KR101721228B1 - Composition for Promoting Hair Growth Comprising Culture Medium of Adipose-Derived Stem Cells - Google Patents

Abstract

The present invention provides an adipose-derived stem cell culture fluid having hair growth promoting ability, a method for producing the same, and a hair growth promoting composition containing the same. The adipose-derived stem cell culture fluid expressing LL-37 or EGR-1 of the present invention is excellent in hair growth effect and can be very usefully used as a raw material for hair loss prevention and hair growth promoting agent. The composition of the present invention is highly likely to be used as a hair loss treatment agent that maximizes hair growth efficiency and does not show side effects.

Description

TECHNICAL FIELD The present invention relates to a composition for promoting hair growth comprising a culture medium of adipose-derived stem cells as an active ingredient,

The present invention relates to a composition for stimulating hair growth comprising a culture medium of adipose-derived stem cells as an active ingredient.

Minoxidil and Propecia, currently used hair loss treatments, have side effects such as erectile dysfunction and skin troubles accompanied by inflammation, and hair transplantation is also accompanied by pain. The cost is 5,000-7,000 won per hair, There is a problem that it causes an economic burden of about 7 million won and requires continuous management. In order to overcome the side effects of hair loss treatment and hair transplantation, research on the possibility of treating hair loss using an adipose stem cell culture fluid and development of a therapeutic agent are under way. Basically, the use of culture medium of human adipose stem cells is expected to have a small adverse effect, and the possibility of growth as a hair loss therapeutic agent is considered to be very high by treating a root cause rather than a simple scalp treatment. However, since the stem cell culture medium itself has a large difference in the degree of hair regeneration activity depending on the kind of source (donor's age, harvesting site) and culture technique, the systemic and efficient effect of treating hair loss is expected it's difficult.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a stem cell culture solution which can effectively treat hair loss and can be safely applied to human body. As a result, a culture medium of adipose-derived stem cells overexpressing LL-37 or EGR- The present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for promoting hair growth, which comprises a culture medium of adipose-derived stem cells as an active ingredient.

Another object of the present invention is to provide a composition for promoting hair growth comprising LL-37 protein as an active ingredient.

It is still another object of the present invention to provide a hair growth stimulating composition comprising EGR-1 protein as an active ingredient.

It is still another object of the present invention to provide a method for producing a stem-cell culture fluid derived from a fat having hair growth promoting ability.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a hair growth stimulating composition comprising, as an active ingredient, a culture solution of adipose-derived stem cells in which LL-37 (cathelicidin) or EGR-1 (Early growth response protein 1) do.

The present inventors have tried to develop a stem cell culture solution which can effectively treat hair loss and can be safely applied to human body. As a result, a culture medium of adipose-derived stem cells overexpressing LL-37 or EGR- .

As used herein, the term " stem cell " refers to a cell that has the ability to continue proliferation, i. E. Self-replicating, and has the ability to differentiate into different cell types of a particular type.

In the present invention, " adipose-derived stem cells " are stem cells derived from adipose tissue and have pluripotency and autoproducing ability.

According to one embodiment of the present invention, the adipose derived stem cells of the present invention can be obtained through liposuction and various surgical operations.

The culture medium of adipose stem cells in which LL-37 or EGR-1 is highly expressed as an active ingredient of the hair growth stimulating composition of the present invention is transformed with an expression vector containing LL-37 or EGR-1 gene, Derived stem cells.

According to one embodiment of the present invention, the high expression of LL-37 induces an LL-37 expression vector comprising the gene encoding the nucleotide sequence of SEQ ID NO: 3 in a transfected adipose stem cell.

In the present specification, LL-37 is used in combination with CAMP (cathelicidin antimicrobial peptide).

According to another embodiment of the present invention, the high expression of EGR-1 induces an EGR-1 expression vector comprising a gene encoding the nucleotide sequence of SEQ ID No. 4 in a transfected adipose stem cell.

In the present invention, the adipose-derived stem cell into which the gene is introduced is preferably stably transformed into a primary cultured stem cell or a subcultured cell of a primary culture stem cell by introducing a vector containing the gene into a form capable of expressing the gene, ).

In the present invention, the vector used for introducing the therapeutic gene into the stem cell may preferably include, but is not limited to, (i) a lentiviral vector; (Ii) retroviral vectors; (Iii) adeno-associated viral vectors; (Iv) a herpes simplex virus vector; (v) SV40 vector; (Vi) a polyoma virus vector; (Ⅶ) papilloma virus vector; (Vii) picornovirus vectors; (Vii) Vexinia virus vector; (X) adenovirus vectors; () Helper-dependent adenovirus vector.

The origin of replication contained in the vector of the present invention includes, but is not limited to, f1 replication origin, SV40 replication origin, pMB1 replication origin, Adeno replication origin, AAV replication origin, and BBV replication origin.

Promoters that may be used in the vectors of the invention include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter , The SV40 promoter, the cytomegalovirus (CMV) promoter and the tk promoter of HSV) can be used, and the polyadenylation sequence is included as the transcription termination sequence, for example, the SV40 polA sequence and the BGH polA sequence.

The vector used in the present invention is a selection marker and includes an antibiotic resistance gene commonly used in the art, for example, bradisidine, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There are resistance genes for geneticin, neomycin, puromycin and tetracycline.

