allowed to move freely.

Forty-eight hours after stroke induction, rats were randomly assigned to an experimental group (n = 8) and a control group (n = 5) to be implanted with mesenchymal stem cells or mesenchymal stem cell-derived microemboli. The mice were anesthetized with Zoletil (Virbac SA, France, dose: 25 ㎎ / ㎏) and fixed in the brachial plexus. Five hundred thousand mesenchymal stem cells (5 × 10 ⁵ cells / 5 μl PBS) or 50 μg / PBS mesenchymal stem cell-derived microvesicles were transplanted at a rate of 1 μl / min into the stroke-induced striatum (AP + 0.7, ML + 2.0, DV -5.5). The control group was also injected with PBS in the opposite direction of the lesion in the same manner. After cell transplantation, 10 mg / kg of cyclosporine was intraperitoneally administered to all rats daily.

Behavioral experiment

Behavioral capacity tests were performed at 3, 4, 7, 14, 21, and 28 days after the procedure.

(One) Increased Body swing  exam( Elevated body swing test )

The increased body swing test was performed using a known method (Borlongan, CV; Cahill, DW; Sanberg, PR Locomotor and passive avoidance deficits following the occlusion of the middle cerebral artery. Physiol . Behav . 58: 909-917; The rats' tail was moved up to a height of 10 cm from the floor and the plaque motion was examined. The frequency of left or right swing was recorded for 1 minute. The value was calculated as 0 point for the left or right swing of the torso twist, 1 point for the asymmetric twist of 30 degrees or less, and 2 points for the asymmetric twist of 30 degrees or more. The difference in torso twist is an ipsilateral twist in the same direction as the infarction site, a counter-directional twist in the opposite direction.

(2) Beam balance test Beam balance test )

(Goldstein, LB; Davis, JN). In order to evaluate the balance ability and vestibular-motor function of animal models, Methods. 31: 101-107; 1990). The mice were placed on the average scale (100 cm x 5 cm x 2 cm) and evaluated with the following six scales. A balance with a stable posture is 1 point, 2 points of gripping or wobbling of the side of the average team, 3 points when one or more feet fall from the average team, 4 points when falling, 4 points when falling, 5 points, and 6 points if they did not try to balance and fall from the average.

(3) Modified neurobehavioral index ( Modified neurological severity score ; mNSS )

The degree of nerve impairment combined well-known motor neurons, sensory nerve and reflexes (Longa, EZ; Weinstein, PR; Carlson, S.; Cummins, R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84 -91; 1989). The modified neuropsychological behavior index was calculated from the motor neurons, sensory nerves and reflex nerves based on the torso twisting test, the mean band test, the prehensile traction test, the open field test (rotation frequency) and the foot fault test The results of the tests were combined and evaluated. 2 points (3 ≤ defects in the front paw <10), 4 points if the opposite paw can not be straightened (≥10 defects in the front paw), 6 points if the opposite side is light (1 ≤ rotational or asymmetric twist <5), 8 for severely tilted (rotational or asymmetric twist ≥ 5), and 10 for tilted to the opposite side (freehand traction ≤ 2).

Hematoxylin staining and infarction measurement

Brain sections were stained with hematoxylin and observed with a microscope (Zeiss, Germany). Relative infarct area was analyzed with image J software. The indirect lesion area was calculated by separating the intact part of the ipsilateral hemisphere from the contralateral hemisphere area. The relative infarct area represents indirect lesion as a percentage compared to the opposite hemisphere (Swanson, RA; Morton, MT; Tsao-Wu, G. Savalos, RA; Davidson, C .; Sharp, infarct volume, J. Cereb. Blood Flow Metab., 10: 290-293, 1990).

Results and discussion

Mesenchymal stem cell - Isolation and analysis of derived micro-vesicles

Flow cytometry was performed to identify mesenchymal stem cells prior to the isolation of microemboli from human bone marrow-derived mesenchymal stem cells. The cells exhibited immunostaining for negative expression of CD31, CD34, CD45 and CD106 in positive mesenchymal stem cell types and CD29, CD44, CD73, CD90 and CD105 in a uniform fusiform population (FIGS. 1A and 1A). The ability of mesenchymal stem cells to differentiate into various lineages as adipocytes, chondrocytes, and osteocytes was confirmed by known methods to confirm stem cell characteristics. ^In order to isolate the microemboli from mesenchymal stem cells, the cells were cultured in serum-free DMEM containing bFGF and ITS for 24 hours. After removal of cells and cell debris, conditioned medium was concentrated 50-fold by serial centrifugation using a 100-kDa filter and sucrose gradient. ²³ transmission electron microscope. As a result, the purified microfilament was in the form of a round follicle having a diameter of 50-200 nm (FIG. 1d). A maximum of 10 mu g of vesicle protein was obtained from the medium of about 80 million sub-confluent cells cultured for 24 hours. The microvesicles consist of a collection of proteins commonly found in microembro- mytes such as CD63, beta-actin and HSP90 (Fig. 1e). Protein complexes such as insoluble immune complexes are similar in biophysical properties (size, light scattering and deposition) of micro-endoplasmic reticulum affecting the determination of micro-vesicles by flow cytometry and purification by ultrafast centrifugation. To reduce the possibility of contamination of the ⁴² insoluble protein complex, serum-free medium was used to separate the microvesicles from mesenchymal stem cells.

