Method for culturing hair follicle-derived stem cells and their uses

Abstract

The present invention relates to a method for culturing hair follicle-derived stem cells and uses thereof, and to a method for culturing hair follicle-derived stem cells; hair follicle-derived stem cells produced by the method; a pharmaceutical composition for treating hair loss or promoting hair growth, comprising the hair follicle-derived stem cells produced by the method, a culture thereof, or cells differentiated from the hair follicle-derived stem cells as active ingredients; 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 hair follicle tissue. The obtained hair follicle-derived stem cells have excellent pluripotency and self-renewal ability, and are effective in treating hair loss and promoting hair growth, and therefore can be usefully used for improving, preventing or treating hair loss.

Description

[Technical field]

[0001] The present invention relates to a method for culturing hair follicle-derived stem cells and uses thereof, and to a method for culturing hair follicle-derived stem cells; hair follicle-derived stem cells produced by the method; a pharmaceutical composition for treating hair loss or promoting hair growth, comprising the hair follicle-derived stem cells produced by the method, a culture thereof, or cells differentiated from the hair follicle-derived stem cells as active ingredients; a quasi-drug composition; and a cosmetic composition. [Background technology]

[0002] A stem cell is a cell at a preliminary stage before it differentiates into each of the cells that make up 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 existing method for isolating hair follicle stem cells is an enzymatic 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 hair follicle-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 treating hair loss and promoting hair growth, thereby completing the present invention. [Means for solving the problem]

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

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

[0011] Another object of the present invention is to provide a pharmaceutical composition for treating hair loss or promoting hair growth, comprising the hair follicle-derived stem cells, a culture thereof, or cells differentiated from the hair follicle-derived stem cells as an active ingredient.

[0012] Another object of the present invention is to provide a quasi-drug composition for improving hair loss or promoting hair growth, which contains the hair follicle-derived stem cells, their cultures, or cells differentiated from the hair follicle-derived stem cells as an active ingredient.

[0013] Another object of the present invention is to provide a cosmetic composition for improving hair loss or promoting hair growth, which contains the hair follicle-derived stem cells, their cultures, or cells differentiated from the hair follicle-derived stem cells as an active ingredient.

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

[0015] Another object of the present invention is to provide a kit for culturing hair follicle-derived stem cells, comprising 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 hair follicle tissue. The obtained hair follicle-derived stem cells have excellent pluripotency and self-renewal ability and are effective in treating hair loss and promoting hair growth, and therefore can be usefully used for improving, preventing or treating hair loss. [Brief description of the drawings]

