KR101673059B1 - Pharmaceutical composition to promote hair growth or wound healing containing microvesicles from mesenchymal stem cell - Google Patents

Abstract

The present invention relates to a composition for preventing hair loss, promoting hair growth, improving wounds, and treating wounds comprising stem cell-derived micro-vesicles as an active ingredient.

Description

[0001] The present invention relates to a pharmaceutical composition for promoting hair growth or treating wounds comprising mesenchymal stem cell-derived micro-endoplasmic reticulum,

The present invention relates to a composition for preventing hair loss, promoting hair growth, improving wounds, or treating wounds 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. In addition, mesenchymal stem cells have effects for immunomodulatory, 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 offer excellent availability, low ethical issues and immunogenicity , Is an ideal candidate for clinical applications in regenerative medicine. Mesenchymal stem cells can be isolated from the brain, liver, lung, fetal blood, cord blood, kidney, adipose tissue and placenta. This widespread distribution to the whole body implies that mesenchymal stem cells play an important role in maintaining tissue homeostasis. Numerous studies have demonstrated the ability of mesenchymal stem cells to differentiate into mesenchymal lineage cells such as bone, cartilage, tendons and fat. This shows the potential for therapeutic agents in preclinical and clinical studies of bone and cartilage regeneration. In addition, clinical trials of mesenchymal stem cell-based cell therapy have been conducted in allergic bone marrow or hematopoietic stem cell transplantation, myocardial stiffness, systemic lupus erythematosus, cirrhosis, diabetes, acute kidney injury and various degenerative neurological diseases, And Graft-versus-host disease (GvHD) in a wide spectrum of human diseases.

The inventors of the present invention have conducted continuous research on mesenchymal stem cells, and found that when mesenchymal stem cell-derived microemboli is used, neurological diseases can be effectively prevented or treated without showing ethical problems and immunogenicity against conventional cell therapeutic agents (Patent Document 1). However, the research on mesenchymal stem cell-derived micro-endoplasmic reticulum is still insufficient.

1. Korean Patent Publication No. 2013-0116552

The present inventors have made efforts to develop a stem cell treatment agent having no ethical problems and no immunogenicity as a pharmaceutical composition for promoting hair growth and wounds, and as a result, they have found a mesoporous stem cell-derived micro-endoplasmic reticulum and proved the above effect. Adult stem cell culture or enriched cultures of various origins have the potential to cause adverse effects due to the proteins derived from fetal calf serum and animal cells, but they solve the problem of ethical problems and immune rejection by using micro-endoplasmic reticulum.

Accordingly, an object of the present invention is to provide a composition for preventing hair loss, promoting hair growth, improving wound, or treating wounds, which comprises a stem cell-derived micro-vesicle as an active ingredient.

The present invention provides a pharmaceutical composition for promoting hair loss prevention or hair growth comprising stem cell-derived micro-endoplasmic reticulum as an active ingredient.

In addition, the present invention provides a cosmetic composition for promoting hair loss prevention or hair growth comprising stem cell-derived micro-endoplasmic reticulum as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for wound healing or wound healing, which comprises stem cell-derived micro-endoplasmic reticulum as an active ingredient.

Since the composition according to the present invention contains the micro-vesicle as an active ingredient, there is no ethical problem, and there is no immune rejection reaction as compared with the use of adult stem cell culture or enriched culture fluid of various origins. Therefore, hair loss prevention, hair growth promotion, Or for wound healing.

