WO2010107287A2

WO2010107287A2 - Composition for trichogenousness using fetal mesenchymal stem cells from amniotic fluid

Info

Publication number: WO2010107287A2
Application number: PCT/KR2010/001730
Authority: WO
Inventors: 유승권; 윤병선; 정혜연; 전은경; 문재희; 김종건; 이중한; 박을순; 맹이삭; 김준성; 이장호; 이황희; 이종원; 조경식
Original assignee: 주식회사 스템메디언스
Priority date: 2009-03-20
Filing date: 2010-03-19
Publication date: 2010-09-23
Also published as: KR101218101B1; KR20100105167A; WO2010107287A3

Abstract

The present invention relates to a medium for fetal mesenchymal stem cells from amniotic fluid. More specifically, the present invention relates to a composition for trichogenousness or preventing depilation, which comprises the medium for fetal mesenchymal stem cells from amniotic fluid as an active ingredient. In addition, the present invention relates to a preparation method of the composition, which comprises: incubating fetal mesenchymal stem cells from amniotic fluid; and collecting the medium.

Description

Hair growth promoting composition using fetal mesenchymal stem cells derived from amniotic fluid

The present invention relates to a culture solution of amniotic fluid-derived mesenchymal stem cells, and more particularly to a composition for promoting hair growth or preventing hair loss comprising a culture solution of amniotic fluid-derived mesenchymal stem cells as an active ingredient. The present invention also relates to a method for producing the composition comprising culturing the fetal derived mesenchymal stem cells in the amniotic fluid and collecting the culture solution.

Stem cells are capable of differentiating into various cells through suitable environment and stimulation, and have self-proliferation ability. Embryonic stem cells (ES cells) isolated from early embryos Three types of embryonic germ cells (EG cells) isolated from embryonic primordial germ cells and multipotent adult progenitor cells (MAPC cells) isolated from adult bone marrow are best known. Since stem cells have the potential to develop into cells having characteristic phenomena and specialized functions, they have been the subject of research as cell resources for functional recovery of various organs.

To date, adult stem cells are known to have the ability to differentiate into various cells. Adult stem cells include bone marrow ( Science 276, 71-74, 1997; Science 284, 143-147, 1999; Science 287, 1442-1446, 2000), skeletal muscle ( Proc. Natl Acad. Sci USA 96, 14482-14486, 1999; Nature 401, 390-394, 1999) and Fat tissue ( Tissue Eng 7, 211-228, 2001; J. Cell.Physiol . 206, 229-237, 2006 ), Each of which can differentiate into similar lines.

Mesenchymal stem cells derived from bone marrow, a type of adult stem cell, have been used for a long time and have been proven to be effective. In addition, recent studies have shown that cells isolated from adipose or other tissues have similar characteristics to bone marrow-derived mesenchymal stem cells. However, isolation and the acquisition of large amounts of cells are difficult, so there is an urgent need for other alternative sources.

Therefore, the present inventors focused on the amniotic fluid that can be easily separated without harming the mother or the fetus.

After fertilization takes place on the wall of the uterus after fertilization, the embryo is surrounded by amniotic sac filled with amniotic fluid. Since amniotic fluid contains a mixture of substances from the fetus's body, the amniotic fluid can be analyzed to determine whether the fetus's chromosomes are abnormal or not infected with bacteria. Amniotic fluid not only keeps the fetus free to move, but also protects the fetus from external shocks and stimuli, prevents bacterial infections, and helps to regulate the body's body temperature.

Before the fetus is born, the amniotic fluid tests various information on the health of the fetus. At this point, the amniotic fluid can be extracted without harming the mother from the beginning of pregnancy until after the birth. The cells used in the test are discarded after the test. If the patient's consent is obtained, the cells can be used for research purposes. Thus, amniotic cells have the advantage of easily obtaining a large amount of cells compared to other adult stem cells that have been studied previously. However, there has not been yet been disclosed whether phenomena that increase cell growth occur when ammonia-derived fetal-derived mesenchymal stem cells are cultured for a certain period of time, and the medium is extracted and put into fibroblasts.