The specific structure of the expression vector comprising the LL-37 gene of the present invention is shown in Fig. 1 (a), and the specific structure of the expression vector containing the EGR-1 gene is shown in Fig. The nucleotide sequence of the LL-37 gene inserted in the LL-37 expression vector shown in FIG. 1A is the third sequence in the sequence listing, and the nucleotide sequence of the EGR-1 gene inserted in the EGR-1 expression vector shown in FIG. List 4 sequence.

Methods for transfection of the vector of the present invention into a stem cell can be carried out by a known transfection method, for example, microinjection (Capecchi, MR, Cell 22, 479 (1980)), calcium phosphate precipitation EMBO J. 1, 841 (1982)), liposome-mediated transfection (Wong, TK et al., EMBO J., (Gopal, Mol. Cell Biol. 5, 1188-1190 (1985)) and the gene bend buddhite (Yang et al., Proc. Natl. Acad. Sci. USA 87, 9568-9572 (1990)), but are not limited thereto.

The step of selecting the transformed stem cells by the introduction of the vector can be easily carried out by using a phenotype expressed by the above-described selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

According to another aspect of the present invention, there is provided a hair growth promoting composition comprising, as an active ingredient, a culture solution of adipose derived stem cells in which LL-37 or EGR-1 is highly expressed by treatment with Lipo-PGE1 (Prostaglandin E1) to provide.

Lipo-PGE1 used in the present invention was previously used as a therapeutic agent for vasodilators and erectile dysfunction, and its safety in vivo has been verified. In the present invention, by activating adipose stem cells using Lipo-PGE1, efficiency is maximized and side effects As a treatment for hair loss.

According to one embodiment of the present invention, when Lipo-PGE1 is treated to adipose-derived stem cells, mRNA and protein levels of LL-37 are increased, and EGR-1 is highly expressed by the increase of LL-37 expression. The conditioned media of lipo-PGE1-containing adipose-derived stem cells inhibits proliferation and cell death of dermal papilla cells, thereby effectively promoting hair growth.

According to another aspect of the present invention, there is provided a composition for promoting hair growth comprising LL-37 protein represented by the amino acid sequence of the first sequence of Sequence Listing as an active ingredient.

The LL-37 protein of the present invention has the amino acid sequence of SEQ ID NO: 1. The nucleic acid molecule encoding the LL-37 protein of the present invention is most preferably the nucleotide of SEQ ID NO: 3. The nucleic acid molecule of the present invention encoding the LL-37 protein is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art. Homology, more preferably at least 90% homology, most preferably at least 95% homology.

According to another aspect of the present invention, there is provided a composition for promoting hair growth comprising EGR-1 protein represented by the amino acid sequence of the second sequence of Sequence Listing as an active ingredient.

The EGR-1 protein of the present invention has the amino acid sequence of SEQ ID NO: 2. The nucleic acid molecule encoding the EGR-1 protein of the present invention is most preferably the nucleotide of SEQ ID NO: 4. The nucleic acid molecule of the present invention encoding EGR-1 protein is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art. Homology, more preferably at least 90% homology, most preferably at least 95% homology.

The composition of the present invention may be prepared from a pharmaceutical composition or a cosmetic composition.

When the composition of the present invention is manufactured 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 is 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, percutaneous administration, mucosal administration, or topical administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, 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 dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person 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 cosmetic composition, it may be prepared into any of the formulations conventionally produced in the art, and examples thereof include solutions, suspensions, emulsions, pastes, gels, creams, lotions, , Surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, but are not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present 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.

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 cell culture fluid and carrier component as the active ingredient, and examples thereof include antioxidants, stabilizers, solubilizers, vitamins, , ≪ / RTI > and the like.

According to another aspect of the present invention, the present invention provides a method for producing a stem-cell culture medium having a hair growth promoting ability, comprising the steps of:

(a) separating and culturing adipose derived stem cells by treating the subcutaneous fat tissue isolated from the subject with collagenase enzyme and centrifuging;

(b) a step of (i) transfecting the expression vector of the LL-37 gene or the expression vector of the EGR-1 gene into the adipose stem cells cultured in the step (a), or (ii) ) To produce adipose-derived stem cells highly expressing LL-37 or EGR-1; And

(c) culturing the prepared adipose-derived stem cells to obtain a culture solution.

The method for producing the adipose-derived stem cell culture liquid of the present invention will be described in detail as follows.

The step (a) is a step of isolating and culturing adipose stem cells from subcutaneous adipose tissue isolated from an individual. First, the collagenase enzyme is mixed with fat tissue at a weight ratio of 1: 1, and then 40- Treated for 90 minutes, and centrifuged to isolate adipose derived stem cells.

The " collagenase enzyme " is an enzyme that promotes the hydrolysis of collagen, and serves to separate collagen of adipose tissue to separate adipose stem cells.

The centrifugation can be performed at 1,000-2,000 rpm for 3-15 minutes, preferably at 1,500 rpm for 5 minutes to obtain a supernatant, remove lipid and fat layer suspended in the supernatant, .

The isolated adipose-derived stem cells were cultured in DMEM (Dulbecco's Modified Eagle Media) supplemented with 10% FBS (Fetal Bovine Serum), 100 units / ml penicillin and 100 μg / ml streptomycin at 37 ° C in a 5% CO ₂ incubator , And subcultured when the confluence of the cells reaches 80-100%. As the culture method, a cell culture method known in the art can be applied.

The step (b) is a step of producing an adipose-derived stem cell highly expressing the LL-37 gene or the EGR-1 gene, wherein the LL-37 expression vector or the EGR-1 expression vector is cultured in the step (a) Derived stem cells highly transfected with LL-37 or EGR-1 by transfecting stem cells or by treating Lipo-PGE1 with adipose-derived stem cells cultured in step (a) above.

