EXOSOMES ISOLATED FROM DERMAL PAPILLA PROGENITOR CELLS, AND USE THEREOF

Abstract

The present invention relates to exosomes isolated from dermal papilla progenitor cells, specifically, the exosomes isolated from the dermal papilla progenitor cells which are excellent in prevention, improvement and treatment of hair loss (alopecia) and are also excellent in terms of skin improvement and wound healing effects, as well as various uses thereof.

Claims

1. An exosome isolated from dermal papilla progenitor cells or from a culture product of dermal papilla progenitor cells.

2. The exosome according to claim 1, wherein the dermal papilla progenitor cells are derived from embryonic stem cells, adult stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells or mesenchymal stem cells.

3. The exosome according to claim 1, wherein the dermal papilla progenitor cells are differentiated or prepared from human-derived stem cells, induced pluripotent stem cells or adipose stem cells.

4. The exosome according to claim 1, wherein the exosome has a size of 50 to 150 nm.

5. The exosome according to claim 1, wherein the exosome expresses CD63, CD9, CD81 or combinations thereof.

6. The exosome according to claim 5, wherein an expression rate of CD63 is at least 80%.

7. (canceled)

8. The exosome according to claim 1, wherein the exosome is obtained by: a) centrifuging a culture of dermal papilla progenitor cells at 200 to 400×g for 5 to 20 minutes to obtain a supernatant; b) removing the remaining cells and cell residues remaining in the centrifuged culture; c) centrifuging the supernatant of a) at 9,000 to 12,000×g for 60 to 80 minutes; d) collecting a supernatant obtained after the centrifuging of c); and e) centrifuging the supernatant collected in d) at 90,000 to 120,000×g for 80 to 100 minutes.

9. The exosome according to claim 1, wherein the dermal papilla progenitor cells are cultured under 1 to 2% oxygen condition.

10. The exosome according to claim 1, wherein the dermal papilla progenitor cells are three-dimensionally cultured or mixed-cultured along with keratinocytes, outer root-sheath cells, melanocytes, fibroblasts or combinations thereof.

11. A pharmaceutical composition for prevention, improvement or treatment of hair loss, the composition comprising the exosome according to claim 1 as an active ingredient.

12. The pharmaceutical composition according to claim 11, wherein the composition comprises 1×10.sup.6 particles to 1×10.sup.12 particles of the exosome.

13. The pharmaceutical composition according to claim 11, wherein the hair loss is androgenetic alopecia, telogen alopecia, chemical hair loss, mechanical hair loss, traumatic alopecia, pressure hair loss, genital alopecia, alopecia areata, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, scarring alopecia, congenital alopecia, circular alopecia, ringworm of the head, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex generalized alopecia or combinations thereof.

14. A method for prevention, improvement or treatment of hair loss, comprising administering the pharmaceutical composition according to claim 11 to a subject in need thereof.

15. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0057] FIG. 1 shows extraction values of exosomes from cells according to the present invention.

[0058] FIG. 2 shows marker expression distributions of the exosomes according to the present invention for each derived cell.

[0059] FIG. 3 shows evaluation result of cell proliferation of the exosomes according to the present invention.

[0060] FIGS. 4A and 4B show evaluation result of effects (hair follicle length growth) of the exosomes according to the present invention on the hair follicles.

[0061] FIG. 5 shows evaluation result of effects of the exosomes according to the present invention on the hair growth cycle.

[0062] FIG. 6 shows evaluation result of cell proliferation of the exosomes according to the present invention in the hair follicles.

[0063] FIG. 7 shows evaluation result of growth factor expression of the exosomes according to the present invention in the dermal papilla cells.

[0064] FIGS. 8A, 8B and 8C show evaluation result of hair growth-related gene expression of the exosomes according to the present invention.

[0065] FIGS. 9A and 9B show evaluation result of aging recovery of the exosomes according to the present invention in aged dermal papilla cells.

[0066] FIG. 10 shows evaluation result of cell mobility of the exosomes according to the present invention.

MODE FOR CARRYING OUT INVENTION

[0067] Hereinafter, the present invention will be described in detail by way of examples and experimental examples.