[0017] [Figure 1] (a) Results of slice formation of hair follicle tissue, (b) schematic diagram of hair follicle tissue incorporated into hydrogel for culture, and (c) results of cell migration and proliferation into the hydrogel at different culture periods. [Diagram 2] These are the results of confirming the immunophenotype of HB-hair follicle-derived stem cells. [Diagram 3] These are the results of confirming the proliferation ability of HB-hair follicle-derived stem cells through subculture. [Figure 4] These are the results of confirming the colony-forming ability of HB-hair follicle-derived stem cells. [Figure 5a] These results confirm the ability of HB hair follicle-derived stem cells to differentiate into fat cells. [Figure 5b] These results confirm the differentiation ability of HB-hair follicle-derived stem cells into bone cells. [Figure 6a] These are the results of a DB-based gene string network analysis related to hair growth function and the results of confirming specific hair growth promoting functional genes of HB-hair follicle-derived stem cells. [Figure 6b] This is the result of a comparative analysis of the expression levels of genes related to the main hair growth functions of HB-hair follicle-derived stem cells. [Figure 6c] This is the result of a comparative analysis of the expression levels of hair growth promoting functional genes specifically expressed in HB-hair follicle-derived stem cells compared to adipose-derived stem cells. [Figure 6d] This is the result of a comparative analysis of the expression levels of hair growth promoting functional genes specifically expressed in HB-hair follicle-derived stem cells compared to adipose-derived stem cells. [Figure 6e] This is the result of a comparative analysis of the expression levels of hair growth-promoting functional genes specifically expressed in HB-hair follicle-derived stem cells compared to existing hair follicle-derived stem cells. [Figure 7a]These results confirm the hair growth promoting effect of HB-hair follicle-derived stem cells in a mouse model. [Figure 7b] These results confirm the hair growth promoting effect of HB-hair follicle-derived stem cells in a mouse model. [Figure 8a] These results confirm the hair follicle formation ability of HB-hair follicle-derived stem cells in a mouse model. [Figure 8b] These results confirm the hair follicle formation ability of HB-hair follicle-derived stem cells in a mouse model. [Figure 8c] These results confirm the hair follicle formation ability of HB-hair follicle-derived stem cells in a mouse model. [Figure 8d] These results confirm the hair follicle formation ability of HB-hair follicle-derived stem cells in a mouse model. [Figure 9a] These results confirm the mechanism of action of Wnt signaling in HB-hair follicle-derived stem cells in a mouse model. [Figure 9b] These results confirm the mechanism of action of Wnt signaling in HB-hair follicle-derived stem cells in a mouse 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 hair follicle-derived stem cells using hair follicle tissue and a hydrogel, and hair follicle-derived stem cells cultured by said method.

[0020] Specifically, the method for culturing hair follicle tissue-derived stem cells includes: (a) incorporating hair follicle 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 hair follicle tissue into the hydrogel.

[0021] 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 hair follicle tissue into the hydrogel three-dimensionally, the hydrogel can provide physical support for the hair follicle tissue and at the same time provide an extracellular matrix function that enables hair follicle-derived stem cells present in the hair follicle tissue to migrate and proliferate within the hydrogel.

[0022] In addition, the hydrogel of the present invention is preferably a phase transitional hydrogel that exists in a solution state for hydrogel-supported 3D culture and can be converted into a sol and a gel, 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.

[0023] In step (a), the hydrogel incorporating the hair follicle tissue may be cultured after immersing the hydrogel incorporating the hair follicle tissue in a conventional culture medium known in the art to be suitable for culturing stem cells.

[0024] In addition, the step (a) may involve incorporating hair follicle tissue between multiple hydrogel layers and culturing the same. Specifically, the step may involve incorporating hair follicle tissue between two hydrogel layers and culturing the same, but is not limited thereto.

[0025] In one embodiment of the present invention, when incorporating hair follicle tissue into the hydrogel, the hydrogel was added in two stages, and the hair follicle tissue was cultured so that it was incorporated between the sandwich-type hydrogel bilayer.

[0026] As described above, when hair follicle tissue is incorporated between multiple hydrogel layers, the hydrophobicity of the surface layer of the hair cuticle remaining on the hair follicle tissue slice can prevent the tissue slice from floating without being hydrated, and the two-stage hydrogel treatment has the advantage that the hair follicle tissue can be sufficiently incorporated into the hydrogel between the hydrogel bilayers.

[0027] In step (a), the hair follicle tissue may be a section obtained by removing a portion corresponding to the follicular infundibulum from the epidermal. The section may be 0.01 to 10 mm, 0.05 to 5 mm, or 0.1 to 3 mm in size, but the size of the section is not limited.

[0028] In one embodiment of the present invention, a 1-3 mm hair follicle tissue slice was prepared by removing the area corresponding to the infundibulum of the epidermal region during the preparation of the hair follicle tissue slice, and used for the culture.

[0029] In addition, in step (a), the culture period of the hair follicle tissue may be, but is not limited to, 3 to 20 days, 3 to 18 days, 3 to 16 days, 3 to 14 days, or 4 to 12 days.

