Method for culturing skin-derived stem cells and their uses

Abstract

The present invention relates to a method for culturing skin-derived stem cells and uses thereof, and to a method for culturing skin-derived stem cells; skin-derived stem cells produced by the method; a pharmaceutical composition for skin regeneration or wound treatment comprising the skin-derived stem cells produced by the method, a culture thereof, or cells differentiated from the skin-derived stem cells as an active ingredient; a quasi-drug composition; and a cosmetic composition. According to the culture method of the present invention, stem cells can be effectively isolated and obtained from skin tissue. The obtained skin-derived stem cells have excellent pluripotency and self-renewal ability, and have excellent skin regeneration and wound treatment effects, and can therefore be usefully used for skin regeneration and wound treatment or improvement applications.

Description

[Technical field]

[0001] The present invention relates to a method for culturing skin-derived stem cells and uses thereof, and to a method for culturing skin-derived stem cells; skin-derived stem cells produced by the method; a pharmaceutical composition for skin regeneration or wound treatment comprising the skin-derived stem cells produced by the method, a culture thereof, or cells differentiated from the skin-derived stem cells as an active ingredient; a quasi-drug composition; and a cosmetic composition. [Background technology]

[0002] A stem cell is a cell at a pre-differentiation stage before it differentiates into each cell that constitutes a tissue. It is capable of infinite proliferation in an undifferentiated state and has the potential to differentiate into cells of various tissues when stimulated by specific differentiation stimuli.

[0003] Stem cells are largely divided into embryonic stem cells (ES cells) and adult stem cells (tissue-specific stem cells) based on their differentiation potential. Embryonic stem cells are stem cells isolated from the inner cell mass (ICM) of the blastocyst embryo, which is the early stage after the formation of a fertilized egg and before it implants in the uterine lining, and have the potential to differentiate into cells of all tissues.

[0004] On the other hand, tissue-specific stem cells are stem cells specific to each organ that appear when the embryonic development process progresses and each organ of the embryo is formed, and their differentiation ability is generally limited to only the cells that make up that tissue (multipotent). Representative tissue-specific stem cells include hematopoietic stem cells present in the bone marrow and mesenchymal stem cells that differentiate into connective tissue cells other than blood cells. Hematopoietic stem cells differentiate into various blood cells such as red blood cells and white blood cells, and mesenchymal stem cells differentiate into osteoblasts, chondroblasts, adipocytes, myoblasts, etc.

[0005] Recently, following the successful isolation of human embryonic stem cells, there has been growing interest in their clinical application, with the most notable application of stem cells being as a cell source for cell replacement therapy. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Publication No. 10-2014-0075469 A Summary of the Invention [Problem to be solved by the invention]

[0007] The current method for isolating skin stem cells is an enzymatic isolation method using collagenase (Korean Patent Publication No. 10-2014-0075469 A), which has the disadvantage of inevitably damaging proteins on the cell surface, affecting cell performance. Therefore, there is a need for research into methods for isolating and culturing skin stem cells.

[0008] Against this background, the inventors conducted extensive research to develop new stem cells, and as a result, confirmed that skin-derived stem cells produced by the culture method of the present invention have excellent in vitro proliferation ability, cellular performance, and differentiation ability, and can be used for skin regeneration and wound treatment, thereby completing the present invention. [Means for solving the problem]

[0009] One object of the present invention is to provide a method for culturing skin-derived stem cells using skin tissue and a hydrogel.

[0010] Another object of the present invention is to provide skin-derived stem cells produced by the above method.

[0011] Another object of the present invention is to provide a pharmaceutical composition for skin regeneration or wound treatment, comprising the skin-derived stem cells, a culture thereof, or cells differentiated from the skin-derived stem cells as an active ingredient.

[0012] Another object of the present invention is to provide a quasi-drug composition for skin regeneration or wound improvement, which contains the skin-derived stem cells, their cultures, or cells differentiated from the skin-derived stem cells as an active ingredient.

[0013] Another object of the present invention is to provide a cosmetic composition for skin regeneration or wound improvement, which contains the skin-derived stem cells, their cultures, or cells differentiated from the skin-derived stem cells as an active ingredient.

[0014] Another object of the present invention is to provide a composition for culturing skin-derived stem cells, comprising skin tissue and a hydrogel.

