Composition for improving, preventing or treating skin diseases comprising induced pluripotent stem cell-derived mesenchymal stem cell and exosome derived therefrom

Abstract

The present invention may provide a pharmaceutical composition for preventing or treating skin diseases, a cosmetic composition for preventing or improving skin diseases, and a stem cell therapeutic agent, which comprise an induced pluripotent stem cell-derived mesenchymal stem cell (iMSC), a culture thereof, or an exosome isolated from an iMSC or iMSC culture as active ingredients. When the composition of the present invention, etc. is used, it is possible to provide a composition for improving, preventing or treating skin diseases and a stem cell therapeutic agent which have an improved immunomodulating function over conventional mesenchymal stem cells.

Claims

1. A method for treating or alleviating a skin disease in a subject in need thereof, comprising: administering to the subject a composition comprising an effective amount of one or more exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells or a culture thereof, wherein the skin disease is selected from the group consisting of atopic dermatitis and contact dermatitis.

2. The method of claim 1, wherein the subject is a mammal or a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically shows the preparation of induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) and exosomes derived from the mesenchymal stem cells (iMSC-exo).

(2) FIG. 2 shows analysis results of surface antigens on MSC, iPSC, and iMSC.

(3) FIG. 3 shows expression levels of IDO in MSC and iMSC.

(4) FIG. 4 shows levels of cytokine secretion from MSC and iMSC.

(5) FIG. 5 shows sizes of exosomes isolated from mesenchymal stem cells (MSC-Exo) and from induced pluripotent stem cell-derived mesenchymal stem cells (iMSC-exo).

(6) FIG. 6 shows the expression of CD63 and CD9 in MSC-Exo and iMSC-exo.

(7) FIG. 7 shows electron microscope images of MSC-Exo and iMSC-Exo.

(8) FIG. 8 is a scheme for constructing an atopic dermatitis animal model.

(9) FIG. 9 shows effects of iMSC and iMSC-exo on atopic lesions of atopic dermatitis animal models.

(10) FIG. 10 shows skin clinical scores resulting from iMSC and iMSC-exo treatment in atopic dermatitis animal models.

(11) FIG. 11 shows the transepidermal water loss resulting from iMSC and iMSC-exo treatment in atopic dermatitis animal models.

(12) FIG. 12 shows skin histological changes by iMSC and iMSC-exo treatment in atopic dermatitis animal models. The scale bars mean 200 μm.

(13) FIG. 13 shows serum IgE levels by iMSC and iMSC-exo treatment in atopic dermatitis animal models.

(14) FIG. 14 shows levels of IL-17a secretion from lymphocytes by iMSC and iMSC-exo treatment in atopic dermatitis animal models.

DETAILED DESCRIPTION

(15) Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it would be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

(16) Unless otherwise stated, the “%” used to express the concentration of a specific material refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid throughout the present specification.

Example 1

Preparation and Assay of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cell

(17) Induced pluripotent stem cells (induced pluripotent stem cells (iPSC) originated from fibroblasts, peripheral blood mononuclear cells (PBMC), and mesenchymal stem cells (MSC), established by Asan Medical Center (AMC, Stem Cell Center, Seoul), which were cultured in DMEM/F-12 supplemented with Knockout xeno-free serum replacement, glutamax, non-essential amino acids, beta-mercaptoethanol, antibiotic, and basic fibroblast growth factor (bFGF) without the use of feeder cells, were allowed to adhere to culture dishes previously coated with vitronectin and then induced to differentiate into mesenchymal stem cells in a DMEM medium supplemented with 10% FBS (v/v), 5 ng/ml basic FGF, 0.1 mM MEM NEAA (Minimum Essential Media Non-Essential Amino Acids), β-mercaptoethanol (1×), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37° C. On day 7 of culturing, the cells were dissociated into single cells by treatment with TrypLE express (1×), transferred onto cell culture plates, and then further incubated for 7 days. While the medium was freshly exchanged every two days, an observation was made of the differentiation into mesenchymal stem cells (iMSC) with a flat and elongated appearance (FIG. 1).

(18) After completion of the induction of differentiation into mesenchymal stem cells (iMSC) for 14 days, the cells were assayed for the negative expression of the mesenchymal stem cell-specific surface antigen markers CD34 (BD Biosciences, Catalog No.: 348053) and SSEA4(BD Biosciences, Catalog No.: 560128) and the positive expression of the mesenchymal stem cell-specific surface antigen markers CD73 (BD Biosciences, Catalog No.: 550257) and CD105 (BD Biosciences, Catalog No.: 560839), using flow cytometry. As a result, the iMSC was found to have typical characteristics of mesenchymal stem cells (MSC), that is, negative for CD34 and SSEA4 and positive for CD73 and CD105 (FIG. 2). The mesenchymal stem cells isolated from fetal umbilical tissues and established by AMC Stem Cell Center were used as a control.

(19) In addition, indoleamine 2,3-dioxygenase (IDO) in MSC and iMSC was quantitated using real-time PCR. A higher expression level of IDO was detected in iMSC than MSC (FIG. 3).

