METHOD FOR PREPARING COMPOSITION, INCLUDED WITHIN EXOSOME OBTAINED FROM NANOG-INTRODUCED, FETUS-DERIVED MESENCHYMAL STEM CELL IN AMNIOTIC FLUID, FOR HAIR GROWTH

Abstract

A composition for hair growth and a preparation method therefor through the separation of exosomes from a culture medium of fetus-derived mesenchymal stem cells overexpressing the dedifferentiation factor Nanog in amniotic fluid are described.

Claims

1. A method of producing a mesenchymal stem cell-derived exosome, the method comprising: (a) isolating a fetus-derived cell from amniotic fluid obtained from a pregnant woman; (b) sub-culturing the isolated fetus-derived cell in a medium containing fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF) to obtain a mesenchymal stem cell derived from a fetus in amniotic fluid; (c) introducing Nanog into the mesenchymal stem cell derived from a fetus in amniotic fluid to obtain a Nanog-overexpressing mesenchymal stem cell derived from a fetus in amniotic fluid; (d) culturing the obtained Nanog-overexpressing mesenchymal stem cell derived from a fetus in amniotic fluid for 1 day to 5 days to prepare a conditioned medium; and (e) isolating an exosome from the conditioned medium, wherein the isolated exosome comprises any one or more human growth factors selected from the group consisting of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), Wingless-type MMTV integration site family member 7A (Wnt7a), and platelet-derived growth factor (PDGF)-AA.

2. The method of claim 1, wherein, in process (c), the Nanog is introduced via a retroviral vector.

3. The method of claim 1, wherein the sub-culturing is performed in a medium further comprising selenium and ascorbic acid.

4. A cosmetic composition for promoting hair growth, the cosmetic composition prepared using the method of claim 1 and comprising a mesenchymal stem cell-derived exosome as an active ingredient.

5. A pharmaceutical composition for promoting hair growth, the pharmaceutical composition prepared using the method of claim 1 and comprising a mesenchymal stem cell-derived exosome as an active ingredient.

Description

DESCRIPTION OF DRAWINGS

[0048] FIG. 1 illustrates results of confirming whether Nanog was overexpressed in cell lines according to an embodiment of the present invention.

[0049] FIG. 2 illustrates results of measuring average sizes and numbers of amniotic fluid-derived MSCs and Nanog-introduced amniotic fluid-derived MSCs, according to an embodiment of the present invention.

[0050] FIG. 3 illustrates analysis results of substances inside exosomes through PCR, according to an embodiment of the present invention.

[0051] FIG. 4 illustrates analysis results of substances inside exosomes through ELISA, according to an embodiment of the present invention.

[0052] FIG. 5 illustrates results of confirming functions of exosomes obtained according to an embodiment of the present invention.

[0053] FIG. 6 illustrates AP staining results of confirming changes in hair growth markers along with the growth of hair papillae by exosomes obtained according to an embodiment of the present invention.

[0054] FIG. 7 illustrates results of confirming changes in markers of exosomes obtained according to an embodiment of the present invention.

BEST MODE

[0055] Unless otherwise defined, technical and scientific terms as used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known in the art and commonly used.

Examples

Example 1: Establishment of Nanog-Introduced MSCs Derived from Amniotic Fluid

[0056] To obtain amniotic fluid-derived MSC lines, which are easy for cell expansion, the Nanog gene was introduced into amniotic fluid-derived MSCs using a retroviral vector system to induce Nanog overexpression, thereby establishing Nanog-introduced amniotic fluid-derived MSC lines. A detailed method is as follows.

[0057] Fetus-derived cells isolated from amniotic fluid obtained from pregnant women were sub-cultured in a low glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, 4 ng/ml of basic fibroblast growth factor (bFGF), 5 ng/ml of selenium, and 50 g/ml of ascorbic acid to obtain MSCs derived from a fetus in the amniotic fluid.

[0058] The Nanog gene was introduced into the obtained amniotic fluid-derived MSCs using a retroviral vector system to induce Nanog overexpression, thereby completing the preparation of Nanog-introduced amniotic fluid-derived MSC lines.