Mesenchymal stem cell - Protein Proteomic Analysis of Derived Microemboli

In order to investigate the protein composition of the hepatic stem cell-derived microemboli, three types of microembodiments were independently separated and analyzed by protein proteome analysis, 1D SDS-PAGE, phosphorylase degradation by trypsin and LC-MS / MS analysis Respectively. ²³ &lt; / RTI &gt; three independent samples of mesenchymatous stem cell-derived microembeletic cells were analyzed by LC-MS / MS. Three LC-MS / MS analyzes showed 98 067, 110 051 and 93 134 MS / MS spectra for the samples, respectively. Three MS / MS spectra were searched for the IPI database (v.3.76, 89 378 entries) using the SEQUEST algorithm. For reliable protein identification, peptides with a peptide propetability of at least 0.95 and proteins with a protein propetability of at least 0.99 were identified. Applying this criterion, a total of 730 micro-vesicle proteins were identified from at least twice-specific peptides in three independent experiments (Figs. 2a and 2b). Of the 730 microembossed proteins, 424 proteins were identified by at least two LC-MS / MSs and 236 proteins were identified by three analyzes.

Mesenchymal stem cell - Characteristics of derived micro-endoplasmic reticulum

The inventors previously reported the construction of microembodial protein from human colon cancer and human HT29 colon cancer cells. ^In order to investigate whether the mesenchymal stem cell-derived micro-endoplasmic reticulum is characteristic of micro-endoplasmic reticulum, the reported micro-endoplasmic proteome and mesenchymal stem cell-derived micro-endoplasmic reticulum were compared. Of the 730 mesenchymal stem cell-derived micro-vesicle proteins, 420 proteins previously reported in micro-vesicular proteomes were identified. Microembodial proteomes include proteins that are associated with the essential properties of the microembroine, such as micro-embryonic development and transport. ^44,45 Consistent with this discovery, the 420 protein has been shown to inhibit the docking and fusion of microemboli, as well as the regulation of 15 RAB proteins (eg, RAB1A, RAB2A, RAB5A / B / C, RAB7A and RAB8A). These RAB proteins are involved in vesicular transport, granule release, fusion membrane formation and ligand removal in the plasma membrane.

Mesenchymal stem cell -Derived microembolic proteome Microembolic  Characteristic

Previous studies have shown that the microembodiment proteome reflects the characteristics of its derived cells. ^{19,22-24,31,35,48} Therefore, we investigated whether mesenchymal stem cell-derived microfibrillar proteome contains mesenchymal stem cell related proteins. Based on the surface markers of mesenchymal stem cells sorted by Kolf et al., 730 microembossal proteins were tested for the presence of 5 positive markers (CD13, CD29, CD44, CD73 and CD105) and 2 variable markers CD90) and no speech markers were examined. In addition, the mesenchymal stem cell-derived microsomal proteome was compared with the proteomes of UCB-hMSCs and human mesenchymal stem cells to confirm that 122 proteins were overlapped. These results support the effectiveness of the micro-vesicle separation and proteomic analysis of the present invention. Microembodial proteomes include molecules in the signaling pathway associated with self-renewal and differentiation of mesenchymal stem cells (Fig. 2C). ^49. First, the micro-vesicles proteome is KEGG database path and GOBP signaling pathway of mesenchymal stem cells based on the self-13 protein comprises a related and two signaling pathways involved in reproduction, growth factor receptors and Wnt signaling pathway. Second, it contains 38 proteins involved in the differentiation of mesenchymal stem cells, the five signaling pathways involved in TGFβ, MAPK, PPAR and BMP signaling pathways. The Wnt signaling pathway is reported to be involved in the self-renewal and differentiation of mesenchymal stem cells. These results suggest that the mesenchymal stem cell-derived microfilament is characterized by mesenchymal stem cells and that these microfilaments may affect self-regeneration and differentiation through proteins involved in the signal transduction pathway.

Mesenchymal stem cell - Potential function of the derived microemboli

The results of comparative analysis of mesenchymal stem cell-derived microsomal proteome and other proteomes indicate that the mesenchymal stem cell-derived microsomal proteome has the characteristics of micro-endoplasmic reticulum and mesenchymal stem cells. However, the potential function of mesenchymal stem cell-derived microemboli was not disclosed.

To systematically investigate the function of the mesenchymal stem cell-derived microembellifer, the functional analysis of the 730 microembroite protein was performed using DAVID software. ⁴¹ GOBPs (Gene Ontology Biological Processes, Fig. 3a) showed a significant increase in cell migration and cell morphogenesis (10.7%), cell attachment (8.4%), cell cycle and proliferation (7.9% and 4.7% (4.1%) and developmental processes (e. G., Ectodermal and epidermal development). In addition, GOMFs (Gene Ontology Molecular Functions, Fig. 3B) showed that the microfibrillar proteome had a nucleotide binding (25.5%), calcium ion binding (11.9%), cytoskeletal protein binding (11.0%) and GTPase activity (9.1% Show. Finally, the major GOCCs (Gene Ontology Cellular Components, Fig. 3c) of the microemboli were found in 38.7% of the plasma membrane, 23.1% of the cytoskeleton, 15.6% of the follicles, 27.1% of the cytosol, ). The broad spectrum and molecular functions of cellular processes can predict the therapeutic effect of mesenchymal stem cell-derived microemboli.

Mesenchymal  Of self-renewal and differentiation mRNA Signature  Integration and Mesenchymal stem cell - conditional badge

In order to examine the therapeutic effect of the mesenchymal stem cell-derived micro-endoplasmic reticulum confirmed by the cell process of the mesenchymal stem cell-derived micro-endoplasmic proteome, it is known to be related to self-regeneration and differentiation closely related to the therapeutic effect of mesenchymal stem cells Genes were integrated to investigate the potential relationship between the self-renewal and differentiation of mesenchymal stem cells and mesenchymal stem cell-derived microsomal proteome. ^50,51 Song et al. Obtained the gene expression profile of human mesenchymal stem cells, 3 differentiated cells (adipocytes, chondrocytes and osteoblasts) and cells demolded from differentiated cells in human mesenchymal stem cells. During the differentiation of mesenchymal stem cells, the genes associated with self-renewal, in which the expression level decreased and the expression was increased during the differentiation, were identified, and genes related to differentiation of mesenchymal stem cells were identified (FIG. 4A). Of the 730 mesenchymal stem cell-microembodial proteins, the microembodial proteins overlapped 53 (7.3%) and 25 (3.4%) with the proteins involved in self-renewal and differentiation of mesenchymal stem cells, respectively (FIG. The redundancy of the potential relationship of these mesenchymal stem cell-derived micro-endoplasmic reticulum could be interpreted more meaningfully at the cellular level than at the molecular level.