[0015] Another object of the present invention is to provide a kit for culturing skin-derived stem cells, which comprises the composition for stem cell culture. Effect of the Invention

[0016] According to the culture method of the present invention, stem cells can be effectively isolated and obtained from skin tissue. The obtained skin-derived stem cells have excellent pluripotency and self-renewal ability and have excellent skin regeneration and wound treatment effects, and can therefore be usefully used for skin regeneration and wound treatment or improvement applications. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1A shows the results of tissue culture of HB-skin-derived stem cells of the present invention, and FIG. 1B shows the results of cell isolation and culture. [Diagram 2] 1 shows the results of confirming the immunophenotype of the HB-skin-derived stem cells of the present invention. [Diagram 3] 1 shows the results of confirming the proliferation ability of the HB-skin-derived stem cells of the present invention by subculture. [Figure 4] 1 shows the results of confirming the colony-forming ability of the HB-skin-derived stem cells (HB-SSCs) of the present invention. [Figure 5a] 1 shows the results of confirming the differentiation ability of HB-skin-derived stem cells of the present invention into adipocytes. [Figure 5b] 1 shows the results of confirming the differentiation ability of HB-skin-derived stem cells of the present invention into bone cells. [Figure 6a] This is the result of DB-based gene string network analysis related to skin regeneration function and the result of confirming specific skin regeneration functional genes of HB-skin-derived stem cells (HB-SSC). [Figure 6b] 1 shows the results of a comparative analysis of the expression levels of skin regeneration functional genes specifically expressed in the HB-skin-derived stem cells (HB-SSCs) of the present invention compared to adipose-derived stem cells (ASCs). [Figure 6c] 1 shows the results of a comparative analysis of the expression levels of skin regeneration functional genes specifically expressed in the skin-derived stem cells (HB-SSCs) of the present invention compared to adipose-derived stem cells (ASCs). [Figure 7a] 1 shows the results of confirming the wound healing effect of the HB-skin-derived stem cells (HB-SSCs) of the present invention in a mouse model. [Figure 7b]1 shows the results of confirming the wound healing effect of the HB-skin-derived stem cells (HB-SSCs) of the present invention in a mouse model. [Figure 8] 1 shows the results of confirming the regenerative ability of the HB-skin-derived stem cells (HB-SSCs) of the present invention to regenerate skin and skin structures in a mouse wound model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each different description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Also, the following specific description is not considered to limit the scope of the present invention.

[0019] To achieve the above-mentioned object, one aspect of the present invention provides a method for culturing skin-derived stem cells using skin tissue and a hydrogel, and skin-derived stem cells cultured by said method.

[0020] The term "skin-derived stem cells" as used herein may include all stem cells derived from skin tissue, and may include, by way of example only, adipose stem cells, epidermal stem cells, dermal stem cells, and the like, but is not limited thereto.

[0021] Specifically, the method for culturing skin tissue-derived stem cells includes: (a) incorporating skin tissue into a hydrogel and culturing it to obtain a culture; and (b) decomposing the hydrogel in the culture obtained above, and recovering stem cells that have migrated and proliferated from the skin tissue into the hydrogel.

[0022] In the present invention, the hydrogel refers to a three-dimensional network structure formed by crosslinking hydrophilic polymers by covalent or non-covalent bonds, and by incorporating skin tissue into the hydrogel three-dimensionally, the hydrogel can provide physical support for the skin tissue and simultaneously provide an extracellular matrix function that enables skin-derived stem cells present in the skin tissue to migrate and proliferate within the hydrogel.

[0023] In addition, the hydrogel of the present invention is preferably a phase transitional hydrogel that exists in a solution state and can be converted into a sol and a gel for hydrogel-supported 3D culture, and specifically, the hydrogel may be any one or more selected from the group consisting of collagen, gelatin, chondroitin, hyaluronic acid, arginic acid, Matrigel™, chitosan, peptide, fibrin, PGA (polyglycolic acid), PLA (polylactic acid), PEG (polyethylene glycol), and polyacrylamide, but is not limited thereto. In addition, the step (a) may be a step of incorporating hair follicle tissue between multiple hydrogel layers and culturing the same. Specifically, the step of incorporating hair follicle tissue between two hydrogel layers and culturing the same, but is not limited thereto.

[0024] In step (a), the hydrogel incorporated with the skin tissue may be cultured after immersing the hydrogel incorporated with the skin tissue in a conventional culture medium known in the art to be suitable for stem cell culture.

[0025] In addition, in the step (a), the skin tissue may be cultured by incorporating the skin slice in the hydrogel for 3 to 20 days, 3 to 18 days, or 3 to 16 days. Specifically, the culture may be for 3 to 14 days, but is not limited thereto.

[0026] In one embodiment of the present invention, skin slices were prepared from healthy adult skin samples and then entrapped in hydrogel and cultured for 9 days.