[0030] As the culture period increases, the number of cells separated per tissue weight increases, which is advantageous in terms of cell separation efficiency. However, as the culture period continues, cell confluency in the hydrogel surrounding the tissue increases, which may inhibit cell proliferation through contact inhibition and induce stem cell differentiation. Furthermore, the cell / hydrogel ratio increases and cell aggregation occurs, which inhibits cell release due to selective decomposition of the hydrogel during cell separation. Therefore, since aggregation due to cell overcrowding and turbidity around the tissue are observed around the tissue, it is important to appropriately adjust the culture period during tissue culture within the period during which cell outgrowth is observed.

[0031] In one embodiment of the present invention, the hair follicle tissue was cultured for 4 to 12 days before use.

[0032] In addition, in step (a), the hair follicle tissue may be cultured with a spacing between tissues of 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, or 10 mm or less, but is not limited thereto.

[0033] When seeding tissues during the above-mentioned culture, the spacing between tissues needs to be determined taking into consideration the outgrown distance and the distance of cells that have migrated / proliferated from one tissue.

[0034] In one embodiment of the present invention, the tissue slices were seeded and cultured with a spacing of 5 mm or more between the tissue slices.

[0035] In the step (b), the hydrogel alone can be selectively degraded in order to recover hair follicle-derived stem cells that have migrated / proliferated within the hydrogel after the hydrogel-supported 3D culture step (a).

[0036] 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.

[0037] The hair follicle stem cell isolation / culture technology of the present invention is a technology that isolates stem cells using a hydrogel-based niche-preserving self-renewal induction isolation technology, and is a new technology that allows hair follicle tissue to survive / culture in vitro and isolate highly pure cells directly from the tissue without enzymatic treatment.

[0038] 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.

[0039] 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 excellent 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.

[0040] Meanwhile, hair follicle-derived stem cells produced by the method for producing hair follicle-derived stem cells provided by the present invention exhibit immunological characteristics in which CD73, CD90, CD105, Nestin or Lgr5 is expressed on the cell surface, but CD34, CD45 or HLA-DR is not expressed, and they exhibit multilineage characteristics that enable them to differentiate into fat cells, bone cells, etc., but are not limited thereto.

[0041] The hair follicle-derived stem cells may be mixed with hair follicle-derived stem cells produced by the method for producing hair follicle-derived stem cells provided by the present invention, hair follicle-derived stem cells of the present invention, hair follicle-derived stem cells, HB-hair follicle-derived stem cells, HB-hair follicle-derived stem cells of the present invention, and HB-HFSC.

[0042] In one example of the present invention, the proliferation ability, differentiation ability, and colony formation ability of hair follicle-derived stem cells produced by the method for producing hair follicle-derived stem cells of the present invention were confirmed.

[0043] In still another embodiment of the present invention, the hair growth promoting effect of hair follicle-derived stem cells produced by the method for producing hair follicle-derived stem cells of the present invention was confirmed.

[0044] 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.

[0045] Hair follicle-derived stem cells produced by the method for producing hair follicle-derived stem cells provided by the present invention can express a specific hair growth-promoting functional gene.

[0046] In this case, the hair growth-promoting functional gene may be, specifically, any one or more genes selected from among SANP25 (Synaptosome Associated Protein 25), COL10A1 (Collagen Type X Alpha 1 Chain), TMEM119 (Transmembrane Protein 119), AQP1 (Aquaporin 1), PLCB4 (1-Phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-4), and ITGA8 (Integrin Subunit Alpha 8), but is not limited thereto, and may include any gene known as a hair growth-promoting functional gene.

[0047] In one embodiment of the present invention, it was confirmed that the seven hair growth-promoting functional genes were expressed at levels 10 times higher in hair follicle-derived stem cells produced by the method for producing hair follicle-derived stem cells than in fat-derived stem cells, confirming that hair follicle-derived stem cells produced by the method of the present invention exhibit specific hair growth-promoting functional genes.