To investigate redundancy at the cellular process level, functional enrichment analysis of genes involved in self-renewal and differentiation was performed and compared to that of mesenchymal stem cell-derived microembologic proteome (FIG. 4c). The mesenchymal stem cell-derived microsomal proteome and self-regeneration-related genes are involved in cell proliferation, cell attachment and migration, actin cytoskeletal tissues, developmental processes (epidermis, ectoderm and angiogenesis), Ras signaling pathway and response to hormone stimulation . On the other hand, the mesenchymal stem cell-derived microembologic proteome and differentiation-related genes share the response to oxidative stress and the cofactor and sulfur metabolism. Cell adhesion and migration share genes associated with self-renewal and differentiation. These results indicate that the mesenchymal stem cell-derived microfibrillar proteome affects proteins involved in self-renewal and differentiation of mesenchymal stem cells.

The mesenchymal stem cell-derived micro-spermatocyte has an effect of reducing the infarct size of myocardial ischemia similar to mesenchymal stem cell-derived conditioned medium. ²⁰ Thus, the mesenchymal stem cells were based on the potential role of ER in the treatment of fine aspects proteome-derived micro vesicles and human mesenchymal stem cells - the medium term proteome compared to the mesenchymal stem cell-derived micro vesicles. ^{Of the} 730 mesenchymal stem cell-derived micro-vesicle proteins, 58 protein (8.0%, 55 specific gene product) overlapped mesenchymal stem cell-conditioned medium proteome. For analysis of genes involved in self-renewal and differentiation, functional enrichment analysis of mesenchymal stem cell-conditioned medium protein was performed. The proteins and genes were divided into the following four groups to compare the cell processes (Fig. 4c): (1) mesenchymal stem cell-derived microfibrillar proteome; (2) self-renewal and (3) differentiation-related genes; And (4) mesenchymal stem cell-conditioned medium proteome. The overlapping patterns of cellular processes for the four groups of proteins and genes were classified into the following five groups: (Group 1) Four groups of proteins and gene duplication related to cell adhesion, cell migration and hormone stimulation; (Group 2) Actin cytoskeleton, Ras signaling and 3 developmental processes (blood vessels, epidermis and ectodermal development) and self-regeneration-associated mesenchymal stem cell-derived micro-endoplasmic reticulum and conditioned medium Proteome overlap; (Group 3) overlapping of the corresponding process, cell redox homeostasis, Rho signaling, cell morphology, response to calcium ions, and mesenchymal stem cell-derived microemboli associated with collagen fiber tissue and conditioned medium proteome; Mesenchymal stem cell-derived microsomal proteomes and self-regeneration-related geneticist middle cerebral cells involved in cell proliferation, epithelial development, protein folding and nucleic acid biosynthesis; (5 groups) overlapping of mesenchymal stem cell-derived microembologic proteome and differentiation-related genes involved in peptide / receptor / sulfur metabolism and response to oxidative stress.

The results show that some of the mesenchymal stem cell-derived micro-vesicle proteins through self-regeneration-related processes (group 1 and 2) and other differentiation-related processes (group 1 and group 3) (Group 1, group 2 and group 3) that are shared by mesenchymal stem cell-derived microembodermes and conditional medium proteases, which may be associated with adverse effects.

Mesenchymal stem cell -Derived &lt; / RTI &gt; Mesenchymal  Promoting recovery of ischemic infarction

In order to investigate whether the mesenchymal stem cell-derived microfibrillar body can replace the effect of mesenchymal stem cells on ischemic stroke, the microfibrillar body purified from cultured mesenchymal stem cells is administered to the infarct area of a rat model having permanent brain ischemia Respectively.

Systemic infusion of microemboli or mesenchymal stem cells was performed by injecting directly into the lesion site by insemination of deep brain, since the therapeutic effect would be limited due to premature return to damaged tissue. After 48 hours of pMCAo, mesenchymal stem cells ⁽⁵ × 10 5 cells), or mesenchymal stem cells directly implanted into the stereotactic anthropogenic core insert-derived micro vesicles (50 ㎍) in naenoe to minimize the cells diluted to the half edge striatum of rats Respectively. As a control, PBS was injected into the same position of the rats.

Nerve function test

Before pMCAo, it was confirmed that all the experimental animals did not have neural defects (Fig. 6). However, rats with pMCAo showed severe neurological deficits at 24 hours without significant differences between the groups with cerebral anemia. In the increased body swing test and the mean band test, functional defects of anemic-induced rats transplanted with mesenchymal stem cells were significantly improved compared with the performance of PBS control group at 4 days (total observation period 28 days) 6a and 6b). Rats transplanted with mesenchymal stem cell-derived micro-vesicles showed significant improvement in nerve impairment from the 4th day after pMCAo compared to the PBS-injected group. On the fourth day, there was no significant difference between mesenchymal stem cells and mesenchymal stem cell - microemboli group. However, unlike the mesenchymal stem cell-grafted group, the 14th to 28th day showed less significant functional recovery in mesenchymal stem cell-microemulsion group than in the control group. Similar to the functional test, the modified neuronal activity index (mNSS) test, the increased body swing test, the mean score test and the freehand traction test based on the behavioral process showed significant functional recovery in the mesenchymal stem cell transplanted rat compared with the control (Fig. 6C). In contrast, single injection of mesenchymal stem cell-derived microemboli resulted in short-term (max. 7 days) behavioral recovery (Fig. 6c).