Fig. 1 shows the results of purification and characterization of microembolisms derived from human bone marrow mesenchymal stem cells. Fig. 1A shows the spindle 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. The histograms of the mesenchymal stem cells stained with anti-CD29, anti-CD44, anti-CD73, anti-CD90, anti-CD105, anti-CD31, anti-CD34, anti-CD45 and CD106 are shown overlapping 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 in the microemboli of purified mesenchymal stem cells.
Fig. 2 shows the proteins identified from the mesenchymal stem cell-derived micro-endoplasmic reticulum. Figure 2a shows the spectra, the number of authentic peptides and the number of identified proteins to mesenchymal stem cell-derived microvesicles. Figure 2b identifies a total of 730 proteins with three independent LC-MS / MS analyzes. Figure 2c shows that the mesenchymal stem cell-derived microfibrillar proteome contains surface marker proteins of mesenchymal stem cells and is involved in the self-renewal and differentiation of mesenchymal stem cells.
Fig. 3 shows the functional analysis of mesenchymal stem cell-derived microembologic proteomes. 3B) and GOCCs (Gene Ontology Cellular Components, Fig. 3C) were analyzed by using DAVID software, as shown in Fig. (p < 0.05).
FIG. 4 shows the results of integrating mRNA data of mesenchymal stem cell-derived micro-expanded proteome, conditioned medium and differentiated cells, dedifferentiated cells and human mesenchymal stem cells. 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 the mesenchymal stem cell-derived micro-vesicle protein and the self-renewal and differentiation-related genes. Figure 4c compares mesenchymal stem cell-derived microembodermes, mesenchymal stem cell-conditioned medium proteomes, self-renewal and differentiation related genes.
FIG. 5 is a photograph showing the effect of treatment of PBS, minoxidil and micro-endoplasmic reticulum on the mouse model from which the hair roots have been removed, respectively.
6 and 7 are photographs showing the photographs of the thickness of hair on the head side, trunk side and tail side of the mouse after treating the PBS model, the minoxidil and the micro-endoplasmic reticulum, respectively, Graph.
FIG. 8 is a graph comparing the hair regeneration area with time after treatment of PBS, minoxidil, and micro-endoplasmic reticulum, respectively, on a mouse model from which hair roots have been removed.
FIGS. 9 and 10 show the results of comparing the hair follicle formation by treating H & E staining of the dorsal skin tissues of the mice at day 0, day 7, and day 14 with PBS, minoxidil, and microvesicle, respectively, Lt; / RTI &gt;
FIG. 11 is a graph showing changes in the number of hair follicles over time by counting the number of hair follicles in the dorsal skin tissue of each group of mice treated with PBS, minoxidil, and micro-endoplasmic reticulum respectively.
FIG. 12 is a graph showing areas of photographed photographs and wound images of a mouse model in which hair roots have been removed and treated with PBS and microvesicles, respectively.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a composition comprising a stem cell-derived micro-vesicle as an active ingredient.

As used herein, the term &quot; stem cell-derived micro-vesicle &quot; is a cell membrane particle derived from stem cells. The microfilament refers to a by-product having a plasma membrane of 100 nm to 1000 nm removed from the cell. The microfilaments mediate the transport of mRNA, miRNA and protein between cells and play an important role in signal transduction and interaction inside and outside the cell. The micro-endoplasmic reticulum exhibits a heterogeneous population depending on the derived cells, the number of cells, the size of the cells and the composition of the antigen.

The term &quot; stem cell &quot; 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.

In the present invention, the stem cells may be mesenchymal stem cells, and the mesenchymal stem cells may be mesenchymal stem cells derived from the brain, liver, lung, cord blood, fetal blood, kidney, adipose tissue, placenta or bone marrow .

According to one embodiment, the stem cell-derived microcell is a mesenchymal stem cell-derived microcell, and may be a mesenchymal stem cell-derived microcellular material isolated from human bone marrow.

One of the main features of the present invention is to analyze proteome of stem cell-derived micro-endoplasmic reticulum to investigate hair growth promoting effect and wound healing effect. The present inventors have identified 730 micro-vesicle proteins through LC-MS / MS (Liquid Chromatography-Mass Spectrometry) analysis of microsomes derived from stem cells.

According to the analysis results, the microemporrtes exhibited (a) immunological characteristics positive for CD13, CD29, CD44, CD73 and CD105 surface antigens; And (b) variable immunological properties for CD10 and CD90.

According to one embodiment, the proteome of the stem cell-derived microemboli includes molecules of the signal transduction pathway associated with self-renewal and differentiation of stem cells. Briefly, first, the microfibrillar proteome is composed of 13 proteins involved in two signaling pathways involved in the self-renewal, growth factor receptor and Wnt signaling pathways of mesenchymal stem cells based on the KEGG pathway database and the GOBP signaling pathway . Second, it includes 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 the microfilament 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 PLAUR (Plasminogen Activator, Urokinase Receptor) Have 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.

In addition, the micro-vesicles can be expressed by 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) (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) At least one signaling molecule.

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.

Also, the microemboli is 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 adhesion molecule.

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.

The microemboli also 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 micro-vesicle of the present invention as an active ingredient can be used as a composition for preventing hair loss or promoting hair growth.