[0048] Another aspect of the present invention provides a pharmaceutical composition for treating hair loss or promoting hair growth, comprising the hair follicle-derived stem cells, a culture thereof, or cells differentiated from the hair follicle-derived stem cells as an active ingredient.

[0049] 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 hair follicle-derived stem cells in a medium.

[0050] The term "hair growth" or "promotion of hair growth" as used herein is a concept that includes not only the effects of promoting hair growth, increasing the number of hairs, and making hair thicker, but also hair follicle regeneration or proliferation of hair follicle cells, and the proliferation of hair follicle cells can include, but is not limited to, the promotion of the conversion of hair follicle cells from the resting phase to the proliferation phase.

[0051] 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.

[0052] In addition, the composition may further contain pharma- ceutically acceptable additives according to the dosage form, and examples of the pharma- ceutically acceptable additives 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, and talc. The pharma- ceutically acceptable additives according to the present invention may be included in an amount of 0.1 to 90 parts by weight based on the composition.

[0053] The pharmaceutical composition may further contain one or more pharma- ceutically acceptable inactive carriers in addition to the hair follicle-derived mesenchymal stem cells, such as a preservative, a pain-relieving agent, a solubilizer, or a stabilizer in the case of an injection, or a base, an excipient, a lubricant, or a preservative in the case of a topical administration preparation.

[0054] 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 provides maximum efficacy at a minimum amount without inducing side effects, and this can be easily determined by those skilled in the art.

[0055] In addition, the pharmaceutical composition provided by the present invention may further contain various ingredients that help treat hair loss or promote hair growth, maintain the activity of the hair follicle-derived stem cells, or promote the differentiation of the hair follicle-derived stem cells, in addition to the hair follicle-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.

[0056] The pharmaceutical composition of the present invention can be used for the purpose of improving, preventing or treating hair loss.

[0057] The hair loss of the present invention includes both non-scarring alopecia, which is temporary hair loss, and cicatricial alopecia, which occurs when hair follicles or hair roots are permanently destroyed. Non-scarring alopecia includes infectious alopecia, traumatic alopecia, inflammatory alopecia, congenital alopecia, endocrine alopecia, tumor-related alopecia, nutritional deficiency alopecia, drug-induced alopecia, and hair loss due to structural abnormalities of the hair, as well as male pattern hair loss, female pattern hair loss, and alopecia areata.

[0058] The term "amelioration" as used herein means any action of administering a composition according to the present invention to an individual to slow the progression of hair loss or reduce the symptoms of hair loss.

[0059] The term "prevention" as used herein means any action of administering a composition according to the present invention to an individual to inhibit or delay the occurrence of hair loss.

[0060] The term "treatment" as used herein means any act of administering the composition of the present invention to an individual suspected of experiencing hair loss to reverse or benefit the hair loss condition.

[0061] Another object of the present invention is to provide a method for promoting hair growth or preventing or treating hair loss, comprising the step of administering to an individual a composition containing, as an active ingredient, the hair follicle-derived stem cells, a culture thereof, or cells differentiated from the hair follicle-derived stem cells.

[0062] The hair follicle-derived stem cells, cultures thereof, or cells differentiated from the hair follicle-derived stem cells, and the promotion of hair growth, hair loss, prevention, and treatment are as described above.

[0063] 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 hair follicle-derived stem cells of the present invention, the culture thereof, or the 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 hair loss site, but is not limited thereto.

[0064] The composition may also be used in combination with other pharmaceutical compositions used to promote hair growth or to prevent or treat hair loss.

[0065] The term "individual" as used herein means any animal, including humans, that develops or may develop alopecia, such as mice, rats, livestock, etc. The animal may be, but is not limited to, not only humans, but also mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. that require prevention or treatment of symptoms similar to those of humans.

[0066] The compositions of the present invention can be administered in a pharma- ceutically effective amount.

[0067] 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, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0068] 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 in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect at the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0069] 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 the 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.