[0068] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

Examples 1 and 2

[0069] In order to isolate the exosomes of the present invention, dermal papilla progenitor cells were prepared with reference to the following document [Gnedeva et al., PLoS One 10, e0116892, 2015]. Using the dermal papilla progenitor cells prepared according to the above-described method, the cells were cultured in a cell incubator under a hypoxic condition of 1% oxygen in Example 1 and under a normal culture condition of 5% carbon dioxide, 37° C. in Example 2, respectively.

[0070] The dermal papilla progenitor cells were cultured on an ultra-low cluster, 96-well culture dish at 10.sup.4 cells per well in 10% DMEM/F12+Glutamax (1:1) medium for 24 hours. After the culture, the above medium was replaced with DMEM/F12+Glutamax (1:1) serum-free medium, followed by culturing for 48 hours. Thereafter, the medium was collected and centrifuged at 300×g for 10 minutes to remove remaining cells and cell residues, while the supernatant was taken, filtered using a 0.22 μm filter, and then centrifuged at 10,000×g and 4° C. for 70 minutes using a high speed centrifuge. The centrifuged supernatant was collected again and centrifuged at 100,000×g and 4° C. for 90 minutes using an ultracentrifuge in order to remove the supernatant, thereby isolating and obtaining the exosomes remaining in the lower layer.

Comparative Examples 1 and 2

[0071] The exosome isolation method was the same as in Examples 1 and 2, except that the exosomes were isolated and obtained using fibroblasts in Comparative Example 1 or dermal papilla cells in Comparative Example 2 instead of the dermal papilla progenitor cells.

<Experimental Example 1> Evaluation of Characteristics (Exosome Size) of the Exosomes According to the Present Invention

[0072] In order to evaluate the characteristics of the exosomes of the present invention, the size of the exosomes isolated from dermal papilla progenitor cells was analyzed using the examples 1 and 2. As a result, the exosomes of the present invention were distributed between 50 and 150 nm, and the average size was found to be 63.6 nm.

<Experimental Example 2> Evaluation of Characteristics (Number of Exosomes Extracted Per Cell) of the Exosomes According to the Present Invention

[0073] In order to evaluate the characteristics of the exosomes of the present invention, the number of exosomes secreted per cell was comparatively evaluated using the examples and Comparative Example 2. As a result, the exosomes secreted from the dermal papilla progenitor cells according to the present invention were 5×10.sup.16/cell, and the exosomes secreted from the dermal papilla cells of Comparative Example 2 were 2×10.sup.16/cell, such that the number of exosomes secreted from the dermal papilla progenitor cells according to the present invention was found to be high 2.5 times or more (FIG. 1).

<Experimental Example 3> Evaluation of Characteristics of the Exosomes According to the Present Invention (Confirmation of Expression Markers

[0074] In order to evaluate the characteristics of the exosomes of the present invention, a distribution of markers expressed in the exosomes was evaluated using the examples and Comparative Examples 1 and 2. As a result of the evaluation, exosome expression markers CD63, CD9 and CD81 were commonly expressed in each cell, but expression rates for each marker were different. With regard to the exosomes of dermal papilla progenitor cells, which is the example of the present invention, the expression rate of CD63 was about 90-95%. On the other hand, with regard to the exosomes of the fibroblasts of Comparative Example 1, the expression rates of CD63, CD81 and CD9 were about 80%, 19% and 1%, respectively. With regard to the exosomes of the dermal papilla cells of Comparative Example 2, CD63 was found to be 98% or more, and the expression rates of CD9 and CD81 were found to be very low (FIG. 2).

<Experimental Example 4> Evaluation of Cell Proliferation

[0075] In order to evaluate hair growth and skin improvement effects of the exosomes according to the present invention, dermal papilla cells (DPCs) which are known to be important for hair follicle formation and hair growth, outer root-sheath cells (ORSCs), and keratinocytes (KCs) as skin cells were subjected evaluation of cell proliferation.

[0076] The control did not have any treatment, the positive control was treated with minoxidil used for hair loss treatment, and the comparative example was treated with fibroblast-derived exosomes. Evaluation results thereof are shown in FIG. 3. An experimental method used herein was conducted as follows: 5×10.sup.3 dermal papilla cells per well were cultured on a 96-well culture dish (well plate) in 10% DMEM medium for 24 hours; after 24 hours, the medium was replaced with a serum-free DMEM medium while the cells were treated with the exosomes (1.0×10.sup.9 particles) of Example 1 and minoxidil (1 μM) as a positive control; and, after 48 hours of the culture, 75 μl of serum-free medium and 25 μl of MTT solution were added, followed by further culturing for 3 hours and then measuring absorbance at 570 nm in order to confirm cell proliferation. A cell proliferation rate refers to an increase rate of cell proliferation indicated on a graph by comparing measurement results of the absorbance to those of the negative control.

