KR101943203B1 - Process for differentiation into dermal papilla cells from adipose-derived stem cells and uses thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising (a) an adipocyte stem cell comprising a sex-determining region (Y-box2), beta-catenin and a platelet-derived growth factor-A Introducing at least one of the genes selected from the group; And (b) culturing the cell into which the gene has been introduced in a medium to obtain a papilloma-like cell; To a method for differentiating adipose-derived stem cells into dermal papilla cells.

Description

TECHNICAL FIELD The present invention relates to a method for differentiating dermal papilla cells into adipose-derived stem cells and uses thereof,

The present invention relates to a pharmaceutical composition comprising (a) an adipocyte stem cell comprising a sex-determining region (Y-box2), beta-catenin and a platelet-derived growth factor-A Introducing at least one of the genes selected from the group; And (b) culturing the cell into which the gene has been introduced in a medium to obtain a papilloma-like cell; To a method for differentiating adipose-derived stem cells into dermal papilla cells.

As interest in cosmetics has recently increased, interest in the treatment of alopecia has also increased. Hair loss refers to the state of hair where hair normally should be. Hair does not have important physiological functions directly related to life, but it plays a very important role from a cosmetic point of view. In addition, it has UV blocking function and hair protection function. If hair loss is severe, there may be a problem in social life, and it can have a serious psychological impact, which is important in terms of quality of life.

Hair loss can be divided clinically into scarring hair loss accompanied by scarring and non-scarring hair loss that only hair falls. In case of cicatricial hair loss, the hair does not come back because the hair follicles are destroyed. Hair is made in a place called a hair follicle, and each hair follicle periodically goes through stages of activity and suspension. The temporal interval of the hair cycle varies depending on the body part. In the case of the hair, it enters the telogen of about 3 ~ 4 months through the anagen of 2 ~ 6 years and the catagen of 2-4 weeks. do. Each hair follicle has a hair follicle growth cycle of 10 to 20 times a lifetime.

Currently, hair transplantation is the most commonly used surgical procedure for hair loss treatment, and drug therapy using propecia and minoxidil is widely practiced. In the case of medication, the hair growth effect appears at the time of administration, but when the treatment is stopped, hair loss resumes. Especially, in case of minoxidil, there are side effects such as sexual dysfunction. Recently, gene therapy related to hair loss is transferred to hair follicles or gene therapy is blocked. However, since the efficacy, cost, and stability of treatment are uncertain, clinical application is not easy .

In addition to gene therapy, stem cell-based hair loss treatment methods have been highlighted. This method utilizes the pluripotency of stem cells, which induces hair cell differentiation into hair follicles by injecting stem cells into hair loss or hairless symptoms.

It has been reported that adipose stem cells (ASC) secrete growth factors and induce anagen of the hair cycle. Recently, fat transplantation using adipose stem cells has been actively performed on the face, but the adipose stem cells can not form new hair follicles at the site of transplantation of the face. On the other hand, hair growth of adipose stem cells can increase the density and thickness of hair by acting as a source of stimulation of the substances secreted by adipose stem cells in existing hair follicles. However, it is reported that adipose stem cells are less able to regenerate hair than dermal papilla cells in patch assay. The main growth factors secreted by adipose stem cells are effective for alopecia, but not for male hair loss caused by hair follicle damage There is a problem.

Korean Patent No. 10-1425653 discloses that a cell treatment agent prepared by mixing an adult tissue-derived adult stem cell and a scalp tissue-derived hair follicle cell in an appropriate ratio can be applied to symptoms such as alopecia and asphyxia, Korean Patent No. 10-1721228 discloses that a fat-derived stem cell culture fluid expressing LL-37 or EGR-1 is excellent in hair growth effect and thus can be very useful as a raw material for hair loss prevention and hair growth promoting agent There is a bar.

However, transfection of a vector containing Sox2, beta-catenin, and PDGF-A into a virus, followed by transfection of adipose derived stem cells to differentiate into adipocytes directly from adipose-derived stem cells And the effect of treating hair loss in differentiated dermal papilla cells has never been reported.