[0027] In addition, prior to step (a), a pretreatment step of the skin tissue may include a step of treating the skin tissue sample at a temperature of 50 to 70° C. for 30 to 90 seconds; a step of immersing the skin tissue in alcohol or a surfactant for 1 to 10 minutes; and a step of washing.

[0028] At this time, the temperature in the skin tissue treatment step may be, but is not limited to, 50 to 70°C, 52 to 68°C, 54 to 66°C, 56 to 64°C, 58 to 62°C, or 60°C.

[0029] Additionally, the skin tissue processing step may be for, but is not limited to, 30 seconds to 90 seconds, 40 seconds to 80 seconds, 50 seconds to 70 seconds, 55 seconds to 65 seconds, 58 seconds to 62 seconds, 59 seconds, 60 seconds, or 61 seconds.

[0030] In addition, the alcohol used in the step of immersing in the alcohol or surfactant during the pretreatment step may include an alcohol having 1 to 4 carbon atoms, specifically, ethanol, methanol, propanol, isopropyl alcohol, etc., but is not limited thereto.

[0031] The surfactant may also include an amphoteric surfactant or a non-ionic surfactant.

[0032] The amphoteric surfactant may include, but is not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like.

[0033] The nonionic surfactant may include fatty acid esters of glycerol, fatty acid esters of sorbitol Spans or Tweens, and specifically, may be glycerol monostearate, glycerol monolaurate, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, Tween 20, Tween 40, Tween 60, Tween 80, and the like, but is not limited thereto.

[0034] In addition, since the step of immersing in alcohol or a surfactant is for removing the fat layer of the skin tissue, any method of contacting the tissue with alcohol or a surfactant, such as immersion, spraying, coating, etc., used in the art, is included, and is not particularly limited.

[0035] Furthermore, the step of immersing in the alcohol or surfactant may be for 1 to 10 minutes, 2 to 9 minutes, 3 to 8 minutes, 4 to 7 minutes, 4 minutes 30 seconds to 6 minutes 30 seconds, 5 minutes to 5 minutes 30 seconds, or 5 minutes, but is not limited thereto.

[0036] In addition, the cleaning step of the pretreatment step is not particularly limited as long as it is used as a cleaning agent in the present technical field.

[0037] In one specific example, the solution may be PBS, and the number of washings may be 1 to 10 times, 2 to 8 times, 3 times, 4 times, 5 times, 6 times, or 7 times, but is not limited thereto.

[0038] Furthermore, the skin tissue sample may be a skin tissue sample extracted from an animal or donated from a human, but is not limited thereto.

[0039] In one embodiment of the present invention, a skin tissue sample was treated at about 60° C. for about 1 minute to separate the stratum corneum, and then immersed in isopropyl alcohol for 5 minutes to remove the fat layer of the skin, followed by a pretreatment step of a washing step.

[0040] In step (b), the hydrogel can be selectively degraded in order to recover skin-derived stem cells that have migrated / proliferated within the hydrogel after the hydrogel-supported 3D culture step in step (a).

[0041] The hydrogel can be degraded by one or more enzymes selected from the group consisting of collagenase, gelatinase, urokinase, streptokinase, TPA (tissue plasminogen activator), plasmin, and hyaluronidase.

[0042] The skin stem cell isolation / culture technology of the present invention is a technology for isolating stem cells as a hydrogel-based niche-preserving self-renewal induction isolation technology, and is a new technology that allows skin tissue to be survived / cultured in vitro and highly pure cells to be isolated directly from the tissue without enzymatic treatment.

[0043] Unlike existing methods, the present isolation method is differentiated from existing methods and has the advantages of being superior in terms of in vitro proliferation ability and cell performance.

[0044] Cells isolated by existing methods can only be expanded in vitro for 2-3 passages (maximum 5 passages), but cells isolated by the method of the present invention have superior performance, such as not undergoing cellular senescence even after being subcultured for 8 passages or more, and can be subcultured for a long period of time, making mass production possible.

[0045] In addition, it was confirmed that the skin-derived stem cells produced by the above method maintained their cell morphology and size up to the ninth subculture after tissue culture and cell separation.

[0046] Meanwhile, the skin-derived stem cells produced by the method for producing skin-derived stem cells provided by the present invention exhibit immunological characteristics in which CD29, CD44, CD73, CD90 or CD105 is expressed on the cell surface, but CD14, CD34 or CD45 is not expressed, and they exhibit multilineage characteristics that enable them to differentiate into fat cells, bone cells, etc., but are not limited thereto.

[0047] The skin-derived stem cells may be mixed with skin-derived stem cells produced by the method for producing skin-derived stem cells provided by the present invention, the skin-derived stem cells of the present invention, skin-derived stem cells, HB-skin-derived stem cells, the HB-skin-derived stem cells of the present invention, and HB-SSCs.