The " vector " is a DNA capable of introducing a desired DNA fragment into a host cell or the like in a DNA recombination experiment. The vector DNA is cleaved with a restriction enzyme or the like, inserted into a target DNA fragment, Cells. The vector DNA that connects the target DNA fragments is inserted into the chromosomal DNA of the host cell and is distributed to each cell along with the division of the host to maintain the desired DNA fragment for generations.

According to one embodiment of the present invention, the plasmid expression vector is referred to as " pLenti6 / V5-hCAMP " and " pLenti6 / V5-hEGRl ", which means a plasmid comprising a nucleic acid sequence encoding LL-37 and EGR- do.

The term " transfection " means introducing a gene DNA, a plasmid DNA, a viral DNA or RNA into a cell through a suspension of a cell culture or a cell to induce gene introduction and infection. Specifically, the transfection of the expression vector can be performed using all available methods including calcium phosphate transfection, electroporation, microinjection, and liposome injection, which are well known in the art. Transfection methods can be used. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers.

The step (c) is a step of culturing adipose derived stem cells to obtain a culture solution. The culture solution is centrifuged to remove cell debris, only a supernatant is obtained, and filtering is performed.

The culture medium of the adipose derived stem cells of the present invention produced by the above method is excellent in the hair growth effect in the hair loss mouse model. Therefore, the culture solution of the adipose-derived stem cells of the present invention can be usefully used as a raw material for hair loss prevention and hair growth promoting agent.

According to another aspect of the present invention, there is provided a method of producing a stem-cell culture fluid for fat-derived hair having a hair growth promoting ability, comprising the steps of:

(a) separating and culturing adipose derived stem cells by treating the subcutaneous fat tissue isolated from the subject with collagenase enzyme and centrifuging;

(b) treating the adipose-derived stem cells with an LL-37 protein represented by the amino acid sequence of SEQ ID NO: 1 or an EGR-1 protein represented by the amino acid sequence of SEQ ID NO: 2; And

(c) obtaining a culture from the result of (b) above.

According to another aspect of the present invention, there is provided an adipose-derived stem cell culture solution produced by the above-described method.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides an adipose-derived stem cell culture fluid having hair growth promoting ability, a method for producing the same, and a hair growth promoting composition containing the same.

(Ii) The adipose-derived stem cell culture solution expressing LL-37 or EGR-1 of the present invention is a culture solution of adipose stem cells highly expressing LL-37 or EGR-1 of the present invention, And can be very useful as a raw material for the hair growth promoting agent.

(Iii) The composition of the present invention is highly likely to be used as a hair loss treatment agent that maximizes hair growth efficiency and does not exhibit side effects.

Figure 1 is a map of an expression vector containing LL-37 or EGR-1 gene.
FIG. 2 shows the results of measuring the proliferation effect of dermal papilla cells (DPC) in a conditioned medium (CM) conditioned medium containing adipose-derived stem / stromal cells containing Lipo-PGE1.
FIG. 3 shows the results of confirming the hair growth effect of Lipo-PGE1 CM in C57BL / 6 mice.
FIG. 4 shows the results of measurement of LL-37, EGR1 mRNA and protein expression in adipose-derived stem cells according to the treatment time and concentration of Lipo-PGE1.
FIG. 5 shows anti-apoptotic and proliferative effects of ASC CM overexpressing hCAMP (LL-37) on DPC.
FIG. 6 shows the result of measuring the hairiness effect of ASC-hCAMP CM on C57BL / 6.
FIG. 7 shows the result of measuring the hair regeneration effect of human ASC on EGR-1, which is a gene that is up-regulated by LL-37.
FIG. 8 shows the results of measurement of changes in mRNA and protein levels of EGR-1 by LL-37.
FIG. 9 shows the result of the hair regeneration effect of ASC CM overexpressing EGR-1 in C57BL / 6.
FIG. 10 shows the results of measurement of changes in the secretion of hair regrowth growth factors (thymosin-beta4, VEGF, SDF-1, and MCP-1) by EGR-1.
11 is a schematic diagram showing the hair regeneration effect by Lipo-PGE1.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Methods and Materials

Experimental animal and hair growth test

Five week old C57BL / 6 mice were purchased (Jackson Laboratory) and adapted for 2 weeks. Hair and hair follicles were removed from 7-week-old mice and the like. Each conditioned media was topically applied until day 18.

Cell culture

Human HFDPC (hair follicle dermal papilla cells) was purchased from Promo Cell (Heidelberg, Germany). After primary cell culture, the cells were frozen using serum-free freezing medium and then cryopreserved (500,000 cells per ml). HFDPC medium was prepared and additional kit (Promo Cell, Heidelberg, Germany), basal medium, FCS (fetal calf serum), basic FGF and insulin were additionally ordered for cell culture. A kit containing HBSS (HEPES buffered saline solution), trypsin / EDTA solution and neutralization solution (Promo Cell, Heidelberg, Germany) was used for subculture. The cells were cultured in a 100-mm dish with 10 ml of the medium until the confluency reached about 85% at 37 ° C and 5% CO ₂ humidified conditions.