The present invention relates to a pharmaceutical composition comprising (a) an adipocyte stem cell comprising a sex-determining region (Y-box2), beta-catenin and a platelet-derived growth factor-A Introducing at least one of the genes selected from the group; And (b) culturing the cell into which the gene has been introduced in a medium to obtain a papilloma-like cell; Derived stem cells into dermal papilla cells.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention relates to a pharmaceutical composition comprising (a) an adipocyte stem cell comprising a sex-determining region (Y-box2), beta-catenin and a platelet-derived growth factor-A Introducing at least one of the genes selected from the group; And (b) culturing the cell into which the gene has been introduced in a medium to obtain a papilloma-like cell; The present invention provides a method of differentiating adipose-derived stem cells into dermal papilla cells.

(c) culturing the mammary epithelial cells in step (b) three-dimensionally.

The dermal papilla cells may be transdifferentiated from the fat-derived stem cells of step (a).

In step (a), the introduction of the gene encoding the Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A May be transformed into a recombinant expression vector into which the polynucleotide has been inserted.

The recombinant expression vector may be a recombinant viral vector, a plasmid, or a transposon.

The recombinant viral vector may be selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpesvirus vector, a lentivirus vector, An avipox virus vector, and the like.

One embodiment of the present invention is directed to a method of treating a disease selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) The present invention provides a cell therapy composition for prevention or treatment of hair loss comprising an adipose-derived stem cell overexpressing any one or more of the genes.

Another embodiment of the present invention is a method for the treatment and / or prophylaxis of a disease selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A A stem cell culture solution in which at least one of the genes is overexpressed, as an active ingredient, is provided.

Wherein the overexpression is selected from the group consisting of genes selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and PDGF-A (platelet-derived growth factor-A) One or more of which may be induced by transfection into adipose-derived stem cells.

The culture medium may be a supernatant obtained by culturing the adipose-derived stem cells for 1 to 7 days, collecting the culture medium, and centrifuging the culture medium.

The composition may be a cosmetic composition.

The cosmetic composition of the present invention can be used for hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair book, hair treatment, eyebrow hair extender, Shampoo or a rinse for a pet.

The present invention relates to a method for the detection of any one of genes selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) Derived stem cells are differentiated into dermal papilla cells. The adipose derived stem cells are characterized in that they have superior proliferation power and do not age as compared to undifferentiated stem cells. In addition, when the adipose-derived stem cells are transplanted into a dormant-induced mouse, hair follicles are formed by inducing a growth period to induce hair follicles. By transplanting the adipose-derived stem cells into a hair follicle damaged area, Can be easily used for treating hair loss.

In addition, any one or more of genes selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and PDGF-A (platelet-derived growth factor-A) The over-expressed adipose-derived stem cell culture fluid can secrete various growth factors from the adipose-derived stem cells, thereby inducing the growth phase of the mother cycle, and thus can be used for preventing hair loss or promoting hair growth.

1 is a schematic diagram showing a method for differentiating adipose-derived stem cells into dermal papilla cells.
FIG. 2 shows the cell shape of the expressed papilloma-like cells after culturing the cells transduced with the virus and then colonizing the cells and subculturing the cells.
Fig. 3 shows the appearance of a cell through continuous passage of fat-derived stem cells.
Figure 4 shows the expression levels of CORIN, NES, ACTA1, ALP and VCAN in adipose derived stem cells, dermal papilloma-like cell-colony and dermal papillary-50% dermal papilla cell culture medium.
FIG. 5 shows changes in the expression level of versican while 14-fold subculture of adipose stem cells.
Fig. 6 (a) shows the results of weighing hair (b) hair after 12 days of injecting fat-derived stem cells and epidermoid-like cells into epilated mouse.
FIG. 7 shows (a) the results of follicle production of the patch assay (b) according to Experimental Example 1-2.

The present inventors transduced a vector containing Sox2, beta-catenin and PDGF-A into a virus and then transfected into adipose-derived stem cells to directly differentiate into adipocytes from adipose-derived stem cells And confirming the effect of treating hair loss in differentiated dermal papilla cells.