[0048] In one embodiment of the present invention, the proliferation ability, differentiation ability, and colony formation ability of skin-derived stem cells produced by the method for producing skin-derived stem cells of the present invention were confirmed.

[0049] In still another embodiment of the present invention, the skin wound healing effect of skin-derived stem cells prepared by the method of preparing skin-derived stem cells of the present invention was confirmed.

[0050] In another embodiment of the present invention, it was further confirmed that the cells have a long in vitro survival period and excellent viability through evaluation of cell growth ability / cell senescence (in vitro proliferation ability) by subculture and purity of primary cells.

[0051] The skin-derived stem cells produced by the method for producing skin-derived stem cells provided by the present invention can express a specific skin regeneration functional gene.

[0052] In this case, the skin regeneration functional gene may be, specifically, any one or more genes selected from OSR2 (odd-skipped-related 2), DMKN (Dermokine), SBSN (Suprabasin), and GAS1 (Growth Arrest Specific 1), but is not limited thereto, and may include any gene known as a skin regeneration functional gene.

[0053] In one embodiment of the present invention, it was confirmed that the skin-derived stem cells produced by the skin-derived stem cell production method showed significant expression levels of the four skin regeneration functional genes, at least 10 times higher, compared to adipose-derived stem cells, confirming that the skin-derived stem cells produced by the present method exert specific skin regeneration functional genes.

[0054] Another aspect of the present invention provides a pharmaceutical composition for skin regeneration or wound treatment, comprising the skin-derived stem cells, a culture thereof, or cells differentiated from the skin-derived stem cells as an active ingredient.

[0055] The term "skin regeneration" as used herein means the process of recovery of skin tissue from damage caused by external and internal factors. Damage caused by external factors includes ultraviolet rays, pollutants, wounds, trauma, etc., and internal factors include genetic and psychological factors, but are not limited thereto.

[0056] In addition, the skin regeneration is a concept that may include hair follicle regeneration or proliferation of hair follicle cells, and the proliferation of hair follicle cells may include, but is not limited to, the promotion of the conversion of hair follicle cells from the resting phase to the proliferation phase.

[0057] The term "culture" or "culture medium" as used herein may refer to a culture or its supernatant, a concentrate, or a lyophilized product thereof obtained during or after culturing skin-derived stem cells in a medium.

[0058] The term "pharmaceutical composition" as used herein means a substance manufactured for the purpose of preventing or treating a disease, and may be administered in various oral and parenteral dosage forms in actual clinical administration. When formulated, the substance may be formulated using a diluent or excipient commonly used, such as a filler, extender, binder, wetting agent, disintegrant, or surfactant.

[0059] Also, depending on the dosage form, a pharma- ceutically acceptable additive may be further included, and in this case, examples of the pharma- ceutically acceptable additive include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicone dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, talc, etc. The pharma- ceutically acceptable additive according to the present invention may be included in an amount of 0.1 to 90 parts by weight based on the composition.

[0060] The pharmaceutical composition may further include one or more pharma- ceutically acceptable inactive carriers in addition to the skin-derived mesenchymal stem cells, such as a preservative, a pain-relieving agent, a solubilizer, or a stabilizer in the case of an injection, and a base, an excipient, a lubricant, or a preservative in the case of a topical administration preparation.

[0061] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be administered singly or multiple times. Taking into consideration all of the above factors, it is important to administer an amount that will obtain maximum effect with the minimum amount without inducing side effects, and this can be easily determined by those skilled in the art.

[0062] In addition, the pharmaceutical composition provided by the present invention may further contain various ingredients that aid in skin regeneration or wound treatment, maintain the activity of the skin-derived stem cells, or promote the differentiation of the skin-derived stem cells, in addition to the skin-derived stem cells. For example, the pharmaceutical composition may further contain an anti-inflammatory agent, a stem cell mobilizing factor, a growth-inducing factor, etc.

[0063] The term "amelioration" as used herein means any action of administering the composition according to the present invention to an individual to slow the progression of skin damage or reduce the symptoms.

[0064] The term "prevention" as used herein means any action of administering the composition according to the present invention to an individual to inhibit or delay the occurrence of skin damage.

[0065] The term "treatment" as used herein means any action of administering the composition of the present invention to an individual suspected of developing skin damage to improve or benefit the symptoms of skin damage.

[0066] Another object of the present invention is to provide a method for skin regeneration or wound improvement or treatment, comprising the step of administering to an individual a composition containing, as an active ingredient, the skin-derived stem cells, a culture thereof, or cells differentiated from the skin-derived stem cells.