[0077] The outer root-sheath cells and keratinocytes of the hair follicles were cultured in Epilife medium at 1×10.sup.4 cells per well in a 96 well plate for 24 hours. After culturing for 24 hours, the medium was replaced with a medium without supplements while the cells were treated with the exosomes (1.0×10.sup.9 particles) of Example 1 and minoxidil (1 μM) as a positive control, followed by the above evaluation process under the same conditions as above.

[0078] As a result of the evaluation, with regard to Example 1 according to the present invention, proliferation was active in all dermal papilla cells, outer root-sheath cells and keratinocytes, in particular, it could be seen that Example 1 has the excellent cell proliferation rate compared to minoxidil used as a hair loss therapeutic agent (FIG. 3).

<Experimental Example 5> Evaluation of Hair Follicle Growth Effects Through Human Hair Follicle Organ Culture Test

[0079] In order to evaluate effects of the exosomes of the present invention on human hair follicle growth, a human hair follicle organ culture test (ex vivo hair follicle organ culture) was conducted. More specifically, scalp tissues were isolated into the unit of hair follicles, and the remaining hair follicles were used after cutting the part below the sebaceous gland of the isolated hair follicles. The prepared hair follicles were treated with Examples 1 and 2, and minoxidil as a positive control, respectively, and lengths of the hair growing on the 3rd and 6th days were measured, followed by comparing the measured results to the negative control in order to evaluate a length growth and a growth cycle of human hair follicles.

5-1. Hair Follicle Length Growth Evaluation 1

[0080] In order to evaluate effects of the dermal papilla progenitor cell-derived exosomes isolated with different cells and culture conditions on the length growth of hair follicles, human hair follicle tissues were cultured in Williams E culture medium containing 2 mM L-glutamine, 100 U/ml streptomycin, 10 ng/ml hydrocortisone and 10 μg/ml insulin, the exosomes (1.0×10.sup.9 particles) of Example 1, and the exosomes (1.0×10.sup.9 particles) of Example 2, minoxidil (1 μM) as a positive control and the exosomes (1.0×10.sup.9 particles) of Comparative Example 2, respectively, were used for treatment of the cells, followed by culturing the cells in a carbon dioxide incubator at 37° C. The lengths of the hair follicles grown on the 3rd and 6th days of the culture, respectively, were measured using a microscope ruler. Evaluation results thereof are shown in FIG. 4A.

[0081] As a result of the evaluation, the control and Comparative Example 1 without any treatment showed the growth of hair by 1 mm for 6 days, while the positive control showed the growth of hair by about 1.5 mm. On the other hand, Example 1 with hypoxic culture showed the growth of hair by about 2.2 mm for 6 days, while Example 2 with general culture showed the growth of hair by 1.8 mm. From the above results, it can be seen that the exosomes isolated after hypoxic culture is more excellent in hair growth effects by 20% or more as compared to the positive control.

5-2. Hair Follicle Length Growth Evaluation 2

[0082] In order to evaluate effects of the dermal papilla progenitor cell-derived exosomes isolated with different cells and culture methods on the length growth of hair follicles, human hair follicle tissues were cultured in Williams E culture medium containing 2 mM L-glutamine, 100 U/ml streptomycin, 10 ng/ml hydrocortisone and 10 μg/ml insulin, the exosomes (1.0×10.sup.9 particles) of Example 2 with three-dimensional culture, the exosomes (1.0×10.sup.9 particles) of Comparative Example 2 with three-dimensional culture, and the exosomes (1.0×10.sup.9 particles) of Example 2 with three-dimensional culture and mixed culture along with the keratinocytes, respectively, were used for treatment of the cells, followed by culturing the cells in a carbon dioxide incubator at 37° C. The lengths of the hair follicles grown on the 3rd and 6th days of the culture, respectively, were measured using a microscope ruler. Evaluation results thereof are shown in FIG. 4B.