Adipose stem cells (conditioned media, CM)

Subcutaneous adipose tissue was secured during surgery under the consent of the patient, which was approved by the institutional ethics committee of Samsung Medical Center. 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. Cell pellets were suspended in DMEM / low glucose medium supplemented with 10% FBS, 100 U / ml penicillin and 100 ug / ml streptomycin and then plated in tissue culture dishes. Invasive adipose stem cells were cultured for 4-5 days until the dish reached saturation. In the experiment, adipose stem cells of passage 4 to 6 were used. In some experiments, conditioned medium (CM) was prepared 48 hours after culture of adipose stem cells and was collected by filtering (0.2 μm filter).

Cell proliferation assays using CCK-8

The cell proliferation rate was measured according to the manufacturer's protocol using Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Japan). The CCK-assay was used to measure cytotoxicity in starved conditions. DPC (5 x 10 ⁴ cells / ml) was dispensed into 96-well plates and cultured in an adipocyte stem cell conditioned medium containing Lipo-PGE1 (0, 50, 150, 250 ng / ml) or 250 ng / ml Lipo-PGE1 (0, 50, 75, 100%) were cultured and cultured for 24 hours. After 24 hours, the cells were washed and the degree of cell growth was assessed using a CCK-8 assay. CCK-8 solution (10 [mu] l) was added to each well and cultured at 37 [deg.] C for 1 hour. The absorbance at 450 nm was then measured using a multiplate reader (Spectramax plus 384; Molecular devices). Lipo-PGE1 (Eglandin) used in the present invention was obtained from " Mitsubishi Tanabe Pharma Korea ".

Real-time PCR

Total RNA was isolated using Trizol reagent (Invitrogen). Using SuperScript II reverse transcriptase, 2 μg of total RNA was synthesized according to the manufacturer's instructions. The primer information used was as follows: GAPDH forward, 5'-ATC ACC ATC TTC CAG GAG CGA-3 '; Reverse, 5'-TTC TCC ATG GTG GTG AAG ACG-3 '; LL-37 forward, 5'-CTG ATG CCT CTT CCA GGT GT-3 '; Reverse direction, 5'-GAG GGA GCC CTT TCT GAA TC-3 '; EGR1 forward, 5'-CTT CAA CCC TCA GGC GGA CA-3 '; Reverse, 5'-GGA AAA GCG GCC AGT ATA GGT-3 '. Quantitative PCR for real-time PCR was performed in the Applied Biosystems (Foster City, Calif.) 7900 Real Time PCR System using the HotStart-IT SYBR Green qPCR master mix kit. Cycling information for real-time PCR (50 cycles) is as follows: 10 min at 95 ° C, 15 sec at 95 ° C and 60 sec at 60 ° C. The Ct (comparative threshold cycle) method, 2 ^-ΔΔCt, was used to calculate the amplification ^factor .

The enzyme-linked immune sorbent assay (ELISA)

Cells were plated in 6-well plates (1,000,000 cells / well) and treated with Lipo-PGE1 at concentrations (0 [control], 50, 150, 250 ng / ml) in serum-free medium. The conditioned media was collected for LL-37 assay after 24 and 48 hours. The LL-37 assay was performed according to the manufacturer's instructions of an ELISA kit (Hycult biotechnology, USA).

Western blot analysis

The total protein of the cell lysate was separated by SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane and incubated with primary antibody [LL-37 (Santa Cruz, USA), EGR-1 (Santa Cruz sc-101033, USA) , And GAPDH (Cell Signaling, USA)] and incubated overnight at 4 ° C. The washed membrane was immersed in an anti-mouse secondary antibody conjugated with HRP (horseradish peroxidase) and incubated for 1 hour. Bands were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Piscataway, NJ) and quantified using Totallab software (Totallab, UK).

Analysis of anti-apoptosis by LL-37

LL-37 (10 or 20 μg / ml) (Phoenix Pharmaceuticals, Inc.) was treated for 30 hours in serum-free medium after incubation for 8 hours with ASC (1 × 10 ⁶ cells / The ASC-CMs were treated with DPC for 48 hours, followed by 10 μM DHT for 1 hour and 30 minutes, followed by Annexin-V and 7-AAD staining. To this end, cells were first obtained and 100 μl of the cell culture (1 × 10 ⁵ cells) was transferred to a culture tube, followed by addition of FITC annexin-V 10 μ and 7 μA of 3 μl, followed by incubation at room temperature for 15 minutes Next, 400 μl of 1X binding buffer was added to the tube, and flow cytometry was performed within 1 hour.

Plasmid construction and stabilization cell selection

Hcamp (LL-37) cDNA clone (pCMV6-AC-GFP-hCAMP) was purchased from OriGene. The result of cleavage of pCMV-AC-GFP-hCAMP with BamH I and SfuI was inserted into multiple cloning sites of the expression plasmid pLenti6 / V5-D-TOPO (Invitrogen) containing the CMV (cytomegalovirus) promoter upstream of the insertion site. The result was named pLenti6 / V5-hCAMP.

To construct EGR1-stabilized cell line, hEGR1 was subcloned into the HindIII / XhoI site of pcDNA3.1 (+) to construct pcDNA3.1 (+) - hEGR1. (+) - hEGRl plasmid was digested with BamHI and SfuI in the multiple cloning site of the expression plasmid pLenti6 / V5-D-TOPO (Invitrogen) containing the CMV promoter upstream of the insertion site. The result was named pLenti6 / V5-hEGR1.

