Patent Application
20220347225
The present disclosure relates to a composition for improving skin condition including skin whitening, which includes stem cell-derived exosomes isolated from skeletal muscle, specifically from orbicularis oculi muscle, as active ingredients.
Orbicularis oculi muscle-derived stem cells (ORM-SCs) have self-renewal, proliferation and multi-lineage differentiation functions [1]. The stem cells located at the junctions of most organs are responsible for the maintenance and recovery of the organs throughout life [2]. According to recent researches, it was reported that the stem cells abundant in the ORM can differentiate into various types (fat, cartilage and bone cells) [3-5].
Ideal eyelid reconstruction should give natural appearance [6]. At present, upper eyelid plastic surgery is the most commonly performed cosmetic ophthalmic surgery in Korea. During the surgery, some of ORM tissues are removed and wasted. Recently, a few surgeons used local flaps instead of skin grafting or Z-plasty for treatment of lagophthalmos. The ORM tissue is used to treat the facial skin defects in the cheek, nose or lower eyelids [7, 8]. For facial burns, the ORM tissue plays an important role in transplantation and recovery [9].
ORM is a very specialized skeletal muscle and can be a good stem cell source owing to active vascularization and excellent accessibility [10]. In general, a sufficient number of ORM-SCs for invasive biopsy processes cannot be obtained because the amount of muscular tissues is limited.
The effect of these cells on tissue regeneration and recovery mostly derives from the factors (secretomes) secreted by them [11]. SC-derived extracellular vesicles (EVs) function usefully as mediators of skin regeneration and reconstruction [12].
Stromal cells and epithelial melanocytes play an important role in regulation of skin pigmentation [13].
The inventors of the present disclosure have isolated ORM-SCs having self-renewal, proliferation and multi-lineage differentiation abilities from orbicularis oculi muscle and have identified that EVs isolated from the ORM-SCs have an important role in regulating pigmentation and inhibiting melanin synthesis.
Throughout the present specification, many literatures and patent documents are cited. The disclosures of the cited literatures and patent documents are incorporated herein by reference in their entirety to describe the level of the art to which the present disclosure belongs and the contents of the present disclosure more clearly.
The inventors of the present disclosure have made consistent efforts to develop skin whitening materials derived from natural products, which have various skin-improving effects such as inhibition of pigmentation, skin tissue regeneration, prevention of aging, etc. without side effects and can be obtained easily. As a result, they have found out that extracellular vesicles secreted by stem cells isolated from skeletal muscle, particularly orbicularis oculi muscle tissue, which have been discarded as byproducts of upper eyelid plastic surgery, not only significantly inhibit melanocyte proliferation, tyrosinase activity and melanogenesis but also induce skin regeneration, wound healing, scar reduction and skin whitening by facilitating collagen secretion, reducing reactive oxygen species and promoting re-epithelialization at wound site, thereby delaying aging, and have completed the present disclosure.
The present disclosure is directed to providing a cosmetic composition for improving skin condition through antioxidation or skin whitening.
The present disclosure is also directed to providing a pharmaceutical composition for preventing or treating a hyperpigmentation disease.
Other purposes and advantages of the present disclosure will become more apparent by the detailed description, claims and drawings of the present disclosure.
In an aspect, the present disclosure provides a cosmetic composition for antioxidation or improvement of skin condition, which contains extracellular vesicles isolated from skeletal muscle-derived stem cells as active ingredients, wherein the improvement of skin condition is selected from a group consisting of skin aging delay, wound healing, scar reduction and skin whitening.
The inventors of the present disclosure have made consistent efforts to develop skin whitening materials derived from natural products, which have various skin-improving effects such as inhibition of pigmentation, skin tissue regeneration, prevention of aging, etc. without side effects and can be obtained easily. As a result, they have found out that extracellular vesicles secreted by stem cells isolated from skeletal muscle, particularly orbicularis oculi muscle tissue, which have been discarded as byproducts of upper eyelid plastic surgery, not only significantly inhibit melanocyte proliferation, tyrosinase activity and melanogenesis but also delay aging caused by oxidative stress by promoting collagen secretion, reducing reactive oxygen species and facilitating re-epithelialization at wound site and induce scar reduction and skin whitening, and thus can be used for a composition for improving skin condition.
In the present specification, the term “stem cell” refers to a cell capable of differentiating into various cell types constituting biological tissues. It refers to a multipotent or pluripotent cell that can regenerate unlimitedly to form specialized cells of tissues and organs. Skeletal muscle-derived stem cells can be obtained by selectively isolating the cells having stemness from a population of inhomogeneous cells obtained form skeletal muscle tissue or a culture thereof according to a common method.