Histological analysis

Tissue defects in each experimental group were defined as infarct regions confirmed by hematoxylin staining of rat brain 28 days after pMCAo (Fig. 7). Infarct lesions were mainly in the cortex and striatum. Relative infarction sites in the mesenchymal stem cell-grafted and mesenchymal stem cell-derived micro-embryo-injected group were decreased compared to the control (28.0 +/- 3.9). Although the difference between the treatment groups of mesenchymal stem cells and mesenchymal stem cell-derived microsomes was not significant, the mesenchymal stem cell-transplantation rats (20.5 ± 4.1) were significantly higher than those of the mesenchymal stem cell- ), Respectively. Thus, improvement in behavioral deficits is accompanied by a reduction in infarct area.

It has been demonstrated that transplantation of mesenchymal stem cells or mesenchymal stem cell-derived micro-ERs in the rat brain in the pMCAo state reduces the infarct area and improves nerve function. During 4 weeks of observation, transplantation of mesenchymal stem cells and single injection of mesenchymal stem cell-derived micro-ERs significantly improved neuronal defects in focal ischemic stroke rats. However, cell tracking analysis shows that transplanted mesenchymal stem cells do not maintain long-term viability in ischemic brain. In particular, the behavioral recovery in the early stage of cell transplantation is that the transplanted cells are induced before the connection of the host's brain to the nerve tissue, so that the transplanted cells themselves do not restore the neural network of the host by replacing the damaged neuron . That is, in a stroke rat, a cell or a micro-vesicle injected from an ischemic brain through mesenchymal stem cell transplantation and mesenchymal stem cell-derived micro-embryo injection provides a growth factor that promotes regeneration of the host's own neural network and improves the microenvironment The results of this study were as follows. Injection of mesenchymal stem cell-derived microvesicles showed functional recovery similar to that at the beginning of mesenchymal stem cell transplantation. Thus, the early functional recovery may not be the result of the introduction and replacement of cells into brain tissue by the transplanted cells (Li, Y., Chen, J .; Wang, L .; Lu, M .; Chopp, M. treatment of stroke in rats with intracarotid administration of marrow stromal cells. Neurology 56: 1666-1672; 2001). This appears to be a strong paracrine effect / nutrient functional recovery in the early stage of transplanted mesenchymal stem cells, rather than replacing the cells in which the damaged cells are restored (Kurozumi, K., Nakamura, K .; Tamiya, T .; Kawano, Y .; Ishii, K .; Kobune, M .; Hirai, S.; Uchida, H.; Sasaki, K .; Ito, Y .; Kato K, ; Hamada H. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat cerebellar artery occlusion model, Mol. Ther. 11: 96-104, 2005; Chen, JR; Cheng, GY; Sheu, CC; Tseng, GF ; Wang, TJ; Huang, YS Transplanted bone marrow stromal cells migrate, differentiate and improve motor function in rats with experimentally induced cerebral stroke J. Anat. 213: 249-258; This suggests that mesenchymal stem cells produce mediators that regulate the microenvironment of the brain, such as inflammation, enhancement of angiogenesis and / or induction of intrinsic neurogenesis (Chen, CP; Lee, YJ; Chiu, ST; Shyu , WC; Lee, MY; Huang, SP; Li, H. The application of stem cells in the treatment of ischemic diseases Hystopathol 21: 1209-1216; Most factors produced by injected mesenchymal stem cells have been reported to induce innate neurogenesis and neuroprotection (Niimura, M .; Takagi, N .; Takagi, K .; Mizutani, R. ; Ishihara, N .; Matsumoto, K .; Funakoshi, H. Nakamura, T .; Takeo, S. Prevention of apoptosis-inducing factor translocation is a possible mechanism for protective effects of hepatocyte growth factor against neuronal cell death in the hippocampus after transient forebrain ischemia, J. Cereb. Blood Flow Metab., 26: 1354-1365; Factors produced from mesenchymal stem cells in ischemic stroke will enhance neuroprotection at the beginning of such transplantation or infusion followed by action from transplanted cells or microemboli, tissue-derived (intrinsic) neurotrophic factors ( Gillardon, F .; Spranger, M. Brain-derived neurotrophic factor, inhibits neuronal death and glial activation after global ischemia in the rat (Kiprianova, I .; Freiman, TM; Desiderato, S .; Schwab, J. Neurosci, Res. 56: 21-27; 1999). Therefore, the infarct area is reduced by suppression of neuronal death by such neuroprotective factors and induction of strong intrinsic nerve development.

Overall, the intracerebral injection of mesenchymal stem cell-derived micro-ERs mediates the microenvironment for intrinsic neuronal development similar to the effect of mesenchymal stem cells, thereby restoring brain function and reducing infarction sites. In terms of cell therapy, such mesenchymal stem cell-derived microfibrillar body is more advantageous than mesenchymal stem cell. Because the microemporal cells are non-cellular, there is no risk of side effects associated with direct transplantation of cells, such as tumor formation and immune rejection. In addition, microvesicles are relatively easy to manufacture and operate in a large quantity and are cost-effective. In this respect, microemboli derived from stem cells or progenitor cells may serve as ideal regenerative drugs for stroke, Alzheimer's disease, central nervous system injury, and neurodegenerative diseases such as Parkinson's disease and neurodegenerative diseases.