The four plasmid-based lentiviral expression systems were developed by Dr. Yang. Briefly, four plasmids containing 2.5 μg of pLP1, 2.5 μg of pLP2, 2.5 μg of pLP / VSVG and 2.5 μg of lentiviral vector pLenti6 / V5-hCAMP were co-transfected into 10 cm plates of HEK293T cells using Fugene6 (Roche) Cotransfection. The medium was replaced every 24 hours, and the supernatant was collected at 48 hours, filtered through a 0.45 μm filter, and ultracentrifuged for 16 hours at 6,000 rpm at 4 ° C. The pellet was dispersed in 1 ml of CM (complete medium) and stored at -80 ° C. Recombinant lentiviruses were seeded into fat-derived stem cells of 10 cm plates. After 48 hours, the cells were replaced with CM containing 10 [mu] g / ml blasticidin. On the 18th day after transfection, cell colonies were selected and separated into 60 mm Petri dishes. The isolated cell colonies were cultured and transferred.

Analysis of genes that are up-regulated by LL-37

RNA was amplified by using Agilent Low Input Quick Amp Labeling Kit, 1-color (Cy5) (Agilent Technologies, CA) with 2 μg of total RNA sample extracted from human stem cells. , And aRNA labeled with the fluorescent incorporation labeling method were hybridized to an Agilent human whole genome 4x44K array (Agilent Technologies, CA) using an Agilent hybridization system.

The analysis of the DNA chip results was performed using the spot intensity obtained by scanning with the signal intensity value using Feature Extraction 10.7.3 (Agilent Technologies, CA). Quantile normalization was performed using a GeneSpring GX 11.0 (Agilent Technologies, CA) program and plotted using a scatter plot. The distribution of all the genes obtained by the experiment is shown by the histogram. The accuracy of the test results is verified by comparing with the distribution of the standardized data. Genes with more than 2-fold (or 1.5-fold) expression mutations relative to the reference sample were selected. Hierarchical clustering analysis was performed using the Euclidean distance and average linkage algorithm with Cluster 3.0 program.

EGR-1 siRNA

Cells were transfected with EGR1 (sc-29303, Santacruz) or negative siRNA using Lipofectamin ^™ 2000. Briefly, when the cell density reaches 60-70%, the final concentration of 100 nM siRNA is mixed with Lipofectamin ^™ 2000 and incubated at room temperature for 20 minutes. The mixture was then applied to ASC and incubated for 6 hours at 37 ° C and maintained in complete medium for 24-36 hours before use in the experiment.

Statistical analysis

Statistical significance was assessed using Student's t-test. When P <0.05, the mean difference was considered significant. The results were expressed as mean ± standard deviation.

Experiment result

A conditioned medium of human adipose-derived stem / stromal cell (ASC) containing Lipo-PGE1 promotes the proliferation of dermal papilla cells (DPC).

ASC-CM (0, 50, 75 and 100%) containing Lipo-PGE1 was treated with DPC for 48 hours. The number of DPCs exposed to Lipo-PGEl-ASC-CM via the CCK assay was measured (Fig. 2a). The proliferation of ASCs was dependent on the concentration of Lipo-PGE1 (* P <0.005) (Figure 2b).

Effect of Lipo-PGE1-ASC-CM on hair regeneration in C57BL / 6

Seven-week-old C57BL / 6 shaved back and ASC-CM, Lipo-PGE1-containing ASC-CM or minoxidil were topically applied daily for 18 days. The left panel in FIG. 3 is a photograph of the degree of hair growth in C57BL / 6. The right graph of FIG. 3 shows the visual scoring results for the hairs in the C57BL / 6 male mouse round hair loss model. When Lipo-PGE1-ASC-CM was applied, remarkable hair regeneration effect was confirmed as compared with the case of treatment with ASC-CM without Lipo-PGE1, and hair growth was promoted to a degree similar to that of minoxidil, a hair growth promoting drug .

Expression of LL-37, EGR1 mRNA and protein in ASC after Lipo-PGE1 treatment

The mRNA level of LL-37 in ASC was increased in a manner dependent on the treatment time of Lipo-PGE1 (Fig. 4A). The expression of LL-37 protein in ASC was increased in a manner dependent on the treatment concentration of Lipo-PGE1 (Fig. 4B). ASC-CM was treated with DPC for 48 hours in the presence of LL-37 and subjected to anti-apoptosis assays via annexin-V and 7-AAD staining (Fig. 4C). As a result, the cell death of the male hormone DHT (Dihydrogentestosterone) induced by dermal papilla cells (DPC) was increased by 21.1% by direct treatment with LL-37 (10 μg / ml) CM 10 μg / ml and 20 μg / ml also increased cell viability. This suggests that LL-37 has a positive effect on both ASC-mediated and non-mediated cell survival of dermal papilla cells.

In addition, the mRNA level (Fig. 4d) and protein expression (Fig. 4e) of EGR-1 in ASC increased during the treatment of Lipo-PGE1.

Effect of ASC-hCAMP (LL-37) on DPC proliferation and anti-apoptosis

The ASC was transfected with the hCAMP plasmid described in Materials and Methods. After transfected cells were lysed, 30 μg of protein was separated by SDS-PAGE and immunoblotting was performed via Western blot. hCAMP expression was measured using each antibody against hCAMP. As a result, it was confirmed that hCAMP was overexpressed in ASC transfected with hCAMP plasmid (Fig. 5A).

It was confirmed that ASC-hCAMP-CM (conditioned medium of ASC transfected with pLenti6 / V5-hCAMP plasmid) induces proliferation of human DPC through CCK-8 assay (Fig. Measurements were expressed as mean ± standard error. * P < 0 . 01, compared with control, § P < 0 . 01, ASC-CM (conditioned medium of ASC) and ASC-pLenti6-CM (conditioned medium of ASC transfected with pLenti6 / V5-D-TOPO plasmid).