[0070] Another object of the present invention is to provide a quasi-drug composition for improving hair loss or promoting hair growth, which contains the hair follicle-derived stem cells, their cultures, or cells differentiated from the hair follicle-derived stem cells as an active ingredient.

[0071] 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.

[0072] Another object of the present invention is to provide a cosmetic composition for improving hair loss or promoting hair growth, which contains the hair follicle-derived stem cells, their cultures, or cells differentiated from the hair follicle-derived stem cells as an active ingredient.

[0073] 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.

[0074] Another object of the present invention is to provide a composition for culturing hair follicle-derived stem cells, which contains hair follicle tissue and a hydrogel, and a kit for culturing hair follicle-derived stem cells, which contains the composition.

[0075] In addition, a kit for culturing hair follicle-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.

[0076] Another object of the present invention is to provide a use of a composition comprising, as an active ingredient, the hair follicle-derived stem cells, a culture thereof, or cells differentiated from the hair follicle-derived stem cells for promoting hair growth or preventing or treating hair loss.

[0077] The present invention will be described in more detail with reference to the following examples. However, these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0078] Example 1. Multilayered hydrogel-supported 3D culture of hair follicle tissue Hair follicle tissue samples, which had been immersed in the provided saline and refrigerated, were washed three times with PBS (Gibco). The hair shaft of the washed hair follicle tissue was cut, and a 0.5-1mm portion of the infundibulum was removed from the cut side to form hair follicle tissue slices. 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 samples were transferred to a 100 mm culture vessel containing the same amount of thrombin solution (0.5 IU / mL thrombin (Green Cross) in DMEM) to allow the hair follicle slices and hydrogel to spread evenly, and then left at room temperature for 10 minutes or less to fix the hair follicle tissue to the hydrogel. 5mL each of fibrinogen solution (0.25% w / v fibrinogen, 200μg / mL tranexamic acid in DMEM) and thrombin solution (0.5IU / mL thrombin in DMEM) were added and left for 10 minutes to form a secondary hydrogel, which was then cultured at 37℃ for 2 hours to allow for sufficient gelation (Fig. 1b). 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 10 mg / mL gentamicin) containing 100μg / mL tranexamic acid was added and cultured for 1 hour with stirring at 10-30 rpm. The growth medium was then replaced every 2-3 days.

[0079] Example 2. Recovery and expansion of HB-hair follicle-derived stem cells migrated and grown in a multilayered hydrogel Hair follicle tissue was cultured for 10 or 14 days as described in Example 1. The hydrogel was washed three times for 3 minutes with warm PBS, then 30% FBS / DMEM was added and agitated at 10-30 rpm on an orbital shaker. After 1 hour, the supernatant was removed, and 10 mL of 30% FBS / DMEM containing 1 μg / mL alteplase 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.

[0080] The results of tissue culture and cell isolation and culture performed according to the above method are shown in FIG.

[0081] Example 3. Confirmation of the immunophenotype of isolated and cultured HB-hair follicle-derived stem cells To confirm the immunophenotype of the HB-hair follicle-derived stem cells isolated and cultured by the method of Example 2, cells cultured in a T75 flask were obtained and suspended in 3% bovine serum albumin (Sigma) / PBS. The cell suspension was divided into nine aliquots and incubated for 1 h at 4°C with the following antibodies: FITC-conjugated CD73 (Abcam, 1:25) antibody, APC-conjugated CD90 (Invitrogen, 1:40), FITC-conjugated CD105 (Abcam, 1:50), FITC-conjugated CD34 (Abcam, 1:10) antibody, FITC-conjugated HLA-DR (Novusbiologicals, 1:20) antibody, Nestin (Novusbiologicals, 1:100) antibody, FITC-conjugated Lgr5 (Origene, 1:100) antibody, and FITC-conjugated mouse IgG isotype control (Novusbiologicals, 1:5). After incubation with either antibody or AlexaFluor594-conjugated CD45 (Novus, 1:20) antibody, the cells were washed by centrifugation at 300g for 5 min, suspended in 3% BSA / PBS, and then analyzed by flow cytometry (BD) (Figure 2).