The composition for suppressing hair loss and promoting hair growth of the present invention can be manufactured as pharmaceutical composition or cosmetic composition, medicine or cosmetic. In the case of pharmaceutical preparations, they are administered orally or intravenously, intravenously, subcutaneously, intranasally or intraperitoneally. Oral administration also includes sublingual administration. Parenteral administration includes injection and drip, such as subcutaneous, intramuscular, and intravenous. In addition, in one embodiment, the external preparation may be prepared as a sheet, a liquid coating, a spray, a lotion, a cream, a puff, a powder, a penetration pad, a spray, a gel, a pasta, a liniment, , Aerosols, powders, suspensions, transdermal absorbers, and the like.

Specifically, the composition according to the present invention may be administered via parenteral administration, for example, by injection.

The composition according to the present invention may be manufactured and administered in the form of various formulations by further comprising a pharmaceutically acceptable carrier. The term &quot; pharmaceutically acceptable &quot; as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like when administered to a human. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter and a perfume in the case of oral administration, and a buffering agent, a preservative, , A solubilizing agent, an isotonic agent and a stabilizing agent may be used in combination. In the case of a preparation for topical administration, a base, an excipient, a lubricant and a preservative may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. in the case of oral administration, and in the case of injections, . In addition, the pharmaceutical composition of the present invention can be prepared according to a conventional technique in the form of various formulations.

The composition for promoting hair loss prevention or hair growth according to the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. Effective dose levels are well known to those skilled in the art, including the type of disease, severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, Can be determined according to the element. The composition of the present invention may be administered alone or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art. In particular, it is preferable that the above-mentioned effective ingredient contains 0.01 to 30 parts by weight, more preferably 0.1 to 20 parts by weight, based on 100 parts by weight of the composition for preventing hair loss or promoting hair growth.

Therefore, the present invention may include a hair loss prevention or hair growth promoter comprising stem cell-derived micro-endoplasmic reticulum as an active ingredient.

In addition, the present invention can include a hair cosmetic or hair cosmetic cosmetic comprising the stem cell-derived micro-endoplasmic reticulum as an active ingredient.

The cosmetic composition of the present invention may further comprise a cosmetically acceptable carrier and may be formulated into conventional formulations such as creams, lotions, tonics, sprays, aerosols, oils, solutions, suspensions, gels, ointments, emulsions, . The composition of the present invention usually contains a base and, if necessary, other ingredients generally used as cosmetic raw materials can be suitably blended. The base of the composition of the present invention can be any of those conventionally used, and examples thereof include purified water, mineral water, ethanol, glycerin, squalene, 1,3-propylene glycol, 1,3-butylene glycol, castor oil, Tsubaki oil and liquid petrolatum. Examples of other ingredients to be incorporated into the composition of the present invention include, but are not limited to, surfactants, emulsifiers, thickeners, preservatives (e.g., p-hydroxybenzoyl methyl), antioxidants, fragrances and the like.

The cosmetic composition of the present invention may contain a component capable of supplying nutrients to the hair follicle or a commonly used hair growth promoting auxiliary component in addition to the above components. For example, vitamins, amino acids, animal and plant oils, sodium chloride But are not limited to these. The amount thereof is preferably 0.0001 to 10% by weight, particularly 0.01 to 1% by weight of the total composition. In this case, the vitamins include, but are not limited to, vitamin A, vitamin B1, vitamin B2, niacin (nicotinic acid), vitamin C, vitamin E, sodium pantothenate, potassium pantothenate and biotin H (vitamin H). Amino acids include, but are not limited to, dopa. Plant and animal oils include, but are not limited to, maize oil, egg oil, olive oil, camellia oil, rapeseed oil, sesame oil, embryo oil and the like.

The cosmetic of the present invention can be used by a method such as directly applying or spreading on hair or scalp. The hair to which the composition of the present invention is applied includes hair follicles and hair follicles, hair and eyelashes and eyelashes, beard, armpits, pubic hair, hair follicles and hair follicles. Accordingly, the present invention relates to hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair extender, eyelash hair extender, , Pet rinse, and the like.

In addition, the composition comprising the stem cell-derived micro-vesicle of the present invention as an active ingredient can be used as a composition for wound healing or treating.