Mesenchymal stem cell - candidate molecules related to the therapeutic effect of the derived microemboli

The microembodial proteins, including groups 1, 2 and 3, are associated with the therapeutic effect of mesenchymal stem cell-derived microemboli. Among the microembodial proteins, the following candidate molecules were focused on: (1) surface receptors (PDGFRB, EGFR and PLAUR); (2) signaling molecules (RRAS / NRAS, MAPK1, GNA13 / GNG12, CDC42 and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins and LGALS1 / LGALS2); And (4) mesenchymal stem cell-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248 and CD276).

First, surface receptors promoted the therapeutic potential of mesenchymal stem cells. The PDGFR signaling pathway is the dominant pathway for supplementation and differentiation of mesenchymal stem cells, which are important for wound regeneration and tissue regeneration. ^In addition, the treatment of soluble and strained EGFs in mesenchymal stem cells increases the proliferation and differentiation of mesenchymal stem cells, thereby enhancing the therapeutic ability of mesenchymal stem cells. ^In addition, PLAUR mediates the mobilization, migration and differentiation of mesenchymal stem cells, thereby improving the therapeutic ability of mesenchymal stem cells. ⁵⁴

Second, signaling molecules can be classified into three signaling pathways: RAS-MAPK (RRAS / NRAS, MAPK1 and VAV2), RHO (GNA13) and CDC42 (GNG12 and CDC42) pathways. Based on the KEGG pathway database (hsa04810: actin cytoskeleton regulation), this pathway is located downstream of surface receptors that promote mobilization, proliferation and differentiation of mesenchymal stem cells, suggesting therapeutic effects of mesenchymal stem cells.

Third, cell adhesion molecules have positive effects on mesenchymal stem cell-based tissue treatment and regeneration-related characteristics. In the absence of growth factors, fibronectin (FN1) regulates the migration of mesenchymal stem cells through the integrin-mediated activity of PDGF-dependent actin reorganization. ⁵⁵ integrin and its associated molecules (CD47, ITGA1 / 2/3/5/7/10/11 / V and ITGB1 / 3/5) are associated with this fibronectin-mediated MSC transfer (KEGG pathway: hsa04810). Furthermore, Galectic-1 (LGALS1) regulates a variety of processes such as embryogenesis, cancer metastasis, cell proliferation, differentiation, migration, attachment, wound healing, angiogenesis and immune regulation. ⁵⁶ For example, LGALS1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration. ⁵⁷ Unlike these molecules, EZR and IQGAP1 do not report any functional relationship with the therapeutic effect of mesenchymal stem cells. However, it regulates endothelial cell proliferation and angiogenesis, indicating a potential implication of mesenchymal stem cell-based tissue regeneration. ^58,59

Finally, mesenchymal stem cell-associated antigens are involved in cell attachment, which is closely related to the therapeutic effect of mesenchymal stem cells. For example, CD9 promotes proliferation and promotes the angiogenic action of human adipose-derived mesenchymal stem cells. ⁶⁰ Furthermore, CD90 plays an important role in cell-cell and cell-extracellular matrix interactions in neurogenesis, anti-inflammation, apoptosis, adhesion, migration, tumor suppression and fibroblast proliferation. ^{61 For} other antigens, the direct role of the therapeutic effect of mesenchymal stem cells during tissue regeneration is unclear. However, the relevance of cell attachment suggests that such molecules will serve as new targets to regulate the therapeutic potential of mesenchymal stem cells during tissue therapy and regeneration.

These candidate molecules (surface receptors, cell adhesion molecules, and signal transduction molecules) are tightly linked through related pathways. To investigate the relationship between candidate molecules and their associated pathways, a network model was constructed for candidate molecules based on protein-protein (Figure 5). The network model shows the potential for candidate molecules and their associated pathways. This implies that the growth factor receptor and fibronectin / integrin activate RAS-MAPK, RHO and CDC42 pathway rearrangements of the actin cytoskeleton for cell attachment and migration. Increased cell migration can lead to the mobilization of mesenchymal stem cells during tissue treatment and regeneration. The results indicate that candidate molecules can contribute to tissue regeneration through mesenchymal stem cell migration.

Proteomic profiling of microemboli provides 730 protein of mesenchymal stem cell-derived microembrooplasm. The integration of several proteomes and transcripts with mesenchymal stem cell-derived microsomal proteome indicates: (1) that the mesenchymal stem cell-derived microsomes have the characteristics of mesenchymal cells and the characteristics of mesenchymal stem cells; (2) the cellular processes exhibited by mesenchymal stem cell-derived microemboli are shared with self-regenerating and differentiating mesenchymal stem cell-conditioned medium and genes; (3) The candidate microtubule protein candidate is related to the therapeutic effect of mesenchymal stem cells, and the related pathway promotes the therapeutic effect of mesenchymal stem cells.

references

(1) Friedenstein, A. J .; Chailakhyan, R. K .; Latsinik, N. V .; Panasyuk, A. F .; Keiliss-Borok, I. V. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues: Cloning in vitro and retransplantation in vivo. Transplantation 1974, 17, 331, 340.

(2) Pittenger, M. F .; Mackay, A. M .; Beck, S. C .; Jaiswal, R. K .; Douglas, R .; Mosca, J. D .; Moorman, M. A .; Simonetti, D. W .; Craig, S.; Marshak, D. R. Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284, 143, 147.

(3) Jiang, Y .; Jahagirdar, B. N .; Reinhardt, R. L .; Schwartz, R. E .; Keene, C. D .; Ortiz-Gonzalez, X. R .; Reyes, M .; Lenvik, T .; Lund, T .; Blackstad, M .; Du, J .; Aldrich, S .; Lisberg, A .; Low, W. C .; Largaespada, D. A .; Verfaillie, C. M. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002, 418, 41.49.