Hereinafter, the present invention will be described in detail.

As used herein, the term " adipose-derived stem cell " means a stem cell derived from adipose tissue and having a pluripotency and autoproducing ability. The adipose-derived stem cell of the present invention is obtained by liposuction and various surgical operations But is not limited thereto.

As used herein, the term " three-dimensional (3D) culture " means a method in which a cell is cultured in a floating state in a liquid medium without adhering to the culture dish.

As used herein, the term " trans-differetiation " is a method of directly differentiating differentiated cells into different cells, and is used in combination with heterologous differentiation.

Of adipose-derived stem cells Dairy cattle  Method of differentiation into cells

The present invention relates to a pharmaceutical composition comprising (a) an adipocyte stem cell comprising a sex-determining region (Y-box2), beta-catenin and a platelet-derived growth factor-A Introducing at least one of the genes selected from the group; And (b) culturing the cell into which the gene has been introduced in a medium to obtain a papilloma-like cell; The present invention provides a method of differentiating adipose-derived stem cells into dermal papilla cells. Specifically, the dermal papilla cells differentiated by the method of the present invention can be differentiated from adipose derived stem cells.

First, the differentiation method according to the present invention is a method for differentiating stem cells from adipose-derived stem cells (Sox2), beta-catenin and platelet-derived growth factor-A (PDGF- (Step (a)) of introducing one or more genes selected from the group consisting of genes. Specifically, the Sox2 gene is a transcription factor essential for maintaining self-renewal or pluripotency of undifferentiated embryonic stem cells, and the beta -catenin is involved in cell-cell adhesion and gene Regulation and regulation of the transcription of the cells. Platelet-derived growth factors promote vascular growth, cell replication and skin formation. Thus, the adipose-derived stem cells into which the gene has been transduced can be transformed into dermal papilla cells, thereby promoting hair follicle formation.

The adipose-derived stem cell into which the gene of the present invention is introduced is a stem obtained by introducing a vector containing the gene in a form capable of expressing the primary cultured stem cell or sub-cultured stem cell of the primary culture stem cell, It is a cell.

In addition, the introduction of the gene into the adipose-derived stem cells of the present invention can be accomplished by introducing a gene encoding Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor (PDGF-A, platelet- ), And the recombinant expression vector may be a recombinant viral vector, a plasmid, or a transposon. However, the recombinant expression vector may be a recombinant viral vector, a plasmid, or a transposon. Specifically, the recombinant viral vector may be an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpesvirus vector, a lentivirus vector, vector, and an avipox virus vector. The vector may be a lentiviral vector, but is not limited thereto.

Furthermore, the vector can be transfected into adipose derived stem cells using known transfection methods such as microinjection (Capecchi, MR, Cell 22, 479 (1980)), calcium phosphate (EMBO J. 1, 841 (1982)), liposome-mediated transfection methods (Wong et al., J. Biol. , DEAE-dextran treatment (Gopal, Mol. Cell Biol. 5, 1188-1190 (1985)), and gene bend buddhism (Yang et al. Proc. Natl. Acad. Sci. USA 87, 9568-9572 (1990)), but are not limited thereto.

Next, the differentiation method according to the present invention includes a step of culturing the cell into which the gene has been introduced in a medium to obtain a papillary-like cell (step (b)). Specifically, an antibiotic resistance gene may be additionally added to the culture medium of the cell into which the gene has been introduced, as a selection marker of the vector, and an antibiotic resistance gene may be added to the culture medium. , Kanamycin, geneticin, neomycin, puromycin and tetracycline, but is not limited thereto.

In addition, the differentiation method according to the present invention may further include a step (c) of three-dimensionally culturing the dermal papillary-like cells of the step (b), wherein the dermal papilloma- ), Hair follicle promotion can be promoted, and hair growth promoting effect can be increased.

Therefore, the method of differentiating adipose-derived stem cells into dermal papilla cells according to the present invention can easily cultivate even a small amount of adipose-derived stem cells and has a high proliferation ability, so that the number of cells capable of being used for treating hair loss can be rapidly secured .