[0059] Specifically, a restriction enzyme was used to isolate the Nanog gene (NCBI GenBank Accession number NM_024865.3 (SEQ ID NO: 1) from a pBS-Nanog vector (Yamanaka lab's plasmid stock # A4, JAPAN). The isolated Nanog gene was ligated to a pMXs vector (Cell Biolab, JAPAN) with T4 ligase to produce pMXs-Nanog. The pMXs-Nanog vector was transfected into 293GPG cells for 6 hours using a transfection reagent. A virus with the Nanog gene was produced for 72 hours, and the supernatant was separated, centrifuged at 2000 rpm for 10 minutes, and then filtered through a 0.45-m filter. The virus was infected into cells by treatment with amniotic fluid-derived MSCs in an 80% confluence state for 6 hours.

Example 2: Verification of Nanog Expression of Nanog-Introduced Amniotic Fluid-Derived MSCs

[0060] For RT-PCR, total RNA was isolated from the Nanog-introduced amniotic fluid-derived MSCs using a TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer's instructions. 500 g of the isolated total RNA was reverse-transcribed with cDNA using a cDNA preparation mix (Bioneer). Test tubes were allowed to stand at 45 C. for 60 minutes and then at 95 C. for 5 minutes, and then stored at 20 C. until use. cDNA was amplified using a Taq polymerase (Promega) and primers specific to Nanog mRNA (Exo-Nanog foward: 5-GCTTGGATACACGCCGC-3 (SEQ ID NO: 2); and Exo-Nanog reverse: 5-GATTGTTCCAGGATTGGGTG-3 (SEQ ID NO: 3)). In the RT-PCR, a cycle of 94 C. for 20 seconds, 60 C. for 30 seconds, and 72 C. for 2 minutes was repeated 35 times, and then final synthesis proceeded at 72 C. for 10 minutes. The PCR products were electrophoresed on 1% agarose gel and analyzed.

[0061] For Western blot experiments to confirm Nanog overexpression, the Nanog-introduced amniotic fluid-derived MSCs were cultured in a DMEM containing 10% FBS to 100% confluency and then obtained, the cells were disrupted, and then 30 g of a protein-containing supernatant was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a nitrocellulose membrane, followed by blocking with 4% skim milk. Subsequently, the membrane was treated with an antibody (AF1997, R&D) for detecting Nanog receptors, as a primary antibody, cultured at 4 C. overnight, and washed with TBST (Tris-buffered saline (TBS) supplemented with 0.1% Tween 20). The membrane was treated with a goat-derived HRP antibody as a secondary antibody (Santa Cruz Biotechnology, USA) along with TBST containing 1% bovine serum albumin (BSA), and a reaction was allowed to occur therebetween for 1 hour, followed by western blotting. As a control for comparison in expression levels, the expression of -tubulin was confirmed using the same method as described above by using an anti--tubulin antibody (R&D, USA) as a primary antibody.

[0062] As a result, it was confirmed that the Nanog-introduced amniotic fluid-derived MSCs exhibited significant increases in an mRNA expression level of the exogenous Nanog gene and a protein expression level of Nanog, as compared to amniotic fluid-derived MSCs (see FIG. 1).

[0063] When the fetus-derived MSCs were cultured in DMEM low Glucose+10% fatal bovine serum+1% penicillin/streptomycin+1% non-essential amino acids+4 ng/ml basic FGF (bFGF)+5 ng/ml selenium+50 g/ml ascorbic acid), continuous cell growth occurred.

Example 3: Analysis of Exosomes in Conditioned Medium Derived from Nanog-Introduced Amniotic Fluid-Derived MSCs

[0064] A cell portion of each of the amniotic fluid-derived MSC lines established in Examples 1 and 2 was selected, 100 ml of a conditioned medium thereof was produced, and then exosomes were isolated therefrom.

[0065] The extraction of exosomes from the conditioned medium was performed using an Amicon Ultra-15 10K centrifugal filter device (Millipore), which was devised to filter certain molecular-weight proteins of the conditioned medium produced from the fetus-derived MSCs. 14.8 ml of the conditioned medium was placed in an upper cap of the device, and then centrifuged at 5,000g for 15 minutes to 40 minutes. 100 ml of the conditioned medium was concentrated in this manner, and then an exosome isolation kit (Invitrogen) was added to the concentrated medium in an amount of of the final volume and then mixed well. The completed samples were stored at 4 C. for 12 hours. Thereafter, each sample was centrifuged at 13,200 rpm for 30 minutes to remove the supernatant, and then the remaining white pellet was dissolved with PBS or DMEM, thereby completing the preparation of exosome samples.