In the present invention, &quot; wound &quot; refers to a state in which a living body is damaged, and a tissue constituting an inner or outer surface of a living body such as skin, muscle, nerve tissue, bone, soft tissue, internal organs, Pathological conditions. Examples of wounds include, but are not limited to, non-healing traumatic wounds, destruction of tissue by irradiation, abrasion, osteogenesis, laceration, avulsion, penetrated wound, Corneal wound, surgical wound, vascular disease, wound due to dermatosis, dermatitis, dermatosis, skin dryness, skin keratosis, cracking, erythema, contusion or bruise, skin dryness, Chronic ulcers, stitches after plastic surgery, spinal trauma wounds, gynecological wounds, chemical wounds and acne, and the like, and the like, including, but not limited to, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, Damage to any part may be included.

The composition for improving or treating wound of the present invention may be manufactured as a pharmaceutical composition, as a pharmaceutical. It is administered orally, intravenously, subcutaneously, intranasally or intraperitoneally, etc., when manufactured into pharmaceutical products. Oral administration also includes sublingual administration. Parenteral administration includes injection and drip, such as subcutaneous, intramuscular, and intravenous.

In one embodiment, the composition may be prepared with an external preparation. The external preparation may be in the form of a sheet, a liquid coating agent, a spray, a lotion, a cream, a puff, a powder, a penetration pad, a spray, a gel, a pasta, a liniment, an ointment, an aerosol, a powder, Of the present invention can be included. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a formulation generally known in all pharmaceutical chemistries.

The composition according to the present invention may be manufactured and administered in the form of various formulations by further comprising a pharmaceutically acceptable carrier. The term &quot; pharmaceutically acceptable &quot; as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like when administered to a human. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter and a perfume in the case of oral administration, and a buffering agent, a preservative, , A solubilizing agent, an isotonic agent and a stabilizing agent may be used in combination. In the case of a preparation for topical administration, a base, an excipient, a lubricant and a preservative may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. in the case of oral administration, and in the case of injections, . In addition, the pharmaceutical composition of the present invention can be prepared according to a conventional technique in the form of various formulations.

The composition for wound healing or wound treatment according to the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. Effective dose levels are well known to those skilled in the art, including the type of disease, severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, Can be determined according to the element. The composition of the present invention may be administered alone or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art. In particular, it is preferable that the above-mentioned effective ingredient contains 0.01 to 30 parts by weight, more preferably 0.1 to 20 parts by weight, based on 100 parts by weight of the composition for preventing hair loss or promoting hair growth.

In addition, the composition can be applied directly to the wound. That is, it can be spread on the wound area. In the case of the sheet form, it is applied to the wound area, where the wound area is appropriately dressed to protect the wound, thereby preventing the therapeutic effect of the active ingredient from being reduced. The dressing may be any commercially available or commonly known dressing. Examples of commercially available dressings include Compeel, Duoderm, Tagaderm and Opsite.

Example

Example 1: Isolation of microembolism

&Lt; 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>

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. Mesenchymal stem cells were seeded into 12-well plates at 5 × 10 ⁴ cells / well for osteocyte and chondrocyte differentiation and seeded at 1 × 10 ⁵ cells / well for adipocyte differentiation. Chondrocytes were cultured by treatment with TGF-β3 (10 ng / ml) in mesenchymal stem cell monolayers. 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.

&Lt; Separation of micro-vesicles >

Microvesicles (MVs) were separated from conditioned media using centrifugation by modifying known methods. free low glucose DMEM containing bFGF (4 ng / ml), 1 x ITS and 2-mercaptoethanol, followed by 10 minutes at 500 g for 10 minutes, 15 minutes at 800 g for 2 hours The culture medium was collected by centrifugation. 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.

&Lt; SDS-PAGE and phosphorus-gel decomposition >

Each of the isolated mesenchymal stem cell-derived microvesicles was separated by SDS-PAGE with 4-12% gradient Novex bis-tris gel (Invitrogen) and the resulting protein bands were analyzed by Gelco-Blue staining reagent (Pierce, USA) Lt; / RTI &gt; The dyed gel was sliced into 10 bands of the same size and in-gel trypsin digestion was performed in a known manner.

&Lt; 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.

&Lt; Identification of peptides and proteins >

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. 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 (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.