Cell Therapeutic Agent composition for preventing or treating hair loss

The present invention relates to a method for the detection of any one of genes selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) Derived stem cell as an active ingredient. The present invention provides a cell therapy composition for prevention or treatment of hair loss. The specific contents of the adipose-derived stem cells are as described above.

Specifically, " hair loss " which can be prevented or treated by the composition of the present invention is a phenomenon in which the hair completely escapes from the scalp and can prevent or treat male pattern hair loss and female pattern hair loss, Prevention or treatment, but is not limited thereto.

Therefore, a gene selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) Derived stem cells are transfected into dermal papilla cells. Thus, as compared with undifferentiated lipid-derived stem cells, it is not only efficient to induce the growth of the mother cycle, but also can form new hair follicles when transplanted into skin , To treat hair loss, or to stimulate hair growth.

Composition for preventing hair loss or promoting hair growth

The present invention relates to a method for the detection of any one of genes selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) Derived stem cell culture liquid as an active ingredient. The composition for promoting hair loss prevention or hair growth is provided. The specific contents of the adipose-derived stem cells are as described above.

Specifically, the overexpression is selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) genes Can be induced by transfecting at least one of the genes into adipose-derived stem cells.

In addition, the adipose-derived stem cell culture solution according to the present invention may be a supernatant obtained by culturing the adipose-derived stem cells for 1 to 7 days, collecting the culture medium and centrifuging, and preferably culturing for 1 to 4 days But is not limited thereto. When the culture period of the adipose-derived stem cells is less than the above range, the yield of the proteins secreted from the adipose-derived stem cells and the cells differentiated from the cells is lowered. If the culture period exceeds the above range, There is a problem that apoptosis of cells and differentiated cells is promoted.

The composition of the present invention may be prepared from a pharmaceutical composition, a quasi-drug composition or a cosmetic composition.

When the composition of the present invention is prepared from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It does not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, mucosal administration, and topical administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . Preferably, the dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg (body weight) on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

When the composition of the present invention is prepared with a quasi-drug composition, the adipose-derived stem cells exhibiting the effect of preventing hair loss or promoting hair growth can be directly added, used in combination with other quasi-drugs or quasi-drugs, have. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition for preventing hair loss or promoting hair growth of the present invention is not particularly limited in its formulation and may be variously formulated in the form of a quasi-drug known in the art to exhibit hair loss prevention or hair growth promoting effect. The formulated quasi-drugs include hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair removal agent, eyelash hair growth agent, Shampoos, pet rinse, hand cleaner, detergent soap, soap, disinfectant cleaner, wet tissue, mask, ointment, patch or filter filler, and all the quasi-drugs in the usual sense.

In addition, the quasi-drug composition for preventing hair loss or promoting hair growth in each formulation can be blended with other components arbitrarily selected depending on the formulation of the other quasi-drugs, the purpose of use, and the like. The amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use, and may include, for example, customary adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

The content of the composition is preferably 0.0001 to 10% by weight based on the total weight of the composition. If it is more than 10% by weight, the color and stability of the composition are poor. When the composition is less than 0.0001% by weight, . The quasi-drug composition containing decane-alkaline as an active ingredient of the present invention has little toxicity and side effects on cells as confirmed by the treatment index, and thus can be useful as a quasi-drug material.

When the composition of the present invention is prepared with a cosmetic composition, the components contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the fat-derived stem cells as an active ingredient. Examples thereof include antioxidants, Customary adjuvants such as solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but are not limited thereto.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

As described above, among the genes selected from the group consisting of Sox2 (sex determining region Y-box2), beta-catenin and PDGF-A (platelet-derived growth factor-A) The adipose-derived stem cell culture solution overexpressing any one or more of the above can secrete various growth factors from the adipose-derived stem cells, thereby inducing the growth phase of the mother cycle, and thus can be used for preventing hair loss or promoting hair growth.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Preparation Example ]