(4) Schwartz, R. E .; Reyes, M .; Koodie, L .; Jiang, Y .; Blackstad, M .; Lund, T .; Lenvik, T .; Johnson, S .; Hu, W. S .; Verfaillie, C. M. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest. 2002, 109, 1291.1302.

(5) Aggarwal, S .; Pittenger, M. F. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005, 105, 1815. 1822.

(6) Caplan, A. I .; Dennis, J. E. Mesenchymal stem cells as trophic mediators. J. Cell. Biochem. 2006, 98, 1076.1084.

(7) van den Berk, L. C. J .; Jansen, B. J. H .; Siebers-Vermeulen, K. G. C .; Huijs, T .; Roelofs, H .; Ko Gler, G .; Figdor, C. G .; Torensma, R. The tissue origin and culture history of mesenchymal stem cells affect their performance in osteoblastic differentiation. J. Tissue Eng. Regener. Med. 2011, 8, 96.105.

(8) Liu, Z. J .; Zhuge, Y .; Velazquez, O. C. Trafficking and differentiation of mesenchymal stem cells. J. Cell. Biochem. 2009, 106, 984.991.

(9) Sensebe, L .; Krampera, M .; Schrezenmeier, H .; Bourin, P .; Giordano, R. Mesenchymal stem cells for clinical application. Vox Sang. 2010, 98, 93.107.

(10) Le Blanc, K .; Rasmusson, I .; Sundberg, B .; Go.therstro.m, C .; Hassan, M .; Uzunel, M .; Ringde.n, O. Treatment of severe acute graftversus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004, 363, 1439, 14411.

(11) Stamm, C .; Westphal, B .; Kleine, H. D .; Petzsch, M .; Kittner, C .; Klinge, H .; Schu.michen, C .; Nienaber, C. A .; Freund, M .; Steinhoff, G. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 2003, 361, 45.46.

(12) Sun, L .; Akiyama, K .; Zhang, H .; Yamazawa, T .; Hou, Y .; Zhao, S.; Xu, T .; Le, A .; Shi, S. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells 2009, 27, 1421, 1432.

(13) Oyagi, S .; Hirose, M .; Kojima, M .; Okuyama, M .; Kawase, M .; Nakamura, T .; Ohgushi, H .; Yagi, K. Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats. J. Hepatol. 2006, 44, 742.748.

(14) Chen, L. B .; Jiang, X. B .; Yang, L. Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells. World J. Gastroenterol. 2004, 10, 3016.3020.

(15) Morigi, M .; Imberti, B .; Zoja, C .; Corna, D .; Tomasoni, S.; Abbate, M .; Rottoli, D .; Angioletti, S .; Benigni, A .; Perico, N .; Alison, M .; Remuzzi, G. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am. Soc. Nephrol. 2004, 15, 1794, 1804.

(16) Phinney, D. G .; Isakova, I. Plasticity and therapeutic potential of mesenchymal stem cells in the nervous system. Curr. Pharm. Des. 2005, 11, 12551265.

(17) Kinnaird, T .; Stabile, E .; Burnett, M. S .; Shou, M .; Lee, C. W .; Barr, S.; Fuchs, S .; Epstein, S. E. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 2004, 109, 1543.1549.

(18) Bruno, S .; Grange, C .; Deregibus, M. C .; Calogero, R. A .; Saviozzi, S.; Collino, F .; Morando, L .; Busca, A .; Falda, M .; Bussolati, B.; Tetta, C .; Camussi, G. Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J. Am. Soc. Nephrol. 2009, 20, 10531067.

(19) Herrera, M. B .; Fonsato, V .; Gatti, S .; Deregibus, M. C .; Sordi, A .; Cantarella, D .; Calogero, R .; Bussolati, B .; Tetta, C .; Camussi, G. Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats. J. Cell. Mol. Med. 2010, 14, 1605.1618.

(20) Lai, R. C .; Arslan, F .; Lee, M. M .; Sze, N. S .; Choo, A .; Chen, T .; Salto-Tellez, M .; Timmers, L .; Lee, C. N .; El Oakley, R .; Pasterkamp, G .; de Kleijn, D .; Lim, S. K. Exosome secreted by MSC reduces myocardial ischemia / reperfusion injury. Stem Cell Res. 2010, 4, 214. 222.

(21) Deregibus, M. C .; Tetta, C .; Camussi, G. The dynamic stem cell microenvironment is orchestrated by microvesicle-mediated transfer of genetic information. Histol. Histopathol. 2010, 25, 397.404.

(22) Muralidharan-Chari, V .; Clancy, J .; Plou, C .; Romao, M .; Chavrier, P .; Raposo, G .; D'Souza-Schorey, C. ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles. Curr. Biol. 2009, 19, 1875.1885.

(23) Choi, D. S .; Lee, J. M .; Park, G. W .; Lim, H. W .; Bang, J. Y .; Kim, Y. K .; Kwon, K. H .; Kwon, H. J .; Kim, K. P .; Gho, Y. S. Proteomic analysis of microvesicles derived from human colorectal cancer cells. J. Proteome Res. 2007, 6, 4646.4655.

(24) Hong, B. S .; Cho, J. H .; Kim, H .; Choi, E. J .; Rho, S .; Kim, J .; Kim, J. H .; Choi, D. S .; Kim, Y. K .; Hwang, D .; Gho, Y. S. Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells. BMC Genomics 2009, 10, 556.

(25) Di Vizio, D .; Kim, J .; Hager, M. H .; Morello, M .; Yang, W .; Lafargue, C. J .; True, L. D .; Rubin, M. A .; Adam, R. M .; Beroukhim, R .; Demichelis, F .; Freeman, M. R. Oncosome formation in prostate cancer: Association with a region of frequent chromosomal deletion in metastatic disease. Cancer Res. 2009, 69, 5601.5609.