<Results and Discussion>

<Isolation and Analysis of Mesenchymal Stem Cell-Derived Microemboli>

Flow cytometry was performed to identify mesenchymal stem cells prior to the separation of the microvesicles from human bone marrow-derived mesenchymal stem cells. The cells exhibited immunostaining for negative expression of CD31, CD34, CD45 and CD106 in the positive mesenchymal stem cell form of a uniform fusiform population and positive for CD29, CD44, CD73, CD90 and CD105 (FIGS. 1A and 1A) . The ability of the mesenchymal stem cells to differentiate into various lineages as adipocytes, chondrocytes, and osteocytes was confirmed by known methods to confirm stem cell characteristics. To separate the microvesicles 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. Transmission electron microscopy revealed that the purified microvesicles were round follicles with 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. In order to reduce the possibility of contamination of the insoluble protein complex, serum-free medium was used to separate the microvesicles from the mesenchymal stem cells.

<Protein Proteome Analysis of Mesenchymal Stem Cell-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. Three independent, isolated mesenchymal stem cell-derived microembeletic samples were analyzed by LC-MS / MS, respectively. 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 with at least 2 LCMS / MS and 236 proteins were identified with 3 analyzes.

<Features of mesenchymal stem cells-derived microemboli>

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 the micro-endoplasmic reticulum, the reported micro-endoplasmic reticulum protein and mesenchymal stem cell-derived micro-endoplasmic reticulum were compared. Of the 730 mesenchymal stem cell-derived microembodial proteins, 420 proteins previously reported in the microembodiment proteome were identified. Microembodial proteomes include proteins that are associated with the essential properties of the microembroine, such as micro-embryonic development and transport. Consistent with this discovery, the 420 protein is able to regulate the docking and fusion of the micro-endoplasmic reticulum as well as the 15 RAB protein (e.g., 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.

<Microembossed Characteristics of Mesenchymal Stem Cell-Derived Microembodiment Proteome>

Previous studies have shown that the microembodiment proteome reflects the characteristics of its derived cells. Therefore, we investigated whether the mesenchymal stem cell-derived microfibrillar proteome contains mesenchymal stem cell related proteins. On the basis of the surface markers of mesenchymal stem cells sorted by Kolf et al., 730 microembossed proteins were tested for the presence of 5 positive markers (CD13, CD29, CD44, CD73 and CD105) and 2 variable markers CD10 and CD90) and no negative markers were detected. In addition, the mesenchymal stem cell-derived microsomal proteome was compared with the proteomes of the umbilical cord blood-derived mesenchymal stem cells (UCB-hMSCs) and human mesenchymal stem cells, and it was confirmed that the 122 proteins were overlapped. These results support the effectiveness of the micro-vesicle separation and proteomic analysis of the present invention. The microfibrillar proteome contains molecules of the signaling pathway involved in self-renewal and differentiation of mesenchymal stem cells (Fig. 2C). First, microfibrillar proteomes include 13 proteins associated with the KEGG pathway database and two signaling pathways involved in self-renewal, growth factor receptors and Wnt signaling pathways of mesenchymal stem cells based on the GOBP signaling pathway. Second, it includes 38 proteins involved in the differentiation of mesenchymal stem cells, the five signaling pathways involved in the 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 microfibrils are characteristic of mesenchymal stem cells and that these microfibrils may affect self-regeneration and differentiation through proteins involved in signal transduction pathways.

<Potential Function of Mesenchymal Stem Cell-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 microembodial 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. GOBPs (Gene Ontology Biological Processes, Fig. 3A) showed a significant increase in cell migration (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 function of cellular processes can predict the therapeutic effect of mesenchymal stem cell-derived microemboli.

<Integration of mRNA signature of self-renewal and differentiation of mesenchymal stem cells and mesenchymal stem cell-conditioned medium>

In order to investigate 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 reticulum, the self-regeneration and differentiation, which is closely related to the therapeutic effect of the mesenchymal stem cell, We investigated the potential relationship between self-renewal and differentiation of mesenchymal stem cells and mesenchymal stem cell-derived microsomal proteome by integrating genes known to be involved. Song et al. Determined the gene expression profiles of three differentiated cells (adipocytes, chondrocytes and osteoblasts) and demyelinated cells from human mesenchymal stem cells, human mesenchymal stem cells, and differentiated 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 the differentiation of mesenchymal stem cells were observed (Fig. 4A). Of the 730 mesenchymal stem cell-microembodial proteins, the microembodial proteins overlapped 53 (7.3%) and 25 (3.4%) of the proteins involved in self-renewal and differentiation of mesenchymal stem cells, respectively (FIG. Duplication 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. In order to investigate redundancy at the cellular process level, functional enrichment analysis of genes involved in self-renewal and differentiation was performed and compared with that of mesenchymal stem cell-derived microembologic proteomes (FIG. 4c). The mesenchymal stem cell-derived microsomal proteome and self-regenerating-related genes are involved in cell proliferation, cell adhesion and migration, actin cytoskeletal tissues, developmental processes (epidermis, ectoderm and angiogenesis), Ras signaling pathway and hormone stimulation Share the reaction. On the other hand, mesenchymal stem cell-derived microembelliferous proteome and differentiation-related genes share responses to oxidative stress and 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 the self-renewal and differentiation process of mesenchymal stem cells.