On the other hand, not only the hair, but also all the hair of the human body after a certain period of growth stops growing, enters the resting phase, hair loss, and repeats the process of hair again. This is known as the hair growth cycle, which is known to have a long hair growth period of 1 to 6 years and a short rest period of 2 to 3 months. Each hair has its own independent growth cycle. In adults, 2 to 5% of the hair is in the resting phase, and the hairs in the resting phase are thin and glossy, hairy, thin and combed easily. However, in addition to entering the resting period by the normal growth cycle, the hair growth of the growing phase suddenly enters the resting period due to febrile disease, pregnancy, mental stress, etc. This occurs when the cause is eliminated.

Baldness does not come out of hair but gradually becomes thinner and fluffy. Unlike molting animals, human hair has its own growth cycle, ie, growth-> degenerative-> resting-> growth hair cycle, so that hair is always kept constant without molting. However, as baldness progresses, the nipples present in the hair roots become smaller, and as the nipples become smaller, the thickness of the hair becomes thinner and the hair growth becomes shorter, and the newly grown hair becomes thinner. Therefore, as bald progresses, the hair turns into fluffy hairs, and the hair cycle becomes shorter, and then grows a little and falls out. The greatest cause of baldness is heredity, and male hormones are known to be involved in the expression of bald genes. As bald progresses, oil increases in the scalp, causing seborrheic dermatitis, and the sebaceous glands attached to the hair roots gradually become larger and produce more sebum, which is not the cause of baldness, but it is the secondary phenomenon of baldness, which promotes hair loss again. It causes.

Even if you are not genetically bald, hair loss is often caused by aging or stress. Hair loss due to aging is caused by the decrease of the scalp cells and the increase of the accumulation of scalp fat, which leads to a decrease in oxygen supply and constrains the capillaries around the pores, resulting in poor circulation. It is known. In addition, stress, irregular life, environmental pollution, etc. are also thought to cause hair loss.

The mental pain of the parties due to baldness or hair loss is very high, but there is no clear way or drug to cure hair loss. Currently there are hair regrowths that are being developed and sold, but most of them have a temporary or limited hair regrowth effect, and thus do not sufficiently satisfy the needs of users. Minoxidil, a topical hair regrowth that has been proven to some extent, has been widely used as an agent for preventing hair loss due to vasodilation, and oral propesia for inhibiting activation of male hormones based on finasteride. However, the above-mentioned hair restorers have some effect on the prevention of hair loss, but the effects related to hair growth are insignificant. In other words, when the use of the hair regrowth stops the hair loss occurs again, there is a high risk of side effects in the long-term use, and there is a cost problem due to long-term use, most patients have given up treatment.

We isolated mesenchymal stem cells from the amniotic fluid of fetal stem cells and characterized them. In addition, it was revealed what components are in the conditioned medium made using amniotic fluid-derived mesenchymal stem cells, and the effect of the conditioned medium on the fibroblasts was confirmed. Furthermore, the present inventors completed the present invention by confirming the hair density and hair growth promoting effect of the conditioned medium composition through mouse in vivo and clinical experiments.

One object of the present invention to provide a composition for promoting hair growth or preventing hair loss comprising a culture medium of fetal-derived mesenchymal stem cells in the amniotic fluid as an active ingredient.

Another object of the present invention is to provide a method of preparing the composition comprising culturing the fetal derived mesenchymal stem cells in amniotic fluid and collecting the culture solution.

The present invention can be obtained by culturing from fetal-derived cells in the amniotic fluid, so that another source of bone marrow mesenchymal stem cells can be obtained. Using medium-mesenchymal stem cells with amniotic fetal derived cells obtained from mothers and using mesenchymal stem cells, the medium conditioned with low glucose DMEM serum-free medium, DMEM / F12-ITS bFGF medium and low glucose DMEM serum free medium. It can provide the production of hair growth promoter. That is, the present invention suggests the feasibility of amniotic fetal-derived cells as multipotent mesenchymal stem cells and enables the production of hair growth promoting agents using conditioned medium of fetal-derived mesenchymal stem cells in amniotic fluid.

Figure 1 shows that the amniotic fetal-derived cell lines show various shapes (heterogenous populations (arrows)) and change into mesenchymal stem cells of the same shape (homogenous enrichment) through two or three sub-cultures. will be.

Figure 2 is the result of karyotype of fetal derived cells in amniotic fluid isolated from mother. The sex chromosome came out as XY, indicating that it came from the amniotic fetus, not from the mother.

Figure 3 shows the results of analyzing the immunological phenotype of the cells through the flow cytometry that the fetal-derived cells in the amniotic fluid (B) has similar characteristics to human bone marrow-derived mesenchymal stem cells (A).