In the present specification, the term “skeletal muscle” refers to a striated muscle tissue which is attached to a bone tissue through a tendon composed of collagen fibers and is controlled voluntarily by the somatic nervous system. The skeletal muscle that may be used in the present disclosure is specifically facial skeletal muscle, more specifically a skeletal muscle around eyes selected from a group consisting of levator palpebrae superioris muscle, corrugator supercilii muscle, depressor supercilii muscle and orbicularis oculi muscle.
Most specifically, the skeletal muscle used in the present disclosure is orbicularis oculi muscle.
In the present specification, the term “isolation” includes not only a process of selectively obtaining a desired substance (e.g., a stem cell or an extracellular vesicle) from a biological sample (e.g., a tissue or a cell culture) (positive isolation) but also a process of selectively removing impurities other than the desired substance (negative isolation). Accordingly, the term “isolation” is used with the same meaning as “obtainment”, “extraction” or “purification”. In the present disclosure, a process of isolating a stem cell from a skeletal muscle tissue includes, for example, separation of an antibody specific for a surface antigen of a target cell (e.g., FACS) separation based on the difference in the specific gravity of a target cell (e.g., phase separation or centrifugation). However, any method commonly used in the art for isolating a specific cell from an inhomogeneous sample may be used without being limited thereto.
In addition, the process of isolating extracellular vesicles from the stem cells includes separation of components in a cell culture based on the difference in specific gravity (e.g., ultrafiltration), separation based on size (e.g., ultracentrifugation or vacuum filtration) and separation based on affinity to a specific substrate (e.g., affinity chromatography). However, any method commonly used in the art for a target substance from an inhomogeneous sample based on the intrinsic physical properties of a target substance may be used without being limited thereto.
In the present specification, the term “extracellular vesicle” refers to a vesicle with a lipid bilayer structure secreted naturally by various cells, which includes specific molecules possessed by a cell from which it is derived, such as a protein, a nucleic acid, a lipid, a carbohydrate, etc. and is released to the extracellular environment through fusion of a multivesicular body with the plasma membrane. The extracellular vesicle has various diameters in a range of approximately 30-1,000 nm. In particular, an exosome is a small membrane vesicle with the smallest size of 50-200 nm.
In a specific exemplary embodiment of the present disclosure, the extracellular vesicle used in the present disclosure has a diameter of 50-200 nm, i.e., an exosome. More specifically, it has a diameter of 70-180 nm. Further more specifically, it has a diameter of 90-160 nm. Even more specifically, it has a diameter of 100-140 nm. Most specifically, it has a diameter of 100-120 nm.
In a specific exemplary embodiment of the present disclosure, the extracellular vesicle is contained in the composition of the present disclosure at a concentration of 5-100 μg/mL, more specifically 10-100 μg/mL, further more specifically 30-100 μg/mL, most specifically 50-100 μg/mL.
In a specific exemplary embodiment of the present disclosure, the stem cell of the present disclosure is positive for three or markers, more specifically four or markers, further more specifically five or markers, most specifically all the six markers, selected from a group consisting of CD105, CD90, CD73, ITGA6 (integrin alpha-6), CD146 and TM4SF1 (transmembrane 4 L6 family member 1).
Also, the stem cell of the present disclosure is negative for CD45 or CD34, specifically both CD45 and CD34.
In a specific exemplary embodiment of the present disclosure, the composition of the present disclosure decreases the activity or expression level of β-galactosidase (β-Gal). In the present specification, the term “decrease of activity or expression level” refers to the decrease of the expression level or intrinsic function of β-Gal in vivo such that cellular aging induced by SA-β-Gal can be inhibited or delayed to a detectable level. The decrease of activity includes not only the simple decrease of function but also the ultimate inhibition of activity caused by decreased stability. It may refer to a state where the activity or expression level has decreased specifically by 20% or more, more specifically by 40% or more, further more specifically by 60% or more, as compared to a control group.
In a specific exemplary embodiment of the present disclosure, the composition of the present disclosure induces collagen synthesis. In an example of the present disclosure, it was confirmed that, when the composition of the present disclosure is administered to a skin site where wound has been induced, the wound site is filled quickly as collagen synthesis is increased remarkably.