The mesenchymal stem cell-derived micro-spermatocyte has the effect of reducing the infarct size of myocardial ischemia similar to the mesenchymal stem cell-derived conditioned medium. Therefore, we examined the potential role of microfibrillar proteomes in mesenchymal stem cell-derived microfibrillar-based therapeutic aspects by comparing mesenchymal stem cell-derived microfibrils with human mesenchymal stem cell-conditioned medium proteomes. Of the 730 mesenchymal stem cell-derived microembodid 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 the 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 microembologue proteome; (2) self-renewal and (3) differentiation-related genes; And (4) mesenchymal stem cell-conditioned medium proteomes. 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 cytoskeletal tissue, Ras signaling and three developmental processes (vascular, epidermal and ectodermal development) and self-regenerating-associated mesenchymal stem cell-derived microembellites and conditioned medium proteomes; (Group 3) overlapping of the corresponding process, cell redox homeostasis, Rho signaling, cell morphology, response to calcium ion, and mesenchymal stem cell-derived microembellites and conditioned media proteases related to collagen fiber tissue; (Group 4) 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; (Group 5) duplication 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 microembellifer proteins through self-regeneration-related processes (group 1 and 2) and other differentiation-related processes (group 1 and group 3) are mesenchymal stem cell- (Group 1, group 2, and group 3) that are shared by the mesenchymal stem cell-derived microembodermes and conditioned medium proteomes that may be associated with the therapeutic effect of the cells.

Experimental Example 1. Confirmation of Hair Promotion Effect Using a Mouse

The epidermal cream of the mouse (C57BL / 6 mouse) (n = 6 per group) was applied to hair removal cream (Veet, Oxy-Riccber Kiezer, Korea) and scraped with a spatula after 3 minutes to remove hair and hair roots and wipe with PBS . The surface of the epidermis of the skin of the mouse was pushed to remove the dead skin cells, and then the dead skin cells were further removed using a transparent tape.

As an experimental group, 10 쨉 g of microvesicles were diluted in PBS and injected (injection day) into 8 injection sites where the skin was removed at a dose of 20 / / injection site. At this time, the injection site is a red portion of the day 0 MVs group in FIG.

As a control group, PBS treatment group (untreated control group) and minoxidil 1% treatment group (positive control group) were also tested at the same time. In the PBS-treated group, PBS (20 μl / injection site) was applied to the skin by the same method as in the case of the micro-vesicle. Minoxidil 1% treatment group was treated with 50 μl of minoxidil for 7 days And then beaten to absorb.

Three days later, the difference in the hair growth between the groups was visually observed.

In the present invention, Fig. 5 shows photographs of the results immediately after the treatment, 3 days, 7 days, 10 days, 12 days, and 14 days after treatment. As shown in Fig. 5, it was found that the mouse coated with micro-endoplasmic reticulum had the highest hair growth effect.

6 and 7 are graphs showing the photographs of the thicknesses of the mice taken from the top, the middle and the bottom of each group and the thicknesses thereof, and it can be seen that the thickness of the fur of the group treated with the micro-endoplasmic reticulum is the thickest.

FIG. 8 is a graph comparing the hair regeneration area with time of each mouse of each group, and the hair regeneration area was measured using Image J. FIG. In the graph, the vertical axis represents the ratio of the black portion to the white portion in the photograph of the mouse with respect to time, and the higher the value, the higher the hair regeneration efficiency. As shown in FIG. 8, the hair regeneration efficiency in the micro-endoplasmic reticulum-treated group was high, indicating that the hair end effect was high in the micro-endoplasmic reticulum-treated group.

Some of the 0 day, 7 day, and 14 day mice that had been observed with the naked eye were sacrificed and tissues were secured for tissue staining and hair follicle counting.