[0067] The skin-derived stem cells, cultures thereof, or cells differentiated from the skin-derived stem cells, and skin regeneration, wound improvement, and treatment are as described above.

[0068] The term "administration" as used in the present invention means introducing the composition into an individual by a suitable method. Specifically, the composition containing the skin-derived stem cells of the present invention, their cultures, or cells differentiated from the skin-derived stem cells as an active ingredient, or the pharmaceutical composition containing the same, can be administered as a topical application or injection to a local site, for example, a skin injury or wound site, but is not limited thereto.

[0069] The composition may also be used in combination with other pharmaceutical compositions used to treat skin injuries or wounds.

[0070] The term "individual" as used herein refers to any animal, including humans, including mice, rats, livestock, etc., that develops or may develop skin lesions or wounds. The animal may be, but is not limited to, a mammal, such as a cow, horse, sheep, pig, goat, camel, antelope, dog, cat, etc., that requires prevention or treatment of symptoms similar to those of humans.

[0071] The compositions of the present invention can be administered in a pharma- ceutically effective amount.

[0072] The term "pharmacologically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the level of an effective dose can be determined based on factors including the type and severity of the disease in an individual, age, sex, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0073] The composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or multiple times. It is important to administer an amount that can obtain the maximum effect at the minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by those skilled in the art.

[0074] The composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dosage varies depending on the patient's condition and weight, the severity of the disease, the drug form, the administration route and time, but can be appropriately selected by those skilled in the art. As a specific example, the composition can generally be administered once or several times a day, and a preferred dosage can be appropriately selected by those skilled in the art depending on the individual's condition and weight, the severity of the disease, the drug form, the administration route and period.

[0075] Another object of the present invention is to provide a quasi-drug composition for skin regeneration or wound improvement, which contains the skin-derived stem cells, their cultures, or cells differentiated from the skin-derived stem cells as an active ingredient.

[0076] The term "quasi-drug composition" in the present invention means a fiber, rubber product or similar used for the purpose of treating, mitigating, treating or preventing a disease in humans or animals, a non-instrument or machine or similar item that has a weak effect on the human body or does not directly act on the human body, or an item that corresponds to one of preparations used for sterilization, insecticide and similar purposes for infection prevention, and means an item that is not an instrument, machine or device among items used for the purpose of diagnosing, treating, mitigating, treating or preventing a disease in humans or animals, and an item that is not an instrument, machine or device among items used for the purpose of pharmacologically affecting the structure and function of humans or animals, and specifically may be a skin topical agent or personal hygiene product.

[0077] Another object of the present invention is to provide a cosmetic composition for skin regeneration or wound improvement, which contains the skin-derived stem cells, their cultures, or cells differentiated from the skin-derived stem cells as an active ingredient.

[0078] The term "cosmetic composition" as used herein can be prepared in any commonly produced formulation, for example, a solution, emulsion, suspension, paste, cream, lotion, gel, powder, spray, surfactant-containing cleanser, oil, soap, liquid cleanser, bath additive, foundation, makeup base, essence, lotion, foam, pack, softener, sunscreen cream, or sun oil.

[0079] Another object of the present invention is to provide a composition for culturing skin-derived stem cells, which comprises skin tissue and a hydrogel, and a kit for culturing skin-derived stem cells, which comprises the composition.

[0080] In addition, a kit for culturing skin-derived stem cells may include the composition for culturing stem cells and various components such as solutions and devices required for culturing the stem cells.

[0081] Another object of the present invention is to provide a use of a composition comprising the skin-derived stem cells, a culture thereof, or cells differentiated from the skin-derived stem cells as an active ingredient for skin regeneration or wound treatment.

[0082] The present invention will be described in more detail with reference to the following examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0083] Example 1. Fibrin hydrogel-supported 3D culture of skin tissue Skin tissue samples were extracted from animals or donated from humans. The skin tissue was treated at 60°C for 1 minute to separate the stratum corneum, immersed in isopropyl alcohol for 5 minutes to remove the fat layer, and then washed three times with PBS (Gibco) to pretreat the skin tissue. The treated tissue was fixed using Forcep, cut into pieces of 1-3 mm2 using a razor blade, and washed three times with PBS. After washing once with DMEM (low glucose, Gibco), the supernatant was removed and 5 mL of fibrinogen solution (0.25% w / v fibrinogen (Green Cross), 200 μg / mL tranexamic acid (Shinpun Pharmaceutical) in DMEM) was added. The same amount of thrombin solution (0.5 IU / mL thrombin (Green Cross) in DMEM) was added, and the tissue slices and hydrogel were immediately transferred to a 100 mm culture dish and gently rolled to spread evenly. If necessary, a forcep was used to ensure spacing between the tissue slices. The cells were incubated at room temperature for 5 minutes and then at 37℃ for 2 hours to allow for sufficient gelation. 10mL of growth medium (90% low DMEM:F12=1:1, 10% fetal bovine serum, 20ng / mL epidermal growth factor, 5ng / mL basic fibroblast growth factor, 10ng / mL insulin-like growth factor, and 10mg / mL gentamicin) containing 100μg / mL tranexamic acid was added and the culture medium was changed every hour for 3 hours. Thereafter, the growth medium was changed every 2~3 days.