[0082] As a result, as shown in Figure 2, it was confirmed that the isolated and cultured HB-hair follicle-derived stem cells expressed CD73, CD90, CD105, Nestin, and Lgr5, but did not express CD34, CD45, or HLA-DR.

[0083] Example 4. Confirmation of proliferation ability and colony formation ability of isolated and cultured HB-hair follicle-derived stem cells To confirm the proliferation ability of the isolated and cultured HB-hair follicle-derived stem cells obtained in Example 2, the cells were initially cultured at a density of 4,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml and subcultured every 4 days. The doubling time of the consortium depending on the number of subcultures was measured based on the cell count obtained.

[0084] 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 shown in Figure 3: PDT = [days / (logN2-logN1)] / log2, (N1 = 4,000 cells, N2 = number of cells after 4 days).

[0085] In addition, to confirm the colony forming ability of the HB-hair follicle-derived stem cells isolated and cultured in Example 2, human hair follicle-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 / PBS (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.

[0086] As shown in the results of Figures 3 and 4, it was confirmed that the HB-hair follicle-derived stem cells obtained by the method of this Example 2 maintained their proliferation ability through subculture, and also had colony formation ability at a certain level or above.

[0087] In other words, hair follicle-derived stem cells were cultured up to the sixth subculture to determine the population doubling time (PDT). The PDT was observed to be an average of 32 hours ± 7 hours, confirming that proliferation ability was maintained through subculture.

[0088] Example 5. Confirmation of differentiation potential of HB-hair follicle-derived stem cells To evaluate the differentiation ability of the obtained HB-hair follicle-derived stem cells into adipocytes, cells were cultured at 1 × 10 4 pieces / cm 2The cells were seeded at a density of 100x and cultured in growth medium until they reached 80% confluency. The medium was replaced using a StemProTM Adipogenesis differentiation kit (Gibco, cat. A1007001), and the cells were cultured for 14 days, changing the medium every 3 days. The cells were fixed with 4% PFA for 15 minutes, reacted with 60% isopropanol for 15 minutes, and then all the solutions were removed and the cells were thoroughly dried. The cells were stained with 60% Oil Red O (sigma) / ethanol for 15 minutes at room temperature to confirm the presence or absence of lipid accumulation in the cytoplasm (Figure 5a).

[0089] In addition, to evaluate the differentiation ability of the HB-hair follicle-derived stem cells into bone cells, 1 × 10 cells were 4 pieces / cm 2 The cells were seeded at a density of 1000 x 1000 and cultured in growth medium until they reached 80% confluency. The medium was then replaced with a StemProTM Osteogenesis differentiation kit (Gibco, cat. A1007201) and cultured for 14 days, with medium replacement 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).

[0090] That is, based on the above results, it was confirmed that the HB-hair follicle-derived stem cells obtained by the culture method of the present invention have pluripotency.

[0091] Example 6. Identification of differentially expressed genes in HB-hair follicle-derived stem cells compared to adipose-derived stem cells Paired-end RNA libraries were constructed from three lots of samples of adipose-derived stem cells (ASC-1, ASC-2, ASC-3) and HB-hair follicle-derived stem cells (HB-HFSC-1, HB-HFSC-2, HB-HFSC-3) 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 the pretreatment process, 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 expression profiles of each transcript were obtained using the StringTie program (version 2.1.3b, https: / / ccb.jhu.edu / software / stringtie / ) (Figure 6a). Gene expression was treated as logarithmic TPM (Transcripts Per Kiobase Million) values ​​(Figure 6a). Differentially expressed genes between the control and comparison groups were obtained using the edgeR package [1] in the R program (version 4.0) (1. 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.). To compare the expression patterns of differentially expressed genes in stem cells with related disease transcriptome data, publicly available transcriptome data were collected in the GEO (Gene Expression Omnibus) database.The differential gene expression analysis of each public data was performed using the limma package [2] of the R program (version 4.0) (2. 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.). The 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 with a P value of more than 0.05 were reported, the P value was reported separately.