In a specific exemplary embodiment of the present disclosure, the composition of the present disclosure reduces reactive oxygen species (ROS) in cells. In an example of the present disclosure, it was observed that the amount of ROS accumulated in the cells treated with the composition of the present disclosure was less than 50% as compared to an untreated control group, confirming that the composition of the present disclosure can effectively protect cells, tissues and a subject from oxidative stress.
The present disclosure also provides a method for preventing oxidation or improving skin condition, which includes a step of administering the cosmetic composition of the present disclosure described above.
When the composition of the present disclosure is prepared as a cosmetic composition, it may contain an ingredient commonly used in a cosmetic composition, e.g., a common adjuvant such as a stabilizer, a solubilizer, a vitamin, a pigment and a fragrance, and a carrier in addition to the extracellular vesicle as an active ingredient.
The cosmetic composition of the present disclosure may be prepared into any formulation commonly prepared in the art. For example, it may be formulated into a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a spray, etc., although not being limited thereto. More specifically, it may be formulated into a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.
When the formulation of the present disclosure is a paste, a cream or a gel, an animal oil, a plant oil, a wax, paraffin, starch, tragacanth, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as a carrier ingredient.
When the formulation of the present disclosure is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier ingredient. In particular, a spray may further contain a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.
When the formulation of the present disclosure is a solution or an emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier ingredient. Examples include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, an aliphatic ester of glycerol, polyethylene glycol or an aliphatic ester of sorbitan.
When the formulation of the present disclosure is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending gent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc. may be used as a carrier ingredient.
In a specific exemplary embodiment of the present disclosure, the stem cells of the present disclosure are obtained by:
(a) a step of treating an isolated human skeletal muscle tissue with a collagenase and then culturing the same;
(b) a step of centrifuging the culture of the step (a), and collecting and suspension culturing pellets; and
(c) a step of seeding the cells in the suspension culture of the step (b) on a culture dish.
According to the present disclosure, the skeletal muscle-derived stem cells of the present disclosure are isolated from skeletal muscle tissue, specifically orbicularis oculi muscle tissue, isolated from the human body by enzymatic digestion. Before treating with the collagenase, the isolated tissue may be cut into a regular size after removing blood and blood vessels. Then, after collecting pellets including stem cells through centrifugation and suspension culturing the same, the stem cells were seeded on a culture dish and then selected while observing the adhesion and morphology of the cells. The culture dish may be a gelatin-coated culture dish.
More specifically, the collagenase may be type II collagenase.
In a specific exemplary embodiment of the present disclosure, the extracellular vesicles of the present disclosure are obtained by:
(a) a step of culturing the skeletal muscle-derived stem cells;
(b) a step of collecting the culture of the step (a) and removing the stem cells through centrifugation;
(c) a step of filtering a supernatant obtained through the centrifugation with a vacuum filter; and
(d) a step of ultrafiltering the filtrate.
More specifically, the vacuum filter has a cut-off value of 0.15-0.3 μm. Further more specifically, it has a cut-off value of 0.18-0.25 μm. Most specifically, it has a cut-off value of 0.22 μm.
In the present specification, the term “ultrafiltration” refers to membrane-based separation by which the components constituting a non-homogeneous mixture solution are separated through a semipermeable membrane according to pressure or concentration gradient. An ultrafiltration membrane has pores with a predetermined cut-off value. In a specific exemplary embodiment of the present disclosure, the ultrafiltration is performed using a membrane having a cut-off value of 5-15 kDa, more specifically 10 kDa.
In the present disclosure, the vacuum filtration and the ultrafiltration may be performed either in order or in reverse order.
In another aspect, the present disclosure provides a pharmaceutical composition for preventing or treating a hyperpigmentation disease, which contains extracellular vesicles isolated from skeletal muscle-derived stem cells as active ingredients.
In another aspect, the present disclosure provides a method for preventing or treating a hyperpigmentation disease, which includes a step of administering the composition described above to a subject.
The detailed description of the skeletal muscle-derived stem cells and the extracellular vesicles isolated therefrom used in the present disclosure will be omitted to avoid redundancy because they were described in detail above.
In the present specification, the term “prevention” refers to suppression of the onset of a disease or a disorder in a subject with the risk of the disease or disorder although the subject has not been diagnosed as having the disease or disorder.
In the present specification, the term “treatment” refers to: (a) suppression of the progress of a disease, a disorder or a symptom; (b) amelioration of a disease, a disorder or a symptom; or (c) removal of a disease, a disorder or a symptom. When the composition of the present disclosure is administered to a subject, it suppresses, removes or ameliorates of the progress of a symptom related with hyperpigmentation by inhibiting melanocyte proliferation, tyrosinase activity and melanogenesis. Accordingly, the composition of the present disclosure may be used as a composition for treating a disease on its own, or may be administered as a therapeutic adjuvant for the disease together with another pharmacological ingredient. Therefore, in the present specification, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic support” or “therapeutic adjuvant”.