[0084] Example 2. Recovery and expansion of HB-skin-derived stem cells migrated and grown in fibrin hydrogel The hair follicle tissue was cultured for about 10 days using the method described in Example 1. The hydrogel was washed three times for 3 minutes with warm PBS, then 30% FBS / DMEM was added and agitated with an orbital shaker (30 rpm). After 1 hour, the supernatant was removed, and 10 mL of 30% FBS / low DMEM containing 3,000 units of urokinase (Green Cross) was added to decompose the gel. When the gel was sufficiently decomposed, it was cultured overnight under static conditions, after which the supernatant was removed, growth medium was added, and cultured in the usual manner. The growth medium was replaced every 2-3 days, and the cells were subcultured every 4-6 days.

[0085] The results of tissue culture and cell isolation and culture performed according to the above method are shown in FIG. Specifically, when the cells were isolated and subcultured after tissue culture, it was confirmed that the cell morphology and size were maintained up to the ninth subculture.

[0086] Example 3. Immune phenotypic confirmation of isolated and cultured HB-skin derived stem cells To confirm the immunophenotype of HB-skin-derived stem cells isolated and cultured according to the method of Example 2, cells cultured in a T75 flask were washed once with PBS and then detached by treatment with 3 mL of TrypleExpress (Gibco) for 3 minutes. The detached cells were added with 6 mL of 10% calf serum / PBS to suppress proteolysis. After centrifugation (300g, 5 min), the supernatant was removed and the cells were suspended in 3% bovine serum albumin (Sigma) / PBS.

[0087] The cell suspension was divided into nine equal portions and incubated for 1 h at 4°C with the following antibodies: FITC-conjugated CD29 (Bio-Rad, 1:10), CD44 (Invitrogen, 1:100), CD90 (Invitrogen, 1:20), CD14 (Invitrogen, 1:20) antibody, AlexaFluor488-conjugated CD105 (Abcam, 1:25), PE-conjugated CD73 (Invitrogen, 1:20) antibody, PerCP-conjugated CD34 (Santacruz, 1:5) antibody, or AlexaFluor594-conjugated CD45 (Novus, 1:20) antibody. After incubation, the cells were washed by centrifugation at 300g for 5 min, suspended in 3% BSA / PBS, and analyzed by flow cytometry (BD) (Figure 2).

[0088] As a result, as shown in Figure 2, it was confirmed that CD29, CD44, CD73, CD90, and CD105 were expressed on the surface of the isolated and cultured HB-skin-derived stem cells, but CD14, CD34, and CD45 were not expressed.

[0089] Specifically, it was confirmed that the mesenchymal stem cell markers CD29, CD44, CD90, CD73, and CD105 were positively expressed (95% or more), while the hematopoietic cell markers CD34, CD45, and HLA-DR were negatively expressed (less than 5%).

[0090] Example 4. Confirmation of proliferation and colony formation ability of isolated and cultured HB-skin derived stem cells To confirm the proliferation ability of the isolated and cultured HB-skin-derived stem cells obtained in Example 2, the cells were seeded at a density of 4,000 cells / cm2 during initial culture and subcultured every 4 days. The population doubling time according to the number of subcultures was measured based on the number of cells obtained.

[0091] The number of cells was determined by counting the number of cells that were not stained by trypan blue staining, and the population doubling times (PDTs) were calculated using the following formula and are shown in Figure 3: PDT = [days / (logN2-logN1)] / log2, (N1 = 4,000 cells, N2 = number of cells after 4 days).

[0092] In addition, to confirm the colony-forming ability of the isolated and cultured HB-skin-derived stem cells obtained in Example 2, human skin-derived stem cells (4 passages) were seeded at 20 cells / well in a 6-well plate. The cells were cultured for 14 days while changing the growth medium every 3 days. After washing with PBS, the cells were fixed in 4% paraformaldehyde (4% PFA, Sigma) at room temperature for 15 minutes. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet / methanol at room temperature for 15 minutes. After removing all residual solution, the cells were washed three times with distilled water, and the results of observing the stained colonies are shown in FIG. 4.