(26) Boulanger, C. M., Amabile, N .; Tedgui, A. Circulating microparticles: A potential prognostic marker for atherosclerotic vascular disease. Hypertension 2006, 48, 180.186.

(27) Quesenberry, P. J .; Aliotta, J. M. The paradoxical dynamism of marrow stem cells: Considerations of stem cells, niches, and microvesicles. Stem Cell Rev. 2008, 4, 137.148.

(28) Marzesco, A. M .; Janich, P .; Wilsch-Brauninger, M .; Dubreuil, V .; Langenfeld, K .; Corbeil, D .; Huttner, W. B. Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells. J. Cell Sci. 2005,118, 2849,2858.

(29) Zitvogel, L .; Regnault, A .; Lozier, A .; Wolfers, J .; Flament, C .; Tenza, D .; Ricciardi-Castagnoli, P .; Raposo, G .; Amigorena, S. Eradication of established murine tumors using a novel cell-free vaccine: Dendritic cell-derived exosomes. Nat. Med. 1998,4, 594,600.

(30) Ginestra, A .; La Placa, M. D .; Saladino, F .; Cassara., D .; Nagase, H .; Vittorelli, M. L. The amount and proteolytic content of vesicles shed by human cancer cell lines correlates with their in vitro invasiveness. Anticancer Res. 1998, 18, 3433, 3437.

(31) Sze, S. K .; de Kleijn, D. P .; Lai, R. C .; Khia Way Tan, E .; Zhao, H.; Yeo, K. S .; Low, T. Y .; Lian, Q .; Lee, C. N .; Mitchell, W .; El Oakley, R. M .; Lim, S. K. Elucidating the secretion proteome of human embryonic stem cell-derived mesenchymal stem cells. Mol. Cell. Proteomics 2007, 6, 1680.1689.

(32) Rubio, D .; Garcia-Castro, J .; Martin, M. C .; de la Fuente, R .; Cigudosa, J. C .; Lloyd, A .; Bernad, A. Spontaneous human adult stem cell transformation. Cancer Res. 2005, 65, 3035.3039.

(33) Thirabanjasak, D .; Tantiwongse, K .; Thorner, P. S. Angiomyeloproliferative lesions following autologous stem cell therapy. J. Am. Soc. Nephrol. 2010, 21, 1218.1222.

(34) Si, Y. L .; Zhao, Y. L .; Hao, H. J .; Fu, X. B .; Han, W. D. MSCs: Biological characteristics, clinical applications and their outstanding concerns. Ageing Res. Rev. 2011, 10,93,103.

(35) Thery, C .; Boussac, M .; Veron, P .; Ricciardi-Castagnoli, P .; Raposo, G .; Garin, J .; Amigorena, S. Proteomic analysis of dendritic cell-derived exosomes: A secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol. 2001, 166, 7309, 7318.

(36) Miguet, L .; Pacaud, K .; Felden, C .; Hugel, B .; Martinez, M. C .; Freyssinet, J. M .; Herbrecht, R .; Potier, N .; van Dorsselaer, A .; Mauvieux, L. Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization. Proteomics 2006, 6,153,171.

(37) Aoki, N .; Jin-no, S .; Nakagawa, Y .; Asai, N .; Arakawa, E .; Tamura, N .; Tamura, T .; Matsuda, T. Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: Redox- and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology 2007, 148, 3850.3862.

(38) Kim, S .; Heo, J. S .; Kim, H. S .; Kim, H. O. Isolation of mesenchymal stem cells from the mononuclear cells remaining in the bone marrow processing kit. Korean J. Blood Transfusion 2010, 21, 280, 288.

(39) Shevchenko, A .; Tomas, H .; Havlis, J .; Olsen, J. V .; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006, 1, 2856.2860.

(40) Keller, A .; Nesvizhskii, A. I .; Kolker, E .; Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS / MS and database search. Anal. Chem. 2002, 74, 5383. 5392.

(41) Huang da, W .; Sherman, B. T .; Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009, 4,44.57.

(42) Gyo.rgy, B .; Modos, K .; Pallinger, E .; Paloczi, K .; Pasztoi, M .; Misja..k, P .; Deli, M. A .; Sipos, A .; Szalai, A .; Voszka, I .; Polgar, A .; Toth, K .; Csete, M .; Nagy, G .; Gay, S .; Falus, A .; Kittel, A .; Buzas, E. I. Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood 2011, 117, e39.e48.

(43) Choi, D. S .; Park, J. O .; Jang, S. C .; Yoon, Y. J .; Jung, J. W .; Choi, D. Y .; Kim, J. W .; Kang, J. S .; Park, J .; Hwang, D .; Lee, K. H .; Park, S. H .; Kim, Y. K .; Desiderio, D. M .; Kim, K. P .; Gho, Y. S. Proteomic analysis of microvesicles derived from human colorectal cancer. Proteomics 2011, 11,2745,2751.

(44) Thery, C .; Zitvogel, L .; Amigorena, S. Exosomes: Composition, biogenesis and function. Nat. Rev. Immunol. 2002, 2, 569, 579.

(45) Fevrier, B .; Raposo, G. Exosomes: Endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 2004, 16, 415, 421.

(46) Seabra, M. C .; Mules, E. H .; Hume, A. N. Rab GTPases, intracellular traffic and disease. Trends Mol. Med. 2002, 8,23.30.

(47) van Niel, G .; Raposo, G .; Candalh, C .; Boussac, M .; Hershberg, R .; Cerf-Bensussan, N .; Heyman, M. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 2001, 121, 337, 369.

(48) Feldmann, R. E. J .; Bieback, K .; Maurer, M. H .; Kalenka, A .; Burgers, H. F .; Gross, B .; Hunzinger, C .; Kluter, H .; Kuschinsky, W .; Eichler, H. Stem cell proteomes: A profile of human mesenchymal stem cells derived from umbilical cord blood. Electrophoresis 2005, 26,2749,2758.