Because the composition of the present disclosure alleviates or reversibly restores skin pigmentation, the term “pharmaceutical composition for preventing or treating a hyperpigmentation disease” used in the present specification has the same meaning as “a pharmaceutical composition for skin whitening”.
In another aspect, the present disclosure provides a pharmaceutical composition for healing skin wound or reducing scar, which contains extracellular vesicles isolated from skeletal muscle-derived stem cells as active ingredients.
In another aspect, the present disclosure provides a method for healing skin wound or reducing scar, which includes a step of administering the composition described above to a subject.
In another aspect, the present disclosure provides a pharmaceutical composition for inhibiting skin aging, which contains extracellular vesicles isolated from skeletal muscle-derived stem cells as active ingredients.
In another aspect, the present disclosure provides a method for suppressing skin aging, which includes a step of administering the composition described above to a subject.
In the present specification, the term “administration” or “administer” refers to administration of a therapeutically effective amount of the composition of the present disclosure directly to a subject such that the same amount is formed in the body of the subject.
In the present disclosure, the term “therapeutically effective amount” refers to an amount of the pharmaceutical composition of the present disclosure wherein the content of the pharmacological ingredient (e.g., exosomes) in the composition is sufficient to provide therapeutic or prophylactic effect for a subject to which the composition is to be administered, and thus includes the meaning of “prophylactically effective amount”.
In the present specification, the term “subject” includes human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey without limitation. Specifically, the subject of the present disclosure is human.
When the composition of the present disclosure is prepared as a pharmaceutical composition, the pharmaceutical composition of the present disclosure contains a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present disclosure includes one commonly used for preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., although not being limited thereto. The pharmaceutical composition of the present disclosure may further contain a lubricant, a wetting agent, a sweetener, a flavorant, an emulsifier, a suspending agent, a preservative, etc. in addition to the above-described ingredients. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present disclosure may be administered orally or parenterally. Specifically, it may be administered parenterally, more specifically subcutaneously or transdermally.
An appropriate administration dosage of the pharmaceutical composition of the present disclosure may be determined variously depending on such factors as preparation method, mode of administration, the age, body weight, sex, pathological condition or diet of a patient, administration time, administration route, excretion rate and response sensitivity. A preferred administration dosage of the pharmaceutical composition of the present disclosure is within a range of 0.001 to 100 mg/kg for an adult.
The pharmaceutical composition of the present disclosure may be prepared as a single-dose or multiple-dose forms using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those having ordinary knowledge in the art to which the present disclosure belongs. It may be formulated as a solution in an oily or aqueous medium, a suspension, a syrup, an emulsion, an extract, a dust, a powder, a granule, a tablet or a capsule, and may further contain a dispersant or a stabilizer.
The features and advantages of the present disclosure may be summarized as follows:
(a) The present disclosure provides a composition for improving skin condition having the effect of skin aging delay, wound healing, scar reduction and skin whitening, which contains extracellular vesicles isolated from skeletal muscle-derived stem cells as active ingredients.
(b) The present disclosure can not only utilize tissues wasted after surgical operation, thus allowing easy supply of raw materials, but also takes advantage of natural substances, thereby being free from the risk of side effects even upon long-term administration.
(c) The composition of the present disclosure promotes the secretion of collagen, reduces reactive oxygen species and promotes re-epithelialization at wound site while remarkably inhibiting melanocyte proliferation, tyrosinase activity and melanogenesis. Accordingly, it can facilitate effective recovery from skin tissue damage caused by oxidative stress, physical wound, intrinsic aging at cell or tissue levels, or hyperpigmentation.
Hereinafter, the present disclosure is described in more detail through examples. The following examples are provided only to illustrate the present disclosure more specifically, and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.
Isolation of ORM-SCs from Human Tissue
Human ORM tissue (diameter 1 cm, weight 0.5 g) was acquired from Konkuk University Hospital. ORM-SCs were isolated by enzymatic digestion. Briefly, the tissue was washed twice with PBS and blood and blood vessels were removed to prevent contamination. Then, the tissue was incised with a knife and incubated with type II collagenase at 37° C. for 1 hour while stirring for 30 minutes with 5-minute intervals. After the incubation, the digested tissue was centrifuged twice at 1,500 rpm for 10 minutes to remove the remaining collagenase. Then, after suspending the tissue pellets in an α-MEM (Gibco) medium containing 10% FBS (fetal bovine serum; Peak Serum) and 1% penicillin/streptomycin (Gibco) and then seeding on a 0.2% gelatin-coated culture dish, the adhesion and morphology of the cells were observed with a microscope while incubating at 37° C. and 5% CO2.