[0083] As a result of the evaluation, the exosomes (1.0×10.sup.9 particles) cultured by a general culture method and then isolated in Example 2 showed the growth of hair by about 0.6 mm for 6 days, while the exosomes (1.0×10.sup.9 particles) three-dimensionally cultured and then isolated in Comparative Example 2 showed the growth of hair by about 1.1 mm. On the other hand, the exosomes (1.0×10.sup.9 particles) three-dimensionally cultured and then isolated in Example 2 showed the growth of hair by 1.2 mm, while the exosomes (1.0×10.sup.9 particles) three-dimensionally cultured and then mixed-cultured along with the keratinocytes and then isolated in Example 2 showed the growth of hair by about 1.4 mm. Therefore, it can be confirmed that, unlike the general culture method, the exosome, which was cultured according to three-dimensional culture and then isolated, is more superior in terms of hair follicle length growth rate. In addition, it can be seen that there is a synergistic effect when treated along with keratinocytes.

5-3. Hair Growth Cycle Evaluation

[0084] In order to evaluate effects of the present invention on the hair growth cycle, only the anagen hair follicles were isolated from the hair follicle tissue and the exosomes (1.0×10.sup.9 particles) isolated in each of Examples 1 and 2, and the exosomes (1.0×10.sup.9 particles) isolated in each of the controls, the positive control (1 μM) and the comparative example were used for treatment of the hair follicles. Then, in order to evaluate the growth cycle of hair follicles, an experiment was conducted with reference to the following document [Langan et al., Experimental dermatology, 2015]. Growth cycle classification criteria were classified by comparing a distance between the hair shaft and dermal papilla cells after observing human hair follicles under a microscope. The growth cycle was divided into anagen and catagen phases 1 to 3 by scores, and measured results are shown in FIG. 5.

[0085] As a result of the evaluation, most of the hair follicles treated with the control were converted into the catagen without the anagen, while the hair follicles treated with the exosomes isolated in Comparative Example 1 had a shorter catagen than the control but did not exhibit the anagen. The hair follicles treated with minoxidil also showed similar results to the hair follicles treated with the exosomes isolated in Comparative Example 1. In contrast, with regard to the hair follicles treated with the exosomes isolated in Example 2 (normal culture condition) of the present invention, the catagen was very short at 20%, and the initial catagen was maintained at 40% or more. The hair follicles treated with the exosomes isolated in Example 1 (hypoxic condition) did not show the catagen at all, but had 60% of initial anagen and 20% of anagen, thereby indicating that the hair continues to grow.

5-4. Evaluation of Hair Matrix Cell Proliferation in Hair Follicles

[0086] In order to evaluate effects of the exosomes of the present invention on the proliferation of hair matrix cells involved in direct length growth of hair, only anagen hair follicles were isolated from the hair follicle tissues and used in the present experiment. The human hair follicle tissues were cultured using Williams E culture medium containing 2 mM L-glutamin, 100 U/ml streptomycin, 10 ng/ml hydrocortisone and 10 μg/ml insulin in a carbon dioxide incubator at 37° C. for 2 days after treating the tissues with a control, a positive control (1 μM), and the exosomes (1.0×10.sup.9 particles) three-dimensionally cultured and then isolated in Examples 1 and 2. After culturing, the human hair follicles were frozen and cut, followed by immune-fluorescence staining for Ki-67. As the number of Ki-67 positive cells in the hair matrix cells is increased, hair follicle growth was evaluated to be increased. Evaluation results thereof are shown in FIG. 6.

[0087] As a result of the evaluation, it can be seen that more Ki-67-positive cells (red) were expressed in the hair follicle tissues treated with the exosomes isolated in Examples 1 and 2 of the present invention as compared to the control and minoxidil as the positive control.

<Experimental Example 6> Evaluation of Hair Growth-Related Growth Factor Expression

[0088] In order to evaluate effects of the exosomes of the present invention on hair growth, the expression of genes important for hair growth in dermal papilla cells was investigated. On a 6-well culture dish (well plate), 5.0×10.sup.4 dermal papilla cells per well were cultured in 10% DMEM culture medium for 24 hours. After washing twice using a serum-free DMEM medium, the cells were cultured in the serum-free DMEM medium for 24 hours. The exosomes (1.0×10.sup.9 particles) isolated in Examples 1 and 2, a control and a positive control (1 μM) were used for treatment of the cells for 2 hours, respectively, followed by extracting RNA and synthesizing cDNA. It was evaluated whether FGF7, FGF10, IGF1 and PDGF genes, which are growth factors involved in hair growth, are expressed or not through real-time PCR. Evaluation results thereof are shown in FIG. 7.