(49) Kolf, C. M .; Cho, E .; Tuan, R. S. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: Regulation of niche, self-renewal and differentiation. Arthritis Res. Ther. 2007, 9, 204.

(50) Song, L .; Webb, N. E .; Song, Y .; Tuan, R. S. Identification and functional analysis of candidate genes regulating mesenchymal stem cell self-renewal and multipotency. Stem Cells 2006, 24, 1707, 1718.

(51) Parekkadan, B .; Milwid, J. M. Mesenchymal stem cells as therapeutics. Annu. Rev. Biomed. Eng. 2010, 12.87.117.

(52) Fiedler, J .; Etzel, N .; Brenner, R. E. To go or not to go: Migration of human mesenchymal progenitor cells stimulated by isoforms of PDGF. J. Cell. Biochem. 2004, 93, 990.998.

(53) Ball, S. G .; Shuttleworth, C. A .; Kielty, C. M. Vascular endothelial growth factor can signal through platelet-derived growth factor receptors. J. Cell Biol. 2007, 177, 489.500.

(54) Vallabhaneni, K. C .; Tkachuk, S .; Kiyan, Y .; Shushakova, N .; Haller, H .; Dumler, I .; Eden, G. Urokinase receptor mediates mobilization, migration, and differentiation of mesenchymal stem cells. Cardiovasc. Res. 2011, 90, 113.121.

(55) Veevers-Lowe, J .; Ball, S. G .; Shuttleworth, A .; Kielty, C. M. Mesenchymal stem cell migration is regulated by fibronectin through alpha 5 beta 1-integrin-mediated activation of PDGFR-beta and potentiation of growth factor signals. J. Cell Sci. 2011, 124, 1288. 1300.

(56) He, J .; Baum, L. G. Galectin interactions with extracellular matrix and effects on cellular function. Methods Enzymol. 2006, 417, 247.256.

(57) Chan, J .; O'Donoghue, K .; Gavina, M .; Torrente, Y .; Kennea, N .; Mehmet, H .; Stewart, H .; Watt, D. J .; Morgan, J. E .; Fisk, N. M. Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration. Stem Cells 2006, 24, 1879.1891.

(58) Kishore, R .; Qin, G. J .; Luedemann, C .; Bord, E .; Hanley, A .; Silver, M .; Gavin, M .; Goukassain, D .; Losordo, D. W. The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNFalpha-induced transcriptional repression of cyclin A. J. Clin. Invest. 2005, 115, 1785.1796.

(59) Meyer, R. D .; Sacks, D. B .; Rahimi, N. IQGAP1-dependent signaling pathway regulates endothelial cell proliferation and angiogenesis. PLoS One 2008, 3, e3848.

(60) Kim, YJ; Yu, JM; Joo, HJ; Kim, HK; Cho, HH; Bae, YC; Jung, JS Role of CD9 in proliferation and proangiogenic action of human adipose-derived mesenchymal stem cells. Pflugers Arch. 2007, 455, 283-296. (61) Rege, TA; Hagood, JS Thy-1 as a regulator of cell-cell and cell-matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis. FASEB J. 2006, 20, 1045.1054.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (14)

Medicament for the prevention or treatment of neural disease selected from the group consisting of neural injury, neurodegenerative disease, and neurological diseases caused by ischemia or reperfusion comprising mesenchymal stem cell-derived micro-vesicle as an active ingredient Composition.
delete The composition according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells derived from brain, liver, lung, cord blood, fetal blood, kidney, adipose tissue, placenta or bone marrow
2. The composition of claim 1, wherein said microemboli comprises (a) an immunological characteristic of positive for CD13, CD29, CD44, CD73 and CD105 surface antigens; And (b) a variable immunological characteristic for CD10 and CD90.
2. The method of claim 1, wherein the micro-vesicle comprises at least one nodule selected from the group consisting of Plasmin-Activated Factor Receptor (PDGFRB), Epidermal Growth Factor Receptor (EGFR) and Plasminogen Activator A stem cell self-regenerating surface receptor.
2. The method of claim 1, wherein the micro-vesicle is selected from the group consisting of RRAS (Ras-related protein R-Ras) / NRAS (Neuroblastoma RAS viral oncogene homolog), Mitogen-activated Protein Kinase 1 (MAPK1), Guanine Nucleotide- (Gamma-12), CDC42 (Cell Division Control protein 42 homolog), and Guanine nucleotide exchange factor VAV2 (Guanine nucleotide exchange factor VAV2). &Lt; / RTI &gt; and at least one signaling molecule selected from the group consisting of: &lt; RTI ID = 0.0 &gt;
2. The method of claim 1, wherein the micro-vesicle comprises FN1 (Fibronectin), EZR (Ezrin), IQGAP1 (IQ motif containing GTPase Activating Protein 1), CD47, Integrin and LGALS1 (Galectin-1) / LGALS3 Lt; RTI ID = 0.0 &gt; 1 &lt; / RTI &gt; cell adhesion molecule.
2. The composition of claim 1, wherein the microvesicle has at least one mesenchymal stem cell-associated antigen selected from the group consisting of CD9, CD63, CD81, CD109, CD151, CD248 and CD276.
delete 8. The composition of claim 1, wherein the nerve injury is a central nervous system injury.
The composition of claim 1, wherein said neurodegenerative disease is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis.
The composition according to claim 1, wherein the disease caused by ischemia or reperfusion is ischemic stroke.
2. The composition of claim 1, wherein the composition is administered in a single dose or repeatedly.
2. The composition of claim 1, wherein the composition is administered by intravenous injection.

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