Preparation Example  1. Isolation of adipose stem cells

Subcutaneous adipose tissue was secured during the operation under the consent of the patient, which was approved by Shinchon Severance Hospital of Yonsei University. To isolate adipose stem cells, adipose tissue was treated with the same amount of 0.1% collagenase type I (Sigma, US) solution and treated at 37 ° C for 60 minutes with intermittent mixing. Suspended adipocytes were isolated from the stromal vascular fraction by centrifugation at 1,500 rpm for 10 min. The cell pellet was suspended in DMEM / low glucose medium supplemented with 10% FBS, 100 U / ml penicillin and 100 [mu] g / ml streptomycin and then plated in tissue culture dishes. Invasive adipose stem cells were cultured for 4 to 5 days until the dish reached saturation, which was set to zero. In the experiment, adipose stem cells of passage 4 to 6 were used.

Preparation Example  2. Experimental animal  breed

2-1) Eighteen male C3H / HeN mice, 5 weeks old, were purchased from Orient Biotechnology. Adapted to the breeding environment for 1 week, the hair was short-pressed using a hair removal machine, After complete removal, it was stabilized for one day and used in the experiment.

2-2) Epidermal and dermal sections were prepared by stripping skin tissue from six male C3H / HeN mice at birth and using 1 mg / ml collagenase / dispase.

[ Example ]

Example  One. Dairy cattle  Cell-differentiable Of adipose-derived stem cells  Produce

Adipose-derived stem cells were added to α-MEM medium and cultured in a 12-well plate at 1 × 10 ⁴ cells / sell for 24 hours. Lenti-virus expressing Sox2, β-catenin and PDGF-A gene was then transduced into the adipose-derived stem cells and further cultured for 24 hours. Then, the medium was changed and cultured for 2 to 3 days. Antibiotics resistant to the respective genes were added to the medium to selectively acquire transduced cells only. Sox2 resistant antibiotic puromycin, β-catenin resistant antibiotic blasticidin, and PDGF-A resistant antibiotic G418 were treated at concentrations of 2 μg / ㎖, 15 μg / ml and 1.5 mg / ml, respectively. Three days later, the virus-free cells died and only the cells with all three genes survived. When the surviving cells are cultured in fresh medium for 3 days, colony-type cells are observed to grow together. When the above phenomenon is observed, the cells are cultured once for 7 to 10 days after subculture. When the cell density was 80 to 90% of the culture dish, subculture was repeated 15 times.

Example  2. Quantitative- PCR  for With RNA securing  cDNA synthesis

The fat-derived stem cells prepared in Example 1 were dissolved in Trizol and stored at room temperature for 2 minutes. Then, a 1/5 volume of chloroform solution was added and shaken vigorously for 15 seconds. The well-mixed solution was centrifuged at 4 to 12,000 g for 15 minutes. Then, the upper layer of the two-layered solution was obtained, and the same volume of isopropanol was added thereto, and the mixture was stored at room temperature for 10 minutes. After centrifugation at 4 to 12,000 g for 10 minutes, the solution was removed, 75% ethanol was added to the pellet that had been submerged, and centrifuged at 4 to 7,500 g for 5 minutes. The ethanol added was removed, and the pellet was dried at room temperature. The pellet was dissolved in DEPC water and quantified.

CDNA was synthesized with 50ng / 의 oligo (dT) primer, 10mM dNTP, and 10,000U reverse transcriptase using 500ng of quantified total RNA. Using the cDNA, the degree of expression of genes such as versican, corin, NES, ACTA1 and ALP (alkaline phosphatase) having characteristics of hair follicle cells was observed by quantitative-PCR.

As a result, as shown in Fig. 4, the expression levels of versican, corin, NES, ACTA1 and ALP genes having the characteristics of hair follicle cells in the adipose-derived stem cells of Example 1 were significantly . Specifically, when the density of the papillary-like cells in the culture medium was 50% as compared with the papillary-like cell-colony-like cells formed by differentiation of the adipose-derived stem cells of Example 1, the expression level of the gene was significantly , Respectively. That is, when the adipose-derived stem cells according to the present invention are cultured, genes expressing hair follicle cell characteristics can be expressed. As a result, the adipose-derived stem cells are differentiated into dermal papilla cells by conversion differentiation.