Cells were stained with annexin V-FITC and 7-AAD and the results were analyzed by flow cytometry. Cell death was induced by DHT in 36.6% of DPC and by 14% by ASC-hEGR1-CM, Approximately 18% cell death was observed by ASC-hCAMP-CM and cell death was inhibited by ASC-hEGR1-CM and ASC-hCAMP-CM (Fig. 5C).

Effect of ASC-hCAMP on hair regeneration in C57BL / 6

Seven week old C57BL / 6 shades were shaved and ASC-CM, ASC-pLenti6-CM (vector only), ASC-hCAMP-CM or minoxidil were topically applied daily for 18 days. 6A is a photograph of the degree of hair regeneration at C57BL / 6. Figure 4b 6b shows visual scoring results for the hairs in the C57BL / 6 male mouse round hair loss model. When ASC-hCAMP-CM was applied, hair regeneration was promoted to a degree similar to that of minoxidil, a hair growth promoting drug.

The gene EGR-1 up-regulated by LL-37

A gene showing increased expression level as compared to control cells through human cDNA microarray analysis is shown in Fig. Among them, EGR-1 showed about 18-fold increased expression level compared to control cells.

LL-37 increases EGR-1 mRNA and protein levels.

FIG. 8A shows the results of measuring the expression level of EGR-1 mRNA by LL-37 in ASC.

The level of EGR-1 protein expression was increased in ASC by LL-37 treatment time (Fig. 8B). Increased EGR-1 secretion by LL-37 was attenuated by EGR-1 siRNA (Figure 8c).

Effect of EGR-1 over-expressing cell line-derived ASC-CM on hairy growth of C57BL / 6

Seven weeks old C57BL / 6 shades were shaved and cultured in ASC-hEGR1-CM (adjusted culture of ASC), ASC-pLenti6-CM (conditioned culture of ASC transfected with pLenti6 / V5-D-TOPO plasmid) conditioned culture of ASC transfected with pLenti6 / V5-hEGRl plasmid) or minoxidil was topically applied daily for 18 days.

FIG. 9A is a photograph of the degree of hair regeneration at C57BL / 6. FIG. Figure 9b shows the visual scoring results for hair growth in the C57BL / 6 male mouse round hair loss model. When EGR-1 over-expressing cell line-derived ASC-CM was applied, hair regeneration was promoted to a degree similar to that of minoxidil, a hair growth promoting drug.

EGR-1 promotes secretion of growth factors involved in hair regeneration.