(20) TABLE-US-00001 TABLE 1 Sequences of primers for detection of  indoleamine 2,3-dioxygenase (IDO) Forward primer 5′-GCCCTTCAAGTGTTTCACCAA-3′ (SEQ ID NO: 1)) Reverse primer 5′-GCCTTTCCAGCCAGACAAATAT-3′ (SEQ ID NO: 2)

(21) Indoleamine 2,3-dioxygenase, which is an enzyme converting tryptophan into kynurenine, is known to suppress inflammation and immune responses by depleting tryptophan in the periphery of cells and inhibiting the proliferation of immune cells. It is thus understood that the iMSC of the present invention with high IDO expression has high inhibitory activity against immune responses.

(22) In addition, the cultures of MSC and iMSCs were separately collected to measure and compare levels of secretion of inflammation-related cytokine proteins therebetween.

(23) Inflammation-related cytokines were measured according to Magnetic Luminex® Screening Assay. First, cultures of MSCs and iMSCs were vortexed and then centrifuged, and the supernatants were ½ diluted with a diluent. Each of the samples thus prepared was incubated with the bead-Ab mixture at room temperature for 2 hours. After completion of the reaction, the cytokines were measured by the Luminex instrument (Luminex, Austin, Tex., USA).

(24) The analytes in the sample reacted with corresponding antibodies attached to particularly numbered beads, respectively and independently while the detection antibody reacted with 2′(streptavidin-PE) (sandwich assay). For the assay results, the fluorescence level of phycoerythrin (PE) attached to the corresponding bead surface in proportion to the amount of each analyte was measured. While flowing the beads one by one, the Luminex instrument checked the bead number and measured the PE fluorescence intensity of the bead surface to obtain a reaction result value (MFI) of each analyte. A standard curve was made by the best fit method in the calculation software “MasterPlex QT 2010 (MiraiBio, Hitachi, Calif., USA)” from the reaction measurement value (MFI) for each standard concentration, and on the basis of the standard curve, the resultant concentration value of the corresponding sample was calculated by reflecting the dilution factor.

(25) As a result, iMSC secreted IFN-γ, TNF-α, IL-1β, IL-6, and IL-6Rα at low levels, compared to MSC (FIG. 4).

(26) From the results, it is understood that the induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) of the present invention are novel mesenchymal stem cells having cytological (immunological) traits distinguished from those of existing MSC.

Example 2

Isolation and Verification of Exosomes from Induced Pluripotent Stem Cell-Differentiated Mesenchymal Stem Cells

(27) iMSC identified to have traits of mesenchymal stem cells was additionally cultured in a culture medium supplemented with 10% exosome-depleted FBS. After incubation for 72 hours, the culture of iMSC was collected and centrifuged at 300×g for 10 minutes to remove cells and cell debris. The supernatant was filtered using a 0.22-μm filter, and then centrifuged at 10000×g for 70 minutes at 4° C. in a high-speed centrifuge. The supernatant thus obtained was centrifuged at 100,000×g and 4° C. for 90 minutes in an ultracentrifuge to obtain exosomes as a pellet. The exosomes were diluted in phosphate buffered saline (PBS) before use.

(28) The exosomes respectively isolated from the cultures of MSC and iMSC were counted and analyzed for size distribution by using nanoparticle tracking assay (NanoSight NS300, Malvern) (FIG. 5). The expression of CD9 and CD63, which are exosome-specific surface antigens, was verified by western blotting (FIG. 6) and morphological observation was made of the exosomes under an electron microscope (FIG. 7).

(29) It could be therefore confirmed that the exosomes respectively derived from MSC and the iMSC of the present invention had the traits of exosomes themselves.

Example 3

Construction of Atopic Disease-Induced Mice and Administration Thereto

(30) As experimental animals, 8-week-old BALB/c female mice were purchased (Orient Bio, South Korea) and acclimated for 1 week before use. For the induction of atopic dermatitis, the backs of the BALB/c mice at 9 weeks of age were shaved to the upper part as much as possible by a shaver. An Aspergillus fumigates (Af) extract (40 μg) was applied to the shaved dorsal skin tissue (1×1 cm.sup.2) at intervals of 24 hours for 5 days. After a 2-week rest period, the extract was repeatedly applied five times at intervals of 24 hours from day 19, to thereby establish atopic dermatitis animal models.

(31) After establishment of atopic dermatitis animal models, MSC, iMSC, MSC-Exo (exosomes from mesenchymal stem cells) or iMSC-Exo (exosomes from induced pluripotent stem cell-derived mesenchymal stem cell) was subcutaneously injected. MSC or iMSC was injected at a dose of 2×10.sup.6 cells per animal, and MSC-exo or iMSC-exo was injected at a dose of 12 μg per animal. The mice were sacrificed and analyzed on day 5 after the injection (FIG. 8).