[0092] A biological network in which marker genes participate was constructed using String Database (version 11.5) [3] (3. SMYTH, Gordon K., et al. LIMMA: linear models for microarray data. In Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Statistics for Biology and Health. 2005.) to construct a protein-protein interaction (PPI) network (Figure 7a, b). The organism reference used to construct the PPI network was Homo sapiens. In the network construction settings, edges connecting nodes representing proteins were connected based on experimental results, biological databases, literature, gene fusion, etc., and an edge score of 0.4 or more was considered significant. After constructing the network, orphan nodes with no connectivity to other nodes were removed. The biological functions to which the node groups belong were extracted based on the KEGG database [4] and were only statistically significant (false discovery rate < 0.05) (4. KANEHISA, Minoru; GOTO, Susumu. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic acids research, 2000, 28.1: 27-30.). T-Tests were performed to test for significant differences between the two groups, and P values ​​< 0.05 were considered significant. The statistical test results provided voluntarily by the analysis program were used as is, and a significance level of less than 0.05 was considered significant. When results with a P value of more than 0.05 were reported, the P value was reported separately.

[0093] Using String DB, genes expressed in HB-hair follicle-derived stem cells were analyzed with the hair growth promotion-related gene network to confirm their relationship with known hair growth promotion pathways, and seven hair growth promotion functional genes (SNAP25, ITGA8, PLCB4, COL10A1, GPNMB, TMEM119, AQP1) were derived (Figure 6a). In addition, it was confirmed that HB-hair follicle-derived stem cells expressed genes related to major hair growth functions (TCF3, PRDM1, WNT5A, WNT5B, WNT9A, FGFR2, etc.) at higher levels than adipose-derived stem cells (Figure 6b). 80 genes were identified that were expressed in HB-hair follicle-derived stem cells with a fold change > 10 compared to adipose-derived stem cells, and of these, seven hair growth promotion functional genes derived in Figure 6a (SNAP25, ITGA8, PLCB4, COL10A1, GPNMB, TMEM119, AQP1) showed differential expression compared to adipose-derived stem cells (Figures 6c and 6d). When comparing the gene expression levels of existing hair follicles or hair follicle-derived stem cells with those of HB-hair follicle-derived stem cells, it was confirmed that the expression of seven hair growth-promoting functional genes (SNAP25, ITGA8, PLCB4, COL10A1, GPNMB, TMEM119, AQP1) was increased by more than 10-fold, indicating significant differential expression (Figure 6e).

[0094] The seven hair growth-promoting functional genes are known to be related to Wnt signaling and PI3K signaling, which are signaling pathways known to be associated with hair follicle formation and growth. Based on this, the hair growth-promoting effect of hair follicle-derived stem cells produced by the hair follicle-derived stem cell production method of the present invention can be inferred.

[0095] Furthermore, decreased expression of the CCN2 and TGFB1 genes, which are known to induce the catagen phase of hair follicles, was confirmed (Figure 6b).

[0096] Example 7. Confirmation of hair growth promoting effect in nude mice The experimental animals, 7-12 week old BALB / c nude mice (center experimental animals), were anesthetized with 3% Isoflurane (Hana Pharmaceuticals) by inhalation. After confirming that anesthesia was complete, HB-hair follicle-derived stem cells (1×106 The cells were suspended in 100 μl of normal saline (NS) and injected intradermally into 4 corners (2 × 2 cm) on the dorsal part using an insulin syringe (30 gauge). After administration of HB-hair follicle-derived stem cells, mice were observed for 14 and 31 days, and the administration sites were photographed with a digital camera immediately after administration (day 0), and on days 3, 7, 14, 28, and 31. 2 The area of ​​hair growth was measured using Image J (NIH) based on the percentage of hair growth (Figures 7a and 7b). As a result, the hair growth promoting effect of the HB-hair follicle-derived stem cells was confirmed.