The secretion of growth factors by EGR-1 through ELISA was measured and the secretion of thymosin-beta4, VEGF, SDF-1 and MCP-1 was increased by EGR-1 (Fig. 10a-d).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Composition for Promoting Hair Growth Comprising Culture Medium          of Adipose-Derived Stem Cells <130> PN140118 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 173 <212> PRT <213> LL-37 Amino Acid <400> 1 Met Gly Thr Met Lys Thr Gln Arg Asp Gly His Ser Leu Gly Arg Trp   1 5 10 15 Ser Leu Le Leu Leu Leu Leu              20 25 30 Ala Gln Val Leu Ser Tyr Lys Glu Ala Val Leu Arg Ala Ile Asp Gly          35 40 45 Ile Asn Gln Arg Ser Ser Asp Ala Asn Leu Tyr Arg Leu Leu Asp Leu      50 55 60 Asp Pro Arg Pro Thr Met Asp Gly Asp Pro Asp Thr Pro Lys Pro Val  65 70 75 80 Ser Phe Thr Val Lys Glu Thr Val Cys Pro Arg Thr Thr Gln Gln Ser                  85 90 95 Pro Glu Asp Cys Asp Phe Lys Lys Asp Gly Leu Val Lys Arg Cys Met             100 105 110 Gly Thr Val Thr Leu Asn Gln Ala Arg Gly Ser Phe Asp Ile Ser Cys         115 120 125 Asp Lys Asp Asn Lys Arg Phe Ala Leu Leu Gly Asp Phe Phe Arg Lys     130 135 140 Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile 145 150 155 160 Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu Ser                 165 170 <210> 2 <211> 543 <212> PRT <213> EGR1 Amino acid <400> 2 Met Ala Ala Ala Lys Ala Glu Met Gln Leu Met Ser Pro Leu Gln Ile   1 5 10 15 Ser Asp Pro Phe Gly Ser Phe Pro His Ser Pro Thr Met Asp Asn Tyr              20 25 30 Pro Lys Leu Glu Glu Met Met Leu Leu Ser Asn Gly Ala Pro Gln Phe          35 40 45 Leu Gly Ala Ala Gly Ala Pro Glu Gly Ser Gly Ser Asn Ser Ser Ser      50 55 60 Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Asn Ser Ser  65 70 75 80 Ser Ser Ser Thr Phe Asn Pro Gln Ala Asp Thr Gly Glu Gln Pro                  85 90 95 Tyr Glu His Leu Thr Ala Glu Ser Phe Pro Asp Ile Ser Leu Asn Asn             100 105 110 Glu Lys Val Leu Val Glu Thr Ser Tyr Ser Ser Gln Thr Thr Arg Leu         115 120 125 Pro Pro Ile Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn     130 135 140 Ser Gly Asn Thr Leu Trp Pro Glu Pro Leu Phe Ser Leu Val Ser Gly 145 150 155 160 Leu Val Ser Met Thr Asn Pro Pro Ala Ser Ser Ser Ala Pro Ser                 165 170 175 Pro Ala Ala Ser Ser Ala Ser Ala Ser Gln Ser Pro Pro Leu Ser Cys             180 185 190 Ala Val Pro Ser Asn Asp Ser Ser Pro Ile Tyr Ser Ala Ala Pro Thr         195 200 205 Phe Pro Thr Pro Asn Thr Asp Ile Phe Pro Glu Pro Gln Ser Gln Ala     210 215 220 Phe Pro Gly Ser Ala Gly Thr Ala Leu Gln Tyr Pro Pro Ala Tyr 225 230 235 240 Pro Ala Ala Lys Gly Gly Phe Gln Val Pro Met Ile Pro Asp Tyr Leu                 245 250 255 Phe Pro Gln Gln Gln Gly Asp Leu Gly Leu Gly Thr Pro Asp Gln Lys             260 265 270 Pro Phe Gln Gly Leu Glu Ser Arg Thr Gln Gln Pro Ser Leu Thr Pro         275 280 285 Leu Ser Thr Ile Lys Ala Phe Ala Thr Gln Ser Gly Ser Gln Asp Leu     290 295 300 Lys Ala Leu Asn Thr Ser Tyr Gln Ser Gln Leu Ile Lys Pro Ser Arg 305 310 315 320 Met Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Pro His Glu Arg                 325 330 335 Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser             340 345 350 Asp Glu Leu Thr Arg His Ile Arg Ile His Thr Gly Gln Lys Pro Phe         355 360 365 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr     370 375 380 Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile 385 390 395 400 Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His Thr Lys                 405 410 415 Ile His Leu Arg Gln Lys Asp Lys Lys Ala Asp Lys Ser Val Val Ala             420 425 430 Ser Ser Ala Thr Ser Ser Ser Ser Ser Tyr Ser Ser Ser Val         435 440 445 Ser Tyr Pro Ser Ser Val Thr Thr Ser Tyr Ser Ser Ala Thr Thr     450 455 460 Ser Tyr Pro Ser Ser Val Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 465 470 475 480 Thr Tyr Pro Ser Pro Val His Ser Gly Phe Pro Ser Ser Ser Val Ala                 485 490 495 Thr Thyr Ser Ser Val Pro Pro Ala Phe Pro Ala Gln Val Ser Ser             500 505 510 Phe Pro Ser Ser Ala Val Thr Asn Ser Ser Ser Ser Thr Gly Leu         515 520 525 Ser Asp Met Thr Ala Thr Phe Ser Pro Arg Thr Ile Glu Ile Cys     530 535 540 <210> 3 <211> 522 <212> DNA <213> LL-37 Nucleotide <400> 3 atggggacca tgaagaccca aagggatggc cactccctgg ggcggtggtc actggtgctc 60 ctgctgctgg gcctggtgat gcctctggcc atcattgccc aggtcctcag ctacaaggaa 120 gctgtgcttc gtgctataga tggcatcaac cagcggtcct cggatgctaa cctctaccgc 180 ctcctggacc tggaccccag gcccacgatg gatggggacc cagacacgcc aaagcctgtg 240 agcttcacag tgaaggagac agtgtgcccc aggacgacac agcagtcacc agaggattgt 300 gacttcaaga aggacgggct ggtgaagcgg tgtatgggga cagtgaccct caaccaggcc 360 aggggctcct ttgacatcag ttgtgataag gataacaaga gatttgccct gctgggtgat 420 ttcttccgga aatctaaaga gaagattggc aaagagttta aaagaattgt ccagagaatc 480 aaggattttt tgcggaatct tgtacccagg acagagtcct ag 522 <210> 4 <211> 1632 <212> DNA <213> EGR1 Nucleotide <400> 4 atggccgcgg ccaaggccga gatgcagctg atgtccccgc tgcagatctc tgacccgttc 60 ggatcctttc ctcactcgcc caccatggac aactacccta agctggagga gatgatgctg 120 ctgagcaacg gggctcccca gttcctcggc gccgccgggg ccccagaggg cagcggcagc 180 aacagcagca gcagcagcag cgggggcggt ggaggcggcg ggggcggcag caacagcagc 240 agcagcagca gcaccttcaa ccctcaggcg gacacgggcg agcagcccta cgagcacctg 300 accgcagagt cttttcctga catctctctg aacaacgaga aggtgctggt ggagaccagt 360 taccccagcc aaaccactcg actgcccccc atcacctata ctggccgctt ttccctggag 420 cctgcaccca acagtggcaa caccttgtgg cccgagcccc tcttcagctt ggtcagtggc 480 ctagtgagca tgaccaaccc accggcctcc tcgtcctcag caccatctcc agcggcctcc 540 tccgcctccg cctcccagag cccacccctg agctgcgcag tgccatccaa cgacagcagt 600 cccatttact cagcggcacc caccttcccc acgccgaaca ctgacatttt ccctgagcca 660 caaagccagg ccttcccggg ctcggcaggg acagcgctcc agtacccgcc tcctgcctac 720 cctgccgcca agggtggctt ccaggttccc atgatccccg actacctgtt tccacagcag 780 cagggggatc tgggcctggg caccccagac cagaagccct tccagggcct ggagagccgc 840 acccagcagc cttcgctaac ccctctgtct actattaagg cctttgccac tcagtcgggc 900 tcccaggacc tgaaggccct caataccagc taccagtccc agctcatcaa acccagccgc 960 atgcgcaagt accccaaccg gcccagcaag acgccccccc acgaacgccc ttacgcttgc 1020 ccagtggagt cctgtgatcg ccgcttctcc cgctccgacg agctcacccg ccacatccgc 1080 atccacacag gccagaagcc cttccagtgc cgcatctgca tgcgcaactt cagccgcagc 1140 gaccacctca ccacccacat ccgcacccac acaggcgaaa agcccttcgc ctgcgacatc 1200 tgtggaagaa agtttgccag gagcgatgaa cgcaagaggc ataccaagat ccacttgcgg 1260 cgaaggaca agaaagcaga caaaagtgtt gtggcctctt cggccacctc ctctctctct 1320 tcctacccgt ccccggttgc tacctcttac ccgtccccgg ttactacctc ttatccatcc 1380 ccggccacca cctcataccc atcccctgtg cccacctcct tctcctctcc cggctcctcg 1440 acctacccat cccctgtgca cagtggcttc ccctccccgt cggtggccac cacgtactcc 1500 tctgttcccc ctgctttccc ggcccaggtc agcagcttcc cttcctcagc tgtcaccaac 1560 tccttcagcg cctccacagg gctttcggac atgacagcaa ccttttctcc caggacaatt 1620 gaaatttgct aa 1632 <210> 5 <211> 21 <212> DNA <213> GAPDH Forward Primer <400> 5 atcaccatct tccaggagcg a 21 <210> 6 <211> 21 <212> DNA <213> GAPDH Reverse Primer <400> 6 ttctccatgg tggtgaagac g 21 <210> 7 <211> 20 <212> DNA <213> LL-37 Forward Primer <400> 7 ctgatgcctc ttccaggtgt 20 <210> 8 <211> 20 <212> DNA <213> LL-37 Reverse Primer <400> 8 gagggagccc tttctgaatc 20 <210> 9 <211> 20 <212> DNA <213> EGR1 Forward Primer <400> 9 cttcaaccct caggcggaca 20 <210> 10 <211> 21 <212> DNA <213> EGR1 Reverse Primer <400> 10 ggaaaagcgg ccagtatagg t 21