[0093] As shown in the results of Figures 3 and 4, the HB-skin-derived stem cells obtained by the method of this Example 2 were cultured up to the 7th subculture to determine the population doubling time (PDT). The PDT was observed to be an average of 35 hours ± 6 hours, demonstrating that proliferation ability was maintained through subculture, and colony formation ability was also confirmed to be at a certain level or above.

[0094] Example 5. Confirmation of differentiation potential of HB-skin derived stem cells To evaluate the differentiation ability of the obtained HB-skin-derived stem cells into adipocytes, the cells were seeded at a density of 1×104 cells / cm2 and cultured in growth medium until they reached 80% confluency, then the medium was replaced using a StemProTM Adipogenesis differentiation kit (Gibco, cat. A1007001) and cultured for 14 days with medium replacement every 3 days. The cells were fixed with 4% PFA for 15 minutes and reacted with 60% isopropanol for 15 minutes, after which all the solutions were removed and the cells were thoroughly dried. The presence or absence of fat accumulation in the cytoplasm was confirmed by staining with 60% Oil Red O (sigma) / ethanol at room temperature for 15 minutes (Figure 5a).

[0095] To evaluate the differentiation ability of the HB-skin-derived stem cells into bone cells, the cells were seeded at a density of 1×104 cells / cm2 and cultured in growth medium until they reached 80% confluency, then the medium was replaced with a StemProTM Osteogenesis differentiation kit (Gibco, cat. A1007201) and cultured for 14 days while changing the medium every 3 days. The cells were fixed with 4% PFA for 60 seconds, treated with BCIP / NBT substrate solution (Sigma), and stained for 10 minutes in the dark to confirm alkaline phosphatase (ALP) activity (Figure 5b).

[0096] That is, from the above results, it was confirmed that the HB-skin-derived stem cells obtained by the culture method of the present invention have pluripotency.

[0097] Example 6. Identification of differentially expressed genes in HB-skin derived stem cells For the adipose stem cell (ASC-1, ASC-2, ASC-3) and HB-skin derived stem cell (HB-SSC-1, HB-SSC-2, HB-SSC-3) samples, paired-end RNA libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's protocol, and sequencing was performed at Macrogen (Seoul, Republic of Korea). After pretreatment, the samples were mapped to the reference genome (version GRCh38) using the HISAT2 program (version 2.1.0, https: / / ccb.jhu.edu / software / hisat2 / index.shtml), and transcript-specific expression profiles were obtained using the StringTie program (version 2.1.3b, https: / / ccb.jhu.edu / software / stringtie / ). Gene expression levels were treated as logarithmic TPM (Transcripts Per Kiobase Million) values. Differentially expressed genes between the control and comparison groups were obtained using the edgeR package [1] in the R program (version 4.0) (1. ROBINSON, Mark D.; MCCARTHY, Davis J.; SMYTH, Gordon K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. bioinformatics, 2010, 26.1: 139-140.) (Figures 6b and 6c). Biological network construction involving marker genes was performed using the String Database (version 11.5) [2] to construct a protein-protein interaction (PPI) network (2. SZKLARCZYK, Damian, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic acids research, 2019, 47.D1: D607-D613).The organism reference used to construct the PPI network was Homo sapiens. In the network construction, edges connecting nodes representing proteins were connected based on experimental results, biological databases, literature, gene fusion, etc., and edge scores of 0.4 or higher were considered significant. After constructing the network, orphan nodes with no connectivity to other nodes were removed. The biological functions to which the node groups belonged were extracted only if they were statistically significant based on the KEGG database [3] (3. KANEHISA, Minoru; GOTO, Susumu. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic acids research, 2000, 28.1: 27-30) (False Discovery Rate < 0.05) (Figure 6a). The significant difference between the two groups was tested by T-Test, and a P value of less than 0.05 was considered significant. Statistical test results provided by the analysis program were used as is, and a significance level of less than 0.05 was considered significant. When results were reported with a P value exceeding 0.05, the P value was reported separately.

[0098] Using String DB, genes expressed in HB-skin-derived stem cells were analyzed as skin regeneration-related gene networks to confirm related signaling pathways, and four skin regeneration functional genes (GAS1, OSR2, DMKN, SBSN) were confirmed to be expressed at high levels (Fig. 6a). 73 genes were confirmed to be expressed in HB-skin-derived stem cells with a fold change > 10 compared to adipose-derived stem cells, and of these, the four skin regeneration functional genes (GAS1, OSR2, DMKN, SBSN) derived in Fig. 6a were confirmed to be expressed at a 10-fold or greater differential expression compared to adipose-derived stem cells (Figs. 6b and 6c).