[0089] As a result of the evaluation, it can be seen that the exosomes isolated in Examples 1 and 2 of the present invention have higher expression rates of all growth factors compared to minoxidil, that is, the positive control. In particular, it could be seen that the expression of the growth factors treated with the exosomes isolated in Example 1 was the highest.

<Experimental Example 7> Evaluation of Gene Expression Related to Hair Follicle Formation Ability

[0090] In order to evaluate how much the exosomes of the present invention maintains hair follicle formation ability, the expression of proteoglycan, a Wnt target gene and a dermal papilla cell marker gene, respectively, which are genes involved in the hair follicle formation ability of dermal papilla cells, was investigated.

[0091] On a 6-well culture dish (well plate), 5.0×10.sup.4 dermal papilla cells per well were cultured in 10% DMEM culture medium for 24 hours. After washing twice using a serum-free DMEM medium, the cells were cultured in the serum-free DMEM medium for 24 hours. The exosomes (1.0×10.sup.9 particles) isolated in Examples 1 and 2, a control and a positive control (1 μM) were used for treatment of the cells for 4 hours, respectively, followed by extracting RNA and synthesizing cDNA. It was evaluated whether each of the genes is expressed or not through real-time PCR. Evaluation results thereof are shown in FIGS. 8A to 8C.

7-1. Evaluation of Proteoglycan-Related Gene Expression

[0092] Proteoglycans are polymorphic macromolecules present in skin and hair, and are known to interact with growth factors and collagen. Further, it has been found that the above macromolecules are decreased in telogen hair follicles while being increased in anagen hair follicles in the hair growth cycle (Couchman, 2017, Journal of Dermatological Science). Using the dermal papilla cells treated with the exosomes isolated in Examples 1 and 2, a control and a positive control, respectively, the expression of Versican, Biglycan and Syndecanl genes involved in proteoglycan expression was evaluated through real-time PCR. Evaluation results thereof are shown in FIG. 8A.

[0093] As a result of the evaluation, the exosomes isolated in Example 2 of the present invention demonstrated the expression of genes at a level equal to or higher than that of the positive control. In particular, it can be seen that, with regard to the exosomes isolated in Example 1 of the present invention, the expression of all genes exhibits higher than that of the positive control.

7-2. Evaluation of Wnt Target Gene Expression

[0094] The Wnt signaling system is a key material in the process of hair follicle formation and differentiation, and it is well known that the activation of related genes is important for entering the anagen from the telogen in the hair follicle growth cycle (Lim & Nusse, 2013). In order to confirm whether the exosomes of the present invention regulate the expression of major target genes in the Wnt signaling system, the dermal papilla cells treated with the exosomes isolated in Examples 1 and 2, the control and the positive control, respectively, were used to evaluate the expression rates of LEF1, Axin2, beta-catenin and Wnt5a gens, which are involved in the expression of Wnt target genes through real-time PCR. The evaluation is to indicate values in proportion to gene expression types of the negative control on a graph. Evaluation results thereof are shown in FIG. 8B.

[0095] As a result of the evaluation, the exosomes isolated in Examples 1 and 2 of the present invention demonstrated higher expression rates of LEF1, Axin2, beta-catenin and Wnt5a as compared to the control and the positive control. In particular, it can be seen that the expression rate of each of LEF1 and Axin2 exhibits high 2 times or more, while beta-catenin and Wnt5a exhibit high 30-40% or more, as compared to the positive control.

7-3. Evaluation of Dermal Papilla Cell Representative Gene Expression

[0096] It is known that, with increase in the expression rates of BMP4, SOX1 and Corin, which are well known as markers of dermal papilla progenitor cells, the hair follicles formation ability is increased. Using the dermal papilla cells treated with the exosomes isolated in Examples 1 and 2, the control and the positive control, respectively, the expression of BMP4, SOX2 and Corin genes, which are representative dermal papilla cell genes, was evaluated through real-time PCR. Results thereof are shown in FIG. 8C.