Claims (14)

A composition for stimulating hair growth comprising a culture medium of adipose-derived stem cells highly expressing LL-37 (cathelicidin) or EGR-1 (Early growth response protein 1) as an active ingredient.
The method according to claim 1, wherein the high expression of LL-37 is induced by transfection of an LL-37 expression vector comprising a gene encoding a nucleotide of the third sequence of SEQ ID NO: 3 into adipose stem cells. / RTI &gt;
2. The method according to claim 1, wherein the high expression of EGR-1 is induced by transfection of an EGR-1 expression vector comprising a gene encoding a nucleotide sequence of SEQ ID NO: 4 into adipose stem cells. / RTI &gt;
A composition for stimulating hair growth comprising a culture medium of lipid-derived stem cells in which LL-37 or EGR-1 is highly expressed by treatment with Lipo-PGE1 (Prostaglandin E1) as an active ingredient.
A composition for promoting hair growth comprising LL-37 protein represented by the amino acid sequence of Sequence Listing 1 sequence as an active ingredient.
A composition for promoting hair growth comprising EGR-1 protein represented by the amino acid sequence of Sequence Listing 2 as an active ingredient.
A method for producing a stem-cell culture fluid of fat origin having a hair growth promoting ability, comprising the steps of:
(a) separating and culturing adipose derived stem cells by treating the subcutaneous fat tissue isolated from the subject with collagenase enzyme and centrifuging;
(b) a step of (i) transfecting the expression vector of the LL-37 gene or the expression vector of the EGR-1 gene into the adipose stem cells cultured in the step (a), or (ii) ) To produce adipose-derived stem cells highly expressing LL-37 or EGR-1; And
(c) culturing the prepared adipose-derived stem cells to obtain a culture solution.
A method for producing a stem-cell culture fluid of fat origin having a hair growth promoting ability, comprising the steps of:
(a) separating and culturing adipose derived stem cells by treating the subcutaneous fat tissue isolated from the subject with collagenase enzyme and centrifuging;
(b) treating the adipose-derived stem cells with an LL-37 protein represented by the amino acid sequence of SEQ ID NO: 1 or an EGR-1 protein represented by the amino acid sequence of SEQ ID NO: 2; And
(c) obtaining a culture from the result of (b) above.
The method according to claim 7 or 8, wherein the fatty tissue and the collagenase are mixed in a weight ratio of 1: 1 in the step (a) and the mixture is treated at 30-40 DEG C for 40-90 minutes. A method for producing a cell culture liquid.
The method according to claim 7 or 8, wherein the centrifugation in step (a) is performed at 1,000-2,000 rpm for 3-15 minutes.
[8] The method of claim 7 or 8, wherein the culturing in step (a) comprises culturing the isolated adipose derived stem cells in DMEM supplemented with 10% FBS (Fetal Bovine Serum), 100 units / ml penicillin and 100 占 퐂 / ml streptomycin (Dulbecco's Modified Eagle Media) at 37 ° C in a 5% CO ₂ incubator.
The method according to claim 7 or 8, wherein the transfection in step (b) is carried out by a method selected from the group consisting of calcium phosphate transfection, electroporation, microinjection and liposome injection. A method for producing a culture medium.
The method according to claim 7 or 8, wherein the culture medium in step (c) is a supernatant obtained by collecting the culture medium after centrifugation and filtering after the density of the cells reaches 70-100% A method for producing a stem cell culture solution.
9. A fat-derived stem cell culture produced by the method of any one of claims 7 to 8.

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