[0066] As a result of measuring the average size and number of exosomes using Nanosight, the average sizes of amniotic fluid-derived MSCs and the Nanog-introduced amniotic fluid-derived MSCs were recorded as diameters of 156 nm and 147 nm, respectively, and the numbers thereof were measured as 20.010.sup.8 and 20.110.sup.8, respectively (see FIG. 2). That is, it was confirmed that the isolation of exosomes from the stem cell lines used in the experiments was normally carried out, and it was also possible to isolate the same amount of exosomes from the Nanog-introduced amniotic fluid-derived MSCs.

Example 4: Analysis of Substances in Exosomes

[0067] To analyze internal substances of the exosomes isolated in Example 3, first, the content of mRNA was examined by RT-PCR.

[0068] For qRT-PCR, RNA was obtained in the same manner as the RNA prep during RT-PCR, and cDNA was prepared in the same manner. The prepared cDNA was subjected to qRT-PCR through a CFX-96 PCR system, and was amplified using primers specific to mRNA of bFGF, IGF, Wnt7a, and PDGF-AA. The bFGF-specific primers used were bFGF forward: 5-CAGATTAGCGGACGCGGTGC-3 (SEQ ID NO: 4) and bFGF reverse: 5-TCACGGATGGGTGTCTCCGC-3 (SEQ ID NO: 5), the IGF-specific primers used were IGF forward: 5-CCATGTCCTCCTCGCATCTCTTCT-3 (SEQ ID NO: 6) and IGF reverse: 5-CCATACCCTGTGGGCTTGTTGAA-3 (SEQ ID NO: 7), the Wnt7a-specific primers used were Wnt7a forward: 5-TCTTTCTCAGCCTGGGCATGGT-3 (SEQ ID NO: 8) and Wnt7a reverse: 5-TCCTATGACGATGATGGCGTCG-3 (SEQ ID NO: 9), and the PDFG-AA-specific primers used were PDGF-AA forward: 5-CTGCCCATTCGGAGGAAGAGAA-3 (SEQ ID NO: 10) and PDGF-AA reverse: 5-TGGCACTTGACACTGCTCGTGTT-3 (SEQ ID NO: 11). Relative quantification of target mRNA was analyzed by a CT method.

[0069] To examine the secretion of bFGF, Wnt7a and PDGF-AA proteins, an enzyme-linked immunosorbent assay (ELISA assay) was performed on exosomes isolated from the Nanog-introduced amniotic fluid-derived MSCs. The contents of bFGF, Wnt7a, and PDGF-AA proteins in the respective exosomes were determined using an ELISA kit (RayBiotech). For the ELISA, exosomes were prepared, a standard and a sample were treated with biotin antibodies for 1 hour and then treated with streptavidin for 45 minutes. Subsequently, the standard and sample were treated with a TMB substrate reagent for 30 minutes, and then a stop solution was added thereto to stop the reaction. The results were quantitatively analyzed by measuring absorbance at 450 nm using a microplate spectrophotometer.

[0070] As a result of comparing the expression levels of hair growth-related genes through RT-PCR, the Nanog-introduced amniotic fluid-derived MSC exosomes exhibited higher expression levels for bFGF, PDGF, and IGF except for Wnt7a. This indicates that the number of exosomes from the Nanog-introduced amniotic fluid-derived MSCs was similar to that of existing stem cells, but there was a slight difference in internal substances of the exosomes between the two cases. To more accurately examine RT-PCR data, quantitative analysis was performed through qRT-PCR. Similar to the case of RT-PCR, it was confirmed that mRNA levels of the other growth factors except for Wnt7a were considerable (see FIG. 3). In conclusion, mRNA of hair growth-related factors is included in exosomes, which may be interpreted as aiding in promoting hair growth. The amounts of the proteins were analyzed by ELISA, and the concentrations of Wnt7a, PDGF-AA, and bFGF were measured (see FIG. 4). According to measurement results, the contents of Wnt7a, PDGF-AA, and bFGF were about 8 pg/mL, 2.5 pg/mL, and about 5.5 pg/mL, respectively. From the results, it was confirmed that the hair growth-related proteins were also included in the exosomes.

Example 5: Growth and Marker Changes of Dermal Papilla Cells Through Exosome Treatment

[0071] After the obtained exosomes were divided into various concentrations, dermal papilla cells were treated therewith to confirm two factors that play a crucial role in hair generation, i.e., the growth of dermal papilla cells and changes in hair growth markers. Concentrations used ranged from 100 to 1 and comparative analysis was carried out using a conditioned medium and an exosome-free conditioned medium.