FIG. 9 shows a photograph (40X) in which the dorsal skin tissues of each group of mice were detached and observed by performing hematoxylin and eosin (H & E) staining. As shown in FIG. 9, H & E staining of the paraffin sections of skin tissues, such as those taken from mice, showed that the micro-endoplasmic reticulum-treated group on day 7 had significantly increased hair follicle number , Indicating a quick hair growth effect. In addition, in the photograph of the 14th day in Fig. 9, it was also confirmed that the number of hair follicles in the group treated with the micro-endoplasmic reticulum still increased significantly compared to the group treated with minoxidil, and it was found that the hair microbial effect of the micro- .

FIG. 10 is a photograph of the result of staining of the entire dermis tissue subjected to the H & E staining. As shown in FIG. 10, in the micro-endoplasmic reticulum treatment group, thickened hypodermis and increased number of hair follicles, which are thought to be caused by injection of micro-

On the other hand, FIG. 11 is a graph showing changes in the number of hair follicles over time by counting the number of hair follicles in the dorsal skin tissue of each group of mice as seen in the photograph of FIG. The number of mice per group was 3, and the number of hair follicles in the skin tissues such as mice was averaged.

Experimental Example 2. Confirmation of wound effect

Sprague Dawley rats at 6 weeks of age were divided into two groups and respiration was anesthetized with 2% isoflurane. The hair was removed with electric clipper and hair removal cream. The exposed area was disinfected with 70% ethanol, and wounded with a 6 mm biopsy punch. The top of the wound was scraped off with scissors to prevent damage to the muscle layer. Group 2 animals were treated with 20 쨉 g microspheres and PBS-induced wound dressing, respectively.

In the present invention, FIG. 12 is a graph of photographs of photographed photographs of the results immediately after the treatment, 3 days, 7 days, 10 days, 12 days and 14 days, and areas of the wound surfaces.

As shown in Fig. 12, the mice treated with the microfibrillar body showed excellent wound healing effect.

Claims (11)

A stem cell-derived micro-vesicle as an active ingredient,
Wherein the micro-vesicle is a by-product having a plasma membrane of 100 nm to 1000 nm removed from the cell, which promotes hair loss prevention or hair growth.
The method according to claim 1,
Wherein the stem cell is a mesenchymal stem cell.
The method according to claim 1,
Wherein the stem cell is a mesenchymal stem cell derived from brain, liver, lung, umbilical cord blood, fetal blood, kidney, adipose tissue, placenta, amniotic membrane or bone marrow.
The method according to claim 1,
The microemboli includes (a) immunological characteristics positive for CD13, CD29, CD44, CD73 and CD105 surface antigens; And (b) have variable immunological properties for CD10 and CD90.
The method according to claim 1,
The micro-endoplasmic reticulum is at least one type of mesenchymal stem cell self-renewal selected from the group consisting of Plasmin-Derived Growth Factor Receptor (PDGFRB), Epidermal Growth Factor Receptor (EGFR) and Plasminogen Activator, Urokinase Receptor A pharmaceutical composition having a surface receptor.
The method according to claim 1,
The micro-endoplasmic reticulum is composed of RRAS (Ras-related protein R-Ras) / NRAS (Neuroblastoma RAS viral oncogene homolog), MAPK1 (Mitogen-activated Protein Kinase 1), GNA13 / GNG12 (Guanine Nucleotide-binding protein subunit Alpha-13 / Guanine Nucleotide- at least one selected from the group consisting of 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) A pharmaceutical composition having signaling molecules.
The method according to claim 1,
The micro-endoplasmic reticulum contains at least one species 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 Of a cell adhesion molecule.
The method according to claim 1,
The micro-vesicle has at least one mesenchymal stem cell-associated antigen selected from the group consisting of CD9, CD63, CD81, CD109, CD151, CD248 and CD276.
A stem cell-derived micro-vesicle as an active ingredient,
Wherein the micro-vesicle is a by-product having a plasma membrane of 100 nm to 1000 nm removed from the cell, which promotes hair loss prevention or hair growth.
10. The method of claim 9,
Cosmetics include hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair extender, eyelash hair extender, eyelash nourishment, pet shampoo or pet A cosmetic composition which is an animal rinse.
A stem cell-derived micro-vesicle as an active ingredient,
Wherein the micro-vesicle is a byproduct having a plasma membrane of 100 nm to 1000 nm removed from the cell.

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