[0099] The four skin regeneration functional genes are known to be genes related to immune regulation and cell proliferation in relation to skin regeneration and wound healing. Based on this, the skin regeneration and wound healing effects of skin-derived stem cells produced by the skin-derived stem cell production method of the present invention can be inferred.

[0100] Example 7. Full-thickness skin wound mouse model and confirmation of wound healing effect The experimental animals, 12-week-old BALB / c nude mice (center experimental animal), were anesthetized by inhalation with 3% Isoflurane (Hana Pharmaceutical), and the surgical site was disinfected with alcohol swab (Meditop). After attaching a scale sticker to the back, the skin was incised into a square shape (5mm x 5mm, 25mm2) with sterilized surgical scissors to the subcutaneous layer, and the subcutaneous tissue was separated to induce a full-thickness skin defect wound. To prevent wound contraction, the wound surrounding area was fixed with suture (ProleneTM, EHICON, cat. W8566). 5 x 105 cells were suspended in 100μl of normal saline (NS) and injected subcutaneously (insulin syringe, 31 gauge) at 25μl / site (4 sites) at the wound surrounding area. After wound induction, the surrounding area was disinfected with alcohol swab to prevent infection, and all experimental groups were separated and observed. After wound induction, the wound site of each group was photographed at the same time on days 0, 1, 3, 5, 7, 12, and 14 using a digital camera, and the wound area was measured using Image J Software (US National Institute of Health Program, USA). The wound area was calculated as a percentage based on the wound area immediately after wound induction (Figures 7a and 7b).

[0101] As a result, the wound healing effect of HB-skin-derived stem cells produced by the production method of the present invention was confirmed.

[0102] Example 8. Confirmation of regenerative ability of skin and skin structures in a mouse model with full-thickness skin wound For histological analysis, the skin tissues were fixed in a fixative (4% formaldehyde, Sigma, USA) for 24 hours. The tissues were then embedded in paraffin, sliced ​​into blocks at 6 μm, and stained with H&E (Hematoxylin & Eosin). The tissues of the experimental groups were observed and compared using an optical microscope (Figure 8).

[0103] As a result, it was confirmed that the HB-skin-derived stem cells produced by the production method of the present invention have a regenerative effect on skin and skin structures.

[0104] From the above description, it will be understood by those skilled in the art to which the present invention pertains that the present invention may be embodied in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. (a) incorporating skin tissue into a hydrogel and culturing it to obtain a culture; and (b) a method for culturing skin-derived stem cells, comprising a step of decomposing the hydrogel in the culture obtained above and recovering stem cells that have migrated and proliferated in the hydrogel from the skin tissue, A method for culturing skin-derived stem cells, wherein the expression of one or more genes selected from GAS1, OSR2, DMKN, and SBSN is increased in the skin-derived stem cells.

2. 2. The method of claim 1, wherein the skin tissue is cultured by incorporating a skin slice into a hydrogel for 3 to 20 days.

3. 2. The method of culturing skin-derived stem cells according to claim 1, further comprising, prior to step (a), a pretreatment step of skin tissue, comprising treating the skin tissue sample at a temperature of 50 to 70°C for 30 to 90 seconds; immersing the skin tissue sample in alcohol or a surfactant for 1 to 10 minutes; and washing the skin tissue.

4. the skin-derived stem cells express CD29, CD44, CD73, CD90, and CD105; The method for culturing skin-derived stem cells according to claim 1, wherein the cells have immunological properties that do not express CD14, CD34, and CD45.

5. 2. The method for culturing skin-derived stem cells according to claim 1, wherein the skin-derived stem cells maintain their cell morphology and size up to the ninth subculture after tissue culture and cell separation.

6. Skin-derived stem cells having increased expression of one or more genes selected from GAS1, OSR2, DMKN and SBSN.

7. the skin-derived stem cells express CD29, CD44, CD73, CD90, and CD105; The skin-derived stem cells according to claim 6, which have the immunological property of not expressing CD14, CD34 and CD45.

8. A pharmaceutical composition for skin regeneration or wound treatment, comprising the skin-derived stem cells according to claim 6, a culture thereof, or cells differentiated from said skin-derived stem cells as an active ingredient.

9. A quasi-drug composition for skin regeneration or wound treatment, comprising the skin-derived stem cells according to claim 6, a culture thereof, or cells differentiated from said skin-derived stem cells as an active ingredient.

10. A cosmetic composition for skin regeneration or wound treatment, comprising the skin-derived stem cells according to claim 6, a culture thereof, or cells differentiated from said skin-derived stem cells as an active ingredient.

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