Colony-forming unit was measured to investigate the self-renewal ability of the ORM-SCs. 1×103 ORM-SCs were seeded on a 6-well plate and cultured at 37° C. and 5% CO2 for 12 days. After the culturing, the cells were washed and stained with 0.15% crystal violet. After washing again with PBS, the colony was imaged.
After lysing the ORM-SCs with a Labozol reagent (LaboPass, CMRZ001), total RNA was isolated according to the manufacturer's instructions. The purified RNA was quantified using a NanoDrop spectrophotometer (ND-ONE). cDNA synthesis was performed using a M-MuLV reverse transcription kit (Labopass, CMRT010) and oligo dT primers. PCR was conducted using a rTaq Plus 5×PCR master mix (ELPIS Biotech, EBT-1319) and the PCR product was visualized using 1-2% agarose gel. The primer sequences described in Tables 1-3 were used.
Immunophenotyping assay of the ORM-SCs was conducted by flow cytometry. Briefly, the cells were trypsinized to obtain a single cell suspension and then reacted with primary and secondary antibodies on ice for 10 minutes. The primary antibodies used in the present disclosure were CD34 (R&D Systems, MAB72271), CD45 PD7/26/16+2B11 (Invitrogen, MA5-13197), CD73/NT5E (Invitrogen, RG235718), CD90/Thy1 (R&D Systems, AF2067) and CD105 (Invitrogen, MA5-11854). The intensity of fluorescence emitted from the labeled antibodies was measured with a flow cytometer (BD Bioscience, San Jose, Calif. USA).
Differentiation into Three Lineages
The ORM-SCs were induced to differentiate into fat, bone and cartilage for 2 weeks. For differentiation into fat, the ORM-SCs were exposed to an adipocyte differentiation medium containing 10% low-glucose DMEM supplemented with 5 μg/mL insulin, 500 μM isobutylmethylxanthine (IBMX) and 1 μM dexamethasone. An osteocyte differentiation medium contained 10% low-glucose DMEM supplemented with 50 μg/mL L-ascorbic acid, 10 nM β-glycerophosphate and 100 nM dexamethasone, and a chondrocyte differentiation medium contained 10% low-glucose DMEM, 100 nM dexamethasone, 10 nM β-glycerophosphate, 50 μg/mL L-ascorbic acid, 10 μg/mL TGF-β3, 1 mM sodium pyruvate, 40 μg/mL proline and 1× insulin transferrin-selenium.
The differentiation into bone, fat and cartilage could be confirmed by staining with alizarin red S, oil red and alcian blue, respectively.
For isolation of EVs, 4×106 ORM-SCs were seeded on a 150-mm cell culture dish together with serum-free α-MEM. The culture was collected and centrifuged differentially at 300 g for 10 minutes to remove the cells. Then, the supernatant was transferred cautiously to a fresh tube and the cell debris was removed by centrifuging at 2000 g for 10 minutes. Then, the supernatant was transferred again to a fresh tube and centrifuged at 2000 g for 1 hour. The obtained supernatant was filtered through a 0.22-μm vacuum filter (EMD Millipore SCGP00525 Steriflip-GP filter) to remove vesicles. EVs were separated from the filtrate by performing ultrafiltration using an Equilibrate Amicon® Ultra-15 filter (#UFC901024, 10 kDa MWCO). Finally, the EVs were concentrated by centrifuging at 4,000 g for 30 minutes.
Proteins in the isolated EVs were quantitated using a BCA protein assay kit (Pierce, Waltham, Mass., USA) according to the manufacturer's protocol. The size of the EVs was investigated by dynamic light scattering (DLS) using Nano Zetasizer (Malvern Instruments, Malvern, UK). The number of the EVs was measured using the nanoparticle tracking analyzer NS300 (Nanosight, Amesbery, UK).