Figure 4 shows that the fetal-derived cells in the amniotic fluid has a differentiation capacity similar to that of bone marrow-derived mesenchymal stem cells with multipotency, through differentiation into fat (A), bone (B), cartilage (C). .

FIG. 5 uses low glucose DMEM serum-free medium to produce conditioned medium using established amniotic fetal derived mesenchymal stem cells (A) and another using DMEM / F12-ITS bFGF. (B). Cells were observed under a microscope according to the number of cells 5x10 ⁴ (a), 1x10 ⁵ (b), 2.5x10 ⁵ (c) and 5x10 ⁵ (d).

Figure 6 shows that a large amount of growth factor is produced through two-dimensional electrophoresis for protein component analysis through the production of conditioned media from established amniotic fetal-derived mesenchymal stem cells.

Figure 7 shows the effect of a large amount of growth factors in the conditionally conditioned medium obtained from the established amniotic fluid-derived mesenchymal stem cells on the growth of fibroblasts.

Figure 8 shows that the effect of promoting hair growth on the in vivo using the conditioned medium obtained from amniotic fluid-derived mesenchymal stem cells.

Figure 9 shows that the effect of promoting hair growth through clinical trials using the conditioned medium obtained from amniotic fluid-derived mesenchymal stem cells.

In order to achieve the above object, the present invention relates to a composition for promoting hair growth or preventing hair loss comprising a culture solution of fetal mesenchymal stem cells in the amniotic fluid as an active ingredient.

In the present invention, the term "mesenchymal stem cells (MSCs)" is a cell that helps to create cartilage, bone, fat, myeloid epilepsy, muscle, nerves, etc., but in adults generally stay in the bone marrow In the umbilical cord blood, peripheral blood, other tissues and the like, it means a cell that can be obtained from them. For the purposes of the present invention, the mesenchymal stem cells of the present invention means mesenchymal stem cells derived from the amniotic fluid.

Amniotic fluid from the mother contains many chemicals from the fetus's body that can produce most of the cells in the body and are easy to harvest. In addition, heterologous (heterogenous) cells exist in the amniotic fluid, and the present inventors have confirmed that there are homogenous mesenchymal stem cells having the same shape as fibroblasts characteristic of mesenchymal stem cells.

The present invention is characterized in that the amniotic cells are derived from the fetus.

As a preferred embodiment of the present invention, it is possible to confirm that the cells in the amniotic fluid are cells derived from the fetus through karyotyping. In the amniotic fluid, not only the fetus but also some maternal cells may be present. In the present invention, it was possible to confirm that the amniotic cells are male-derived cells through chromosome analysis (FIG. 2).

Preferably, the composition of the present invention is preferably transferrin, alpha-2-HS-glycoprotein, collagenase and matrix metalloproteinases (MMPs). It includes.

In addition, the amniotic fluid-derived mesenchymal stem cells prepared by the method of the present invention (a) show positive immunological characteristics with respect to CD13, CD29 and CD44, and (b) are attached to the cell culture dish to grow and grow fibroblasts. It is characterized by the morphological characteristics of the spindle-shape, which is a typical shape of, and (c) having the ability to differentiate into mesodermal derived cells.

As used herein, the term “differentiation” refers to a phenomenon in which a cell's structure or function is specialized during cell division and proliferation. Pluripotent mesenchymal stem cells can differentiate into lineage-defining progenitor cells (eg, mesodermal cells) and then further differentiate into other forms of progenitor cells (eg, osteoblasts, etc.), followed by specific tissues (eg, May be differentiated into terminal differentiated cells (eg, adipocytes, bone cells, chondrocytes, etc.) that play a characteristic role in bones, etc. In a preferred embodiment, the amniotic fluid-derived mesenchymal stem cells of the present invention have the ability to differentiate into adipocytes, osteoblasts and chondrocytes.

In a specific embodiment of the present invention, the differentiation medium into adipocytes is high glucose DMEM, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-caffeine (3-isobutyl-1-methyl-xanthine). , 10 ng / ml insulin, 100 mM indomethacin and 10% FBS.

In a specific embodiment of the present invention, the differentiation medium into osteoblasts may be made of high glucose DMEM, 100 mM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM ascorbate and 10% FBS. have.