Example 4

Evaluation of Therapeutic Effect of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cell (iMSC) and Exosome from Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cell (iMSC-Exo) on Atopic Dermatitis

(32) (1) Atopic Symptom Alleviation

(33) To investigate the atopy alleviation levels of iMSC and iMSC-exo, a negative control group without atopic dermatitis (CONTROL, physiological saline treated), a positive control group with atopic dermatitis (AD, Af-treated), an MSC-treated group, an MSC-Exo-treated group, an iMSC-treated group, and an iMSC-Exo-treated group were observed and compared. As a result, the iMSC-treated group and the iMSC-Exo-treated group showed an atopy alleviation effect at similar levels, compared with the negative control group (FIG. 9).

(34) The data demonstrate that iMSC and iMSC-Exo of the present invention have excellent alleviative or therapeutic effects on skin disease including atopic dermatitis.

(35) (2) Skin Clinical Scores and Transepidermal Water Loss Evaluation

(36) Skin clinical scores were set according to five items: dryness, scaling, erosion, excoriation, and hemorrhage. In each item, scores were assigned: 0 point for lesion-free condition; 1 point for a mild condition, 2 points for a moderate condition; and 3 points for a severe condition. The corresponding points were summed up to calculate a skin clinical score. As a result, the iMSC-treated group and the iMSC-exo-treated group showed lower skin clinical scores compared with the positive control group (AD), the MSC-treated group, or the iMSC-exo-treated group (Table 2 and FIG. 10).

(37) TABLE-US-00002 TABLE 2 Skin clinical scores CONTROL AD MSC MSC-exo iMSC iMSC-exo 0.1 4.1 1.6 2.1 1.6 1.8

(38) To evaluate skin barrier damage in the same administration groups, the transepidermal water loss (TEWL) was measured using VapometerSWL-3® (Delfin technologies). As understood from the data, the iMSC-treated group and the iMSC-Exo-treated group showed remarkably reduced levels of transepidermal water loss (TEWL), compared with the positive control (AD) the negative control (CONTROL), the MSC-treated group, and the MSC-Exo-treated group, indicating the improvement in atopic symptoms (Table 3 and FIG. 11).

(39) TABLE-US-00003 TABLE 3 Transepidermal water loss (TEWL) levels CONTROL AD MSC MSC-Exo iMSC iMSC-Exo 3.4 13.3 4.2 4.4 3.6 3.5

(40) Therefore, the iMSCs and iMSC-Exo of the present invention alleviate skin clinical symptoms and significantly reduce transepidermal water loss levels, thereby showing excellent effects of alleviating or treating of skin diseases including atopic dermatitis.

(41) (3) Skin Histological Observation

(42) Skin tissues were isolated from the negative control group, the positive control group, the MSC-treated group, the MSC-Exo-treated group, the iMSC-treated group, and the iMSC-Exo-treated group. The isolated skin tissues were fixed with 10% formalin solution, embedded in paraffin, and then cut into sections 5 μm thick. To investigate skin histological changes and inflammatory cell invasion, the skin sections were stained with hematoxylin and eosin and observed under a microscope at 400× magnification. As a result, stratum corneum damage and the thickness of epidermal and dermal layers were reduced in the iMSC-treated group and the iMSC-Exo-treated group, compared with the positive control group (FIG. 12).

(43) The data demonstrate that the iMSC and iMSC-Exo of the present invention alleviate stratum corneum damage and reduce the thickness of epidermal and dermal layers, thereby showing excellent effects of alleviating or treating skin diseases including atopic dermatitis.

(44) (4) Serum IgE Measurement

(45) Mice in the negative control group, the positive control group, the MSC-treated group, the MSC-Exo-treated group, the iMSC-treated group, and the iMSC-Exo-treated group were each cut to open the abdomen, and about 0.5-0.7 mL of blood was collected from the postcaval vein after needle insertion. Sera was separated from the isolated blood by centrifugation. The total IgE level in each separated serum was measured using an ELISA test kit (eBioscience). The serum IgE level was reduced in the iMSC-treated group, compared with the positive control group, but with no significant changes in the iMSC-Exo-treated group (FIG. 13).

(46) It is understood from the above results and clinical symptom improvement effects in the above-described Examples that the skin disease alleviating efficacy of the iMSC, and iMSC-Exo of the present invention is not mainly medicated by IgE.

(47) (5) Verification of T Cell Immune Response

(48) To investigate T cell immune responses of iMSC and iMSC-Exo, lymphocytes were isolated from lymph nodes of the mice in the negative control group, the positive control group, the iMSC-treated group, and the iMSC-exo-treated group. After the isolated lymphocytes were stimulated by CD3/CD28, the cultures thereof were collected. Cytokine secretion was measured using an ELISA kit (eBioscience). As for IL-17A, a main cytokine produced in Th17, its expression levels in the iMSC-treated group and the iMSC-Exo-treated group were similar to that in the negative control, but significantly lowered compared to the positive control (FIG. 14).

(49) It is inferred on the basis of the above results and the clinical symptom improvement effects and serum IgE level changes in the above-described Examples that the skin disease alleviation efficacy of the iMSC and iMSC-Exo of the present invention was not mainly mediated by IgE, but by inhibiting immune responses of T cells, such as IL-17A.

(50) This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e).