The morphology and structure of the EVs were analyzed at 80 kV using a transmission electron microscope (TEM, JEM-1010, Nippon Denshi, Tokyo, Japan). The ORM-SC-EVs and ORM-SC lysate were separated by 4-12% SDS-PAGE and transferred onto a PVDF (polyvinylidene difluoride) membrane (ThermoFisher, 1624001). The membrane was blocked using 5% skim milk. After incubation overnight at 4° C. with anti-CD63 (Invitrogen, 10628D), anti-CD81 (Santa Cruz, sc-7637), anti-calnexin (CST, 2679T), anti-GM130 (CST, 12480S) and anti-β-actin (CST, 4970S) antibodies, followed by reaction with a secondary antibody (HRP; horseradish protein) at room temperature, protein signals were detected using an enhanced chemiluminescence kit (Amersham Biosciences, USA) and a ChemiDoc™ imaging system (Bio-Rad, 17001401).
For analysis of cell proliferation after exposure to the EVs, B16F10 cells were seeded on a 96-well plate at 3×103 cells/well and retained in a serum-free RPMI medium for 24 hours. Then, the cells were exposed to the ORM-SC-EVs at different concentrations (2, 5, 10, 30, 50 and 100 μg/mL) for 48 hours. After the incubation, 10 μL of a CCK-8 solution (Dojindo, CK04-05) was added to each well and light was blocked after incubation for 2 hours. Then, absorbance was measured at 450 nm using a Bio-Rad x-Mark™ spectrophotometer (Bio-Rad Laboratories, USA).
B16F10 cells were retained on a 12-well plate at 4×104 cells/well using an RPMI medium containing 10% FBS. After culturing for 24 hours, the cells were treated with 200 nM α-melanocyte-stimulating hormone (α-MSH) (Sigma, M4135) and the ORM-SC-EVs (5, 10, 30, 50 μg/mL) or arbutin (100 μM) (Sigma, A4256) and then cultured for 60 hours. For measurement of extracellular melanin content, 100 μL of the culture medium was transferred to a fresh 96-well plate and absorbance was measured at 405 nm using a Bio-Rad x-Mark™ spectrophotometer. For measurement of intracellular melanin content, each well was washed with PBS and the cells were lysed at 80° C. for 1 hour with 200 μL of 1 N NaOH. Then, absorbance was measured at 405 nm. The extracellular and intracellular melanin contents were normalized to total proteins.
B16F10 cells were cultured for 24 hours on a 12-well plate at a density of 4×104 cells/well. Then, after treating each well with 200 nM α-MSH (Sigma, M4135) and the ORM-SC-EVs (5, 10, 30 or 50 μg/mL) or arbutin (100 μM) (Sigma, A4256), the cells were cultured for 48 hours. After washing each well with PBS, the cells were separated with PBS and suspended in a 50 mM phosphate buffer (pH 6.8) containing 1% Triton X-100. After vortexing, the mixture was incubated at −80° C. for 30 minutes and then thawed at room temperature. After centrifuging at 1000 g for 10 minutes, 40 μL of the supernatant and 100 μL of 10 mM L-DOPA (Sigma, 333786) were added to a 96-well plate. After incubation at 37° C. for 1 hour, absorbance was measured at 405 nm using a Bio-Rad x-Mark™ spectrophotometer. The absorbance value was normalized to total proteins.
For investigation of the antioxidant activity of the orbicularis oculi muscle stem cell-derived extracellular vesicles (OOM-SC-EV) on cellular level, normal human dermal fibroblasts (NHDF, PromoCell, c-23020) were subcultured for 13-15 passages to induce aging. Then, after treating with the OOM-SC-EVs, the cells were stained with senescence-associated β-galactosidase (SA-β-gal) according to a previously reported method (Nature Protocols, 2009. 4(12): p. 1798). Briefly, the cells were seeded on a 4-well cell culture plate (SPL, 30004) and treated with the OOM-SC-EVs at a concentration of 50 μg/mL 12 hours later. After removing the culture, followed by addition of 1 mL of 1×PBS (Veratech) and washing twice at 100 rpm for 5 minutes, the cells were fixed for 15 minutes by adding 1 mL of 2% paraformaldehyde and 0.2% glutaraldehyde. Then, after discarding the fixative solution and adding 1 mL of 1×PBS, the cells were washed twice at 100 rpm for 5 minutes. After adding 1 mL of a previously prepared SA-β-gal staining solution, incubation was conducted for 5 hours at 37° C. under a CO2-free condition. Then, after discarding the SA-β-gal staining solution and adding 1 mL of 1×PBS, the cells were washed twice at 100 rpm for 5 minutes. After adding 1 mL of 100% MeOH (Samjin Industrial), the cells were left alone at room temperature for 30 minutes. Then, after discarding the 100% MeOH and adding 1×PBS, the cells were observed under an optical microscope (Fusion 100, Chemyx). The composition of the SA-β-gal staining solution was as follows: 200 mM citric acid/phosphoric acid, 100 mM K4[Fe(CN)6].3H2O, 100 mM K3[Fe(CN)6], 5 M NaCl, 1 M MgCl2, 50 mg/mL X-gal. The cells positive for SA-β-gal are stained blue.