Example  3. 3D Current  Hanging drop culture

3-1) The adipose-derived stem cells of Example 1 were differentiated into papillary-like cells, and the epidermal cells isolated from the mice of Preparatory Example 2-2 were mixed with the above-described papilloma-like cells. The mixed cells were then cultured for 72 hours.

3-2) The adipose-derived stem cells of Example 1 were differentiated into papillary-like cells, and then the papillary-like cells were cultured for 48 hours. Then, the epidermal cells isolated from the mouse of Preparation Example 2-2 were mixed with the above-cultured cells and further cultured for 48 hours.

[ Experimental Example ]

Experimental Example  1. Animal model experiment

1-1. Hair regeneration experiment

1 x 10 ⁴ cells of differentiated papillary-like cells in the adipose-derived stem cells of Example 1 were mixed with 100 μl of PBS and subcutaneously injected once into the dorsal part of the mouse prepared in Preparation Example 2-1. The amount of hair growth of the mouse was examined for 12 days after the injection of cells. The same number of adipose stem cells before differentiation into the positive control group was injected subcutaneously and PBS was subcutaneously injected into the negative control group. After 12 days, the back of the hair of the mouse was re-shaved using a razor, and hair was obtained and weighed.

As a result, as shown in Fig. 6B, in the case of mice subcutaneously injected with differentiated dermal papillary-like cells in the adipose-derived stem cells of Example 1, the weight of the hair was remarkably increased compared with the negative control group, And 50% p or more when compared with the control group.

1-2. Patch assay

The skin tissue of the mouse obtained in Preparation Example 2-1 was disinfected twice with povidone iodine solution and then washed twice with PBS. The disinfected skin tissue was fragmented and stored at 4 in 1 mg / ml collagenase / Dispase. After 24 hours, the skin tissue was separated into epidermal part and dermal part, each tissue was put into 0.25% Triple E solution, cut into pieces and stored at 37 for 15 minutes. Then, FBS was added and vortexed for 15 minutes. Each of the skin tissues was sequentially filtered through a screen of 100 탆 and 70 탆, centrifuged at 15,000 rpm for 5 minutes, and then the number of cells was measured. Each of 1 × 10 ⁶ epidermal cells and dermal cells was mixed and subcutaneously injected into BALB / c-nude mice. After about two weeks, the presence or absence of hair on the mice was observed.

The cells prepared in Examples 3-1 and 3-2 were subcutaneously injected into BALB / c-nude mice, and after about 4 weeks, the presence or absence of hair was observed in the mice.

Experimental Example  2. Hematoxylin  & Eosin Dyeing

The skin tissues of the cells injected with the mouse of Experimental Example 1-2 were cryo-sectioned and then stuck on a slide glass and stored in a deep freezer (-70). For H & E staining, the tissues stored in the cold freezer were dried at room temperature for 20 minutes. The dried tissue was treated with 95% ethanol for 2 minutes and then with 70% ethanol for 2 minutes. The tissue was rinsed with DW for 3 minutes and stained with hematoxylin for 3-10 minutes. The tissue was then rinsed with DW for 5 minutes and further stained with Eosin for 1 to 5 minutes. The stained tissue is plated in DW for 1 minute, then soaked in 70% ethanol for 1 minute, 95% ethanol for 1 minute and finally 100% ethanol for 3 minutes. After complete drying and mounting, the tissues were observed under a microscope.

As a result, as shown in Fig. 7A, when the cells of Example 3-1 in which epidermal cells of the mammary epithelioid cells and the mouse were mixed were injected subcutaneously into a mouse or the like, after 4 weeks, , And it was confirmed that hair was formed at this site. Further, as shown in FIG. 7B, the black skin of the mouse skin was cut and H & E stained. As a result, hair follicles and hair were newly formed.