[0072] Specifically, skin tissues of mice were collected and cells were isolated therefrom, total RNA was isolated from the cells using the same method as that used in Example 2 and reverse-transcribed with cDNA, and cDNA was amplified using a Taq polymerase (Promega) and primers specific to mRNA of ALP, LEF, and Versican. The ALP-specific primers used were ALP forward: 5-TGGCCCTCTCCAAGACGTACAA-3 (SEQ ID NO: 12) and ALP reverse: 5-TGGTTCACTCTCGTGGTGGTCA-3 (SEQ ID NO: 13), the LEF-specific primers used were LEF forward: 5-CTTCCTTGGTGAACGAGTCTG-3 (SEQ ID NO: 14) and LEF reverse: 5-GTGTTCTCTGGCCTTGTCGT-3 (SEQ ID NO: 15), and the Versican-specific primers used were Versican forward: 5-AACTAGCCGTTGGAGTGGATTC-3 (SEQ ID NO: 16) and Versican reverse: 5-AAATGCTCTGTGGCTCTGGA-3 (SEQ ID NO: 17). The resulting product was analyzed by electrophoresis on 1% agarose gel, and a relative mRNA expression level was quantified based on GAPDH mRNA by CT value-based analysis.

[0073] According to the results, the growth of dermal papilla cells was gradually increased as the concentration was increased, and a much better growth promotion ability was exhibited at a certain concentration or higher, compared to that in a conditioned medium. In addition, it was confirmed that the exosome-removed conditioned medium exhibited significantly reduced functionality, which suggests that the functions of exosomes account for a large part also in the conditioned medium (see FIG. 5).

[0074] In addition to the growth of dermal papillae, changes in hair growth markers were confirmed through qRT-PCR and AP staining. In order for the dermal papilla cells to induce hair growth, it is necessary to maintain intrinsic sternness of dermal papilla cells, and ALP, LEF, and Versican are typically known as markers representing this sternness. Thus, as a result of distinguishing the expression levels of these markers in an exosome-free medium (Control) and various concentrations (10 and 50), the expression levels of ALP and LEF were confirmed to be increased 1.5-fold to 2-fold (see upper side of FIG. 6). In the case of Versican, it seemed to be a small increase but not much different. To obtain reliability of this data, AP staining for confirming ALP by staining was carried out, and it was confirmed that, similar to the case of qRT-PCR, the number of cells stained blue was increased as the concentration of exosomes was increased (see lower side of FIG. 6).

[0075] Additionally, since dermal papilla cells are also known to have some degree of functionality as mesenchymal stem cells, they express the common stem cell markers Oct4 and Sox2. As a result of confirming changes in general stem cell markers, a gradual increase was exhibited, as with ALP and LEF (see FIG. 7). In particular, Oct4 exhibited a 5-fold or higher difference, which may be interpreted that exosomes have a great influence on Oct4 expression.

[0076] From these results, it may be determined that exosomes have an efficient effect on hair growth by promoting a hair growth ability of dermal papilla cells.

<Preparation Example 1> Preparation of Hair Lotion

[0077] According to a conventional hair lotion preparation method, a hair lotion including, as an active ingredient, exosomes in a conditioned medium of Nanog-introduced amniotic fluid-derived MSCs of the present invention was prepared as shown in Table 1.

TABLE-US-00001 TABLE 1 Ingredients to be mixed Content (wt %) Exosomes isolated from Nanog- 5.0 introduced amniotic fluid-derived MSCs resorcinol 2.0 Panthenol 0.5 Pyrrothonol amine 0.1 Pigment Appropriate amount Fragrance Appropriate amount Purified water Balance Total 100

<Preparation Example 2> Preparation of Hydrophilic Ointment

[0078] According to a conventional hydrophilic ointment preparation method, a hydrophilic ointment including, as an active ingredient, exosomes in a conditioned medium of Nanog-introduced amniotic fluid-derived MSCs of the present invention was prepared as shown in Table 2.

TABLE-US-00002 TABLE 2 Ingredients to be mixed Content (wt %) Exosomes isolated from Nanog- 0.5 introduced amniotic fluid-derived MSCs Ethyl (or methyl) p-oxybenzoate 0.25 Lauryl sodium sulfate 15 Propyl p-oxybenzoate 0.15 White vaseline Appropriate amount Stearyl alcohol Appropriate amount Propylene glycol Balance Total 100