A 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) reagent was used for measurement of intracellular accumulation of reactive oxygen species (ROS). NHDFs were treated with the OOM-SC-EVs (50 μg/mL) and incubated at 37° C. in high-glucose DMEM (Sigma, D6429; 1% P/S, 10% FBS). After discarding the culture medium, the cells were washed cleanly 1-2 times with 1×PBS and high-glucose DMEM (1% P/S, w/o FBS) was added. After adding H2DCFDA to a final concentration of 10 μM, the cells were incubated at 37° C. and 5% CO2 for 30 minutes. After discarding the culture medium containing 10 μM H2DCFDA, the NHDFs were washed 1-2 times with 1×PBS and then observed using a fluorescence microscope (Nikon Eclipse TE2000-E).
The change in the mRNA expression level of antioxidation-related genes in NHDFs treated with the OOM-SC-EVs (50 μg/mL) was investigated by performing real-time PCR. NHDFs were treated with the OOM-SC-EV at 50 μg/mL. About 24 hours later, the cells were lysed with a Labozol reagent (LaboPass, CMRZ001) and total RNA was isolated according to the manufacturer's instructions. The purified RNA was quantitated using a NanoDrop spectrophotometer (ND-ONE). cDNA synthesis was conducted using a M-MuLV reverse transcription kit (Labopass, CMRT010) and oligo dT primers. Real-time PCR (Amersham Phamacia Biotech 7500) was conducted using a HiPi real-time PCR 2× master mix (SYBR Green, ROX, 500rxn) (ELPIS Biotech, EBT-1802). The primer sequences described in Table 4 were used.
For evaluation of the effect of the OOM-SC-EVs on the migration of NHDFs, NHDFs were cultured on a 6-well cell culture plate (SPL, 30006) to 95% confluency and the proliferation was stopped by treating with 10 μg/mL mitomycin C (Sigma, M4287) for 2 hours. After washing with PBS, the culture plate was scratched using a 200 μL tip end and then treated with the OOM-SC-EVs at 5 μg/mL and 50 μg/mL. The cells were observed with a microscope with 12-hour intervals.
Real-time PCR was conducted to investigate the change in the mRNA expression level of skin regeneration- and wrinkle improvement-associated genes in NHDFs treated with the OOM-SC-EVs (50 μg/mL). The expression of type 1 collagen (Col1A1), monocyte chemoattractant protein (MCP)-1, MCP-3, chemokine ligand 5 (CCL-5), plasminogen activator inhibitor-1 (PAI-1), TFG-β1 (transforming growth factor beta 1) and TGF-β3 (transforming growth factor beta 3) in NHDFs treated with the OOM-SC-EVs (50 μg/mL) was measured using the primers described in Table 5.
6-week-old female BALB/c nude mice were used for experiments after accustomation for a week. After anesthesia, full-thickness wound was formed on the back using an 8-mm biopsy punch (Kai, BP-80F). Then, the OOM-SC-EVs (50 μg/mL) were injected into the wound site over three times. PBS was injected to a control group. Silicone (0.5 mmT) and Tegaderm (1622W) tapes punched with an 8-mm biopsy punch were used to protect the wound site, and wound site was imaged at regular time intervals using an 8-mm punched silicone tape.
On day 14 after the induction of wound, tissue including the whole wound was taken and fixed for 48 hours in a 4% paraformaldehyde solution. Tissue sections passing through the center of the wound were taken, dehydrated and then embedded in paraffin blocks. After sectioning the tissue with a microtome, H&E (hematoxylin & eosin) staining was conducted after removing paraffin by attaching to a polylysine-coated slide and hydrating the tissue. Meanwhile, the slide was placed in a Weigert's iron hematoxylin solution for 10 minutes and in Biebrich scarlet-acid fuchsin and aniline blue for 5 minutes, respectively, for Masson's trichrome staining.
All statistical analysis was performed using GraphPad Prism (version 7). Most experiments were repeated in triplicate. P-value was calculated by one-way ANOVA and t-test using GraphPad Prism and then statistical significance was evaluated. In all the figures, *p<0.05. **p<0.01, ***p<0.001 and ****p<0.0001.