Experimental Example  3. Prevention of hair loss or promotion of hair growth

3-1. Fat stem cells and Dairy cattle  Concentration of differentiated cell culture

The adipose stem cells and the mammary epithelial differentiation cells were each seeded in a 150 mm dish to 1.2 × 10 ⁶ cells. At this time, a 1: 1 mixture of alpha-MEM without fBS and follicle dermal papilla growth media was used. After culturing for 3 days, the medium was collected in one place and filtered to 0.2 탆. Then, the culture was concentrated with 3,000 MWCO membrane using VIVASPIN 20 (Sartorius), diluted with PBS at 100X, and stored in the refrigerator.

3-2. Organ culture

The beard portion of 4 week old C57BL / 6 male mice was obtained and the hair follicles were individually isolated. The prepared adipose stem cell culture media and the mammary epithelial differentiation cell culture solution were diluted to 5X using Williams E media for organ culture, and each hair follicle was cultured for 3 days. Table 1 shows the parent cycle state of mouse hairy bittern (vibrissae) hair follicle after treatment of cell culture with hair follicles isolated from the mouse hairy area.

Growing season A retrograde / rest period Control group 2 6 Fat stem cell culture fluid 0 8 Differentiation 6 2

As shown in Table 1, it was confirmed that the growth period was maintained better in the hairs cultured in the culture medium of the differentiating cells of the mammary epithelium compared to the hairs cultured in the culture medium of the adipose stem cells. There were 6 growing stages of hair growing in the culture of differentiated female molds, and most of the hair growing in the adipose stem cell culture medium was found to be in the regressive phase and the resting state. That is, since the adipocyte differentiation cell culture fluid according to the present invention differentiates into adipogenic cells, it secretes various growth factors and can induce the growth period of the mother cycle.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (12)

(a) introducing Sox2 (sex determining region Y-box2), beta-catenin and PDGF-A (platelet-derived growth factor-A) gene into adipose derived stem cells; And
(b) culturing the cell into which the gene has been introduced in a medium to obtain a papillary-like cell; Derived stem cells into dermal papilla cells.
The method according to claim 1,
(c) culturing the mammary epithelial cells of step (b) in a three-dimensional manner
A method of differentiating adipose derived stem cells into dermal papilla cells.
The method according to claim 1,
Wherein the dermal papilla cells are trans-differentiated from the fat-derived stem cells of step (a)
A method of differentiating adipose derived stem cells into dermal papilla cells.
The method according to claim 1,
The gene transfection in step (a) is performed using polynucleotides encoding Sox2 (sex determining region Y-box2), beta-catenin and PDGF-A (platelet-derived growth factor-A) Is transformed into an inserted recombinant expression vector.
A method of differentiating adipose derived stem cells into dermal papilla cells.
5. The method of claim 4,
Wherein the recombinant expression vector is a recombinant viral vector, a plasmid or a transposon
A method of differentiating adipose derived stem cells into dermal papilla cells.
6. The method of claim 5,
The recombinant viral vector may be selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpesvirus vector, a lentivirus vector, Wherein the vector is selected from the group consisting of avipox virus vectors
A method of differentiating adipose derived stem cells into dermal papilla cells.
Derived stem cells overexpressed with the gene for Sox2 (sex determining region Y-box2), beta-catenin and PDGF-A (platelet-derived growth factor-A) A cell therapy composition for prevention or treatment of hair loss.
Derived stem cell cultures overexpressing the genes for Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF-A) Or hair growth promoting composition.
9. The method of claim 8,
The overexpression transfected the fat-derived stem cells with the Sox2 (sex determining region Y-box2), beta-catenin and platelet-derived growth factor-A (PDGF- Characterized in that
A composition for preventing hair loss or promoting hair growth.
9. The method of claim 8,
Wherein the culture solution is a supernatant obtained by culturing the adipose-derived stem cells for 1 to 7 days and then centrifuging the culture broth
A composition for preventing hair loss or promoting hair growth.
9. The method of claim 8,
Wherein the composition is a quasi-drug composition
A composition for preventing hair loss or promoting hair growth.
12. The method of claim 11,
The quasi-drug composition may be used for hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair book, hair treatment, eyebrow hair removal agent, eyelash hair growth agent, Shampoo, or a rinse for a pet.
A composition for preventing hair loss or promoting hair growth.

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