[0097] As a result of the evaluation, it could be seen that, with regard to the exosomes isolated in Examples 1 and 2 of the present invention, the expression rates of BMP4, SOX2 and Corin genes exhibited higher at least 3 times or more and at most 10 times or more than that of the control and the positive control.

<Experimental Example 8> Evaluation of Aging Recovery in Aged Dermal Papilla Cells

[0098] As the dermal papilla cells are repeatedly sub-cultured, the cells become aged, that is, senescent (cellular senescence) and thus lose an ability of forming hair follicles. It was investigated whether the exosomes of the present invention are effective in the hair follicle formation ability and cell aging recovery of aged dermal papilla cells that have lost the hair follicle formation ability.

8-1. Evaluation of Hair Follicle Formation Ability (“Rejuvenation”

[0099] Alkaline phosphatase (AP) is a representative marker of hair follicle-formation ability and, when dermal papilla cells become aged, the activity of alkaline phosphatase is decreased. On a 6-well culture dish (well plate), 3.0×10.sup.4 dermal papilla cells were cultured in 10% DMEM for 24 hours. After washing twice with a serum-free DMEM medium, the exosomes (1.0×10.sup.9 particles) isolated in Examples 1 and 2, Comparative Example 1 and the control, respectively, were used for treatment of the cells. After 48 hours, the cells were washed twice with tris-buffered saline (TBS) and fixed with cold acetone for 10 minutes, followed by reaction for 10 minutes while adding a mixed solution of NBT/BCIP in TN (0.01M Tris-HCl and 0.1M NaCl, pH 8.0) in a ratio of 1:50 thereto. After washing twice again with tris-buffered saline (TBS), the product was subjected to observation under a microscope, and the activity of AP could be confirmed by a purple staining extent. Results thereof are shown in FIG. 9A.

[0100] As a result of the evaluation, a ratio of cells stained with purple exhibited higher in the exosomes isolated in Examples 1 and 2 of the present invention than that of the exosomes isolated in the control and Comparative Example 1. Therefore, it can be seen that the exosomes of the present invention increase the activity of AP and also increase the hair follicle formation ability.

8-2. Evaluation of Anti-Aging in Dermal Papilla Cells

[0101] In order to investigate whether the exosomes of the present invention are effective in cellular senescence recovery (rejuvenation) of aged dermal papilla cells, senescence-associated β-galactosidase (SA-β-gal) as a cellular senescence marker was subjected to staining. On a 6-well culture dish (well plate), 3.0×10.sup.4 dermal papilla cells were cultured in 10% DMEM for 24 hours. After washing twice with a serum-free DMEM medium, the exosomes (1.0×10.sup.9 particles) isolated in Example 1, a control and a positive control, that is, minoxidil (1 uM) were used for treatment of the cells for 48 hours. The treated cells were stained according to the protocol using a senescence β-galactosidase staining kit. It can be confirmed that the aging of the cells is increased as the SA-β-gal staining extent (blue) is increased. Results thereof are shown in FIG. 9B.

[0102] As a result of the evaluation, it was confirmed that a ratio of cells stained with SA-β-gal was low in the exosomes isolated in Example 1 of the present invention as compared to the control and the positive control. Therefore, it could be seen that the exosomes isolated in Example 1 has excellent anti-aging effects through suppression of cell aging.

<Experimental Example 9> Evaluation of Cell Motility

[0103] In order to evaluate effects of the exosomes according to the present invention on wound healing and skin improvement, the cell migration ability (“motility”) of keratinocytes was investigated. After placing 1.0×10.sup.5 keratinocytes per well on a 6-well culture dish (well plate), the cells were cultured for 24 hours. The cells in culture were scraped on the culture dish using a sterile 10 μl tip to make a wound, and then washed with phosphate buffer saline (PBS). In Epilife medium, the cells were treated with the exosomes (1.0×10.sup.9 particles) isolated in Example 1, a control and a positive control, that is, epidermal growth factor (EGF), which is an epidermal growth factor, respectively. After 12 hours, a degree of cell motility was observed using a microscope. The cell motility was evaluated by observing a degree of filling the wounded area under the microscope. Results thereof are shown in FIG. 10.

[0104] As a result of the evaluation, it could be seen that the cell motility was increased by the exosomes isolated in Example 1 which in turn reduced the wounded area as compared to the control. Further, it could also be seen that the cell motility was almost similar to that of the positive control, that is, EGF.