[0097] Example 8. Confirmation of hair follicle formation and increased skin thickness in nude mice For histological analysis, the skin tissue was fixed in a fixative (4% formaldehyde, sigma) for 24 hours, then embedded in paraffin, sliced, and stained with H&E (hematoxylin & eosin). Morphological changes in hair follicles and skin layer thickness were observed using an optical microscope. The number of hair follicles formed and the thickness of the epidermis, dermis, and hypodermis were measured using Image J (Figure 8). 14 and 31 days after administration of HB-hair follicle-derived stem cells, hair follicle formation and increased skin thickness were confirmed.

[0098] Example 9. Confirmation of the mechanism of action of Wnt signal transduction in nude mice As with the H&E staining, the tissues of each group were fixed, embedded in paraffin, and sliced ​​to prepare tissue slides. Immunofluorescence (IF) was performed using a secondary antibody, beta-catenin antibody (Invitrogen, MA1-2001) (Figure 9a), and the tissue was reacted with prehybridization solution (50% formamide, 4X SSC, 50 mM DDT, 4X Denhart's solution, 'XTED, 100 ug / ml denatured salmon sperm DNA, 250 ug / ml yeast RNA) at 42°C for 1 hour to bind to CD34 mRNA (Figure 9b). 31 days after administration of HB-hair follicle-derived stem cells, it was confirmed that beta-catenin expression and CD34 gene expression increased.

[0099] 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 and culturing hair follicle tissue between hydrogel bilayers to obtain a culture; and (b) a method for culturing hair follicle-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 hair follicle tissue, A method for culturing hair follicle-derived stem cells, wherein the hair follicle-derived stem cells have increased expression of one or more genes selected from SNAP25, COL10A1, TMEM119, AQP1, PLCB4, and ITGA8.

2. 2. The method for culturing hair follicle-derived stem cells according to claim 1, wherein the hair follicle tissue is a section obtained by removing a portion corresponding to the hair follicle infundibulum from the epidermal.

3. 2. The method of claim 1, wherein the culture of hair follicle tissue in step (a) is for 3 to 20 days.

4. 2. The method of claim 1, wherein the hair follicle tissues are cultured in step (a) by seeding the tissues at intervals of 1 mm to 50 mm.

5. the hair follicle-derived stem cells express CD73, CD90, CD105, Nestin, and Lgr5; The method for producing hair follicle-derived stem cells according to claim 1, which have the immunological property of not expressing CD34, CD45 and HLA-DR.

6. A hair follicle-derived stem cell in which the expression of one or more genes selected from SNAP25, COL10A1, TMEM119, AQP1, PLCB4 and ITGA8 is increased.

7. the hair follicle-derived stem cells express CD73, CD90, CD105, Nestin, and Lgr5; The hair follicle-derived stem cells according to claim 6, which have the immunological property of not expressing CD34, CD45 and HLA-DR.

8. A pharmaceutical composition for treating hair loss or promoting hair growth, comprising the hair follicle-derived stem cells according to claim 6, a culture thereof, or cells differentiated from said hair follicle-derived stem cells as an active ingredient.

9. A quasi-drug composition for improving hair loss or promoting hair growth, comprising the hair follicle-derived stem cells according to claim 6, a culture thereof, or cells differentiated from said hair follicle-derived stem cells as an active ingredient.

10. A cosmetic composition for improving hair loss or promoting hair growth, comprising the hair follicle-derived stem cells according to claim 6, a culture thereof, or cells differentiated from said hair follicle-derived stem cells as an active ingredient.

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