Isolation of ORM-SCs from Patients' Tissues
The ORM is composed of the orbital, septal and tarsal (eyelid) portions [14]. The skeletal muscle samples used in the present disclosure were taken from the eyelids of patients who received eyelid surgery. The patients' tissue samples were obtained through the same surgical procedure [15] and the sex and age of the patients were recorded. After incising the ORM with a laser blade and digesting enzymatically, centrifugation was performed at 1,500 rpm for 10 minutes. Then, cell pellets were seeded on a 0.2% gelatin-coated culture plate (
Population doubling time (PDT) was measured while subculturing the orbicularis oculi muscle-derived stem cells for 13 passages (P13). It was confirmed that PDT was maintained almost constant for 24 hours until passage 10 (P10), but was increased slightly from passage 11 and remarkably from passage 13 (
Cumulative cell number was measured while subculturing the orbicularis oculi muscle-derived stem cells. After calculating an expansion factor by dividing the number of harvested cells by the number of seeded cells, the cumulative cell number was calculated by multiplying the calculated expansion factor by the number of cells harvested in the previous passage. As a result of measuring the cumulative cell number while culturing for 3 days per passage on average, the increase in the cumulative cell number was maintained constant until passage 10 and the increasing rate was decreased from passage 11 (
The isolated ORM-SCs were spindle-shaped. The ORM-SCs the were cultured for 12 days in vitro grew fast and showed high colony-forming ability (
Characterization of ORM-SC-EVs Isolated from ORM-SCs
The yield of the EV particles was the highest for the Amicon 10-k Da (pore size) cellulose membrane as compared to other pore sizes or membranes. The ORM-SCs were cultured in a serum-free medium for 24 hours until 90% confluency. Differential centrifugation and ultrafiltration were performed to remove cells, dead cells and cell debris. Then, the ORM-SC-EVs were collected using a filtration system (
The melanin synthesis inhibition effect of the ORM-SC-EVs was investigated by measuring the viability of B16F10 melanoma cells depending on the concentration of ORM-SC-EVs using CCK-8. As a result, no effect was observed at 50 μg/mL and the viability of melanoma cells was decreased at 100 μg/mL (
For investigation of the antiaging effect of the OOM-SC-EVs on cellular level, senescent NHDFs were stained with β-galactosidase. As a result, the β-galactosidase activity of the group treated with the OOM-SC-EVs was decreased to about 43% with respect to the cells of the control group as 100% (
For investigation of the change in the accumulation of intracellular reactive oxygen species (ROS) by the OOM-SC-EVs, the final concentration of H2DCFDA was measured with a flow cytometer (Beckman Coulter/CytoFLEX). As a result, it was confirmed that, the groups treated with the OOM-SC-EV showed 36.8% staining, whereas the control group showed about 75.9% staining (
After stopping the proliferation of NHDFs with mitomycin C, followed by scratching, the cells were treated with the OOM-SC-EVs. As a result, the group treated with 50 μg/mL OOM-SC-EVs showed significantly improved migration ability as compared to the control group. That is to say, the wound formed by the scratching was filled significantly (
In addition, the size of the wound sites formed on four mice with a biopsy punch was compared. As a result, it was confirmed that the area of the wound site was decreased significantly for the group treated with the OOM-SC-EVs. Accordingly, it can be seen that the composition of the present disclosure can effectively induce wound healing in vivo, too (
In addition, histological analysis was performed on the wound-induced site through H&E staining and Masson's trichrome staining. As a result of the H&E staining, it was confirmed that the group treated with the OOM-SC-EVs showed faster re-epithelialization of the wound as compared to the control group. As a result of the Masson's trichrome staining, it was confirmed that more collagen was synthesized in the group treated with the OOM-SC-EVs (
Real-time PCR was conducted to investigate the change in the mRNA expression level of skin regeneration- and wrinkle improvement-associated genes in the group treated with the OOM-SC-EVs (50 μg/mL). As a result, it was confirmed that the expression of type 1 collagen (Col1A1), which is involved in the facilitation of collagen synthesis and inhibition of collagen degradation, monocyte chemoattractant protein (MCP-1), MCP-3, chemokine ligand 5 (CCL-5) and plasminogen activator inhibitor-1 (PAI-1), which is associated with skin regeneration, was increased in the group treated with the OOM-SC-EVs (
While the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that the foregoing description is merely illustrative and the scope of the present disclosure is not limited thereby. Accordingly, it is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0095934 | Aug 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/010496 | 8/7/2020 | WO |