In a specific embodiment of the present invention, the differentiation medium into chondrocytes is high glucose DMEM, 0.1 M dexamethasone, 50 g / ml AsA, 100 g / ml Sodium pyruvate, 40 g / ml Proline, 10 ng / ml TGF-1 and 50 mg / ml ITS premix [6.25 g / ml insulin, 6.25 g / ml transferrin, 6.25 g / ml selenius acid , 1.25 mg / ml BSA and 5.35 mg / ml linoleic acid].

In the present invention, the mesenchymal stem cells derived from the amniotic fluid in each differentiation medium were cultured for 7 to 28 days, and in this way, the amniotic fetal cells derived from the amniotic fluid have the characteristics of mesenchymal stem cells. (Figure 4). Therefore, in the present invention, the fetal-derived cells in the amniotic fluid are cells having the properties of mesenchymal stem cells, and this can be used as a source of other cells other than bone marrow.

The composition of the present invention is effective in promoting hair growth or preventing hair loss. As used herein, the term "anti-hair loss" or "hair growth promotion" is used in the same sense, which has the same meaning as another term wool or hair growth promotion used in the art.

In a specific embodiment of the present invention, as a result of completely removing the hair of the mouse and the back of the rat and applying the composition of the present invention with a brush, it was confirmed that the hair growth was increased in the treatment group coated with the present composition compared to the control. In particular, in the case of mice, the skin is blackened due to the production of melatinocytes in the skin when the hair is produced. In the treatment group to which the present composition is applied, a change in darker skin color than the control group was confirmed (FIG. 8).

In addition, in the clinical trials, bald patients were mesotherapy-treated with the microneedle, and the miniaturization was improved, the hair density was increased, and the thickness was also increased. By stimulating and treating in the same manner, it was confirmed that the hair density was increased, the hair was thickened, and the hair loss site was reduced (FIG. 9).

Thus, according to a preferred embodiment of the present invention, the composition of the present invention is a cosmetic composition.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions in addition to the culture of fetal-derived mesenchymal stem cells as amniotic fluids, and include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and the like. Conventional adjuvants such as perfumes, and carriers. In addition, the cosmetic composition may further include a skin absorption promoting material to enhance the effect. In particular, in the case of the hair regrowth composition, monosaccharides such as glucose, xylose, mannose, and arabinose and maltose, sucrose, cellobiose, tre, which can supply nutrients to hair follicles in addition to the culture solution of fetal mesenchymal stem cells in the amniotic fluid as an active ingredient, It may further include one or more kinds of sugars such as disaccharides such as halose.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be. 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, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether. When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan. When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used. When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier component is 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 and the like can be used.

In addition, the compositions of the present invention may be prepared as pharmaceutical compositions.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like 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 (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and especially since it is a hair regrowth composition, it can be directly applied or spread on the scalp. It is used by transdermal administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention may be administered once or several times a day in an oral dosage form of 0.1-100 mg / kg on an adult basis. It is recommended to apply 1 to 5 times a day in an amount of 3.0 ml to continue for 1 month or more. However, the dosage does not limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or it may be prepared by incorporation into a multi-dose container. The formulation may be used in any form suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may further comprise a dispersant or stabilizer.

As another aspect, the present invention comprises the steps of culturing the fetal derived mesenchymal stem cells in amniotic fluid; And it relates to a method for producing the composition comprising the step of collecting the culture solution.

More preferably, the present invention comprises the steps of (a) isolating fetal derived cells in amniotic fluid obtained from the mother; (b) passaging in FBS and bFGF-containing medium to obtain fetal mesenchymal stem cells in amniotic fluid; (c) culturing the obtained amniotic fluid-derived mesenchymal stem cells in serum-free medium or DMEM / F12-ITS (Insulin-Transferrin-Selenite) complex medium for 1 to 10 days to prepare a conditioned medium; And (d) relates to a method for producing the composition comprising the step of collecting the conditioned medium culture.

In step (a), amniotic fluid can be extracted without harming the mother from the moment of pregnancy to immediately after childbirth. Amniotic fluid is used to test various information about the health of the fetus before the birth of the fetus. Cells discarded after being used for this examination can be used for research purposes with the consent of the patient. Can be obtained. Amniotic fluid from the mother can be centrifuged to separate fetal stem cells from the amniotic fluid.

In step (b), the cells separated from the amniotic fluid can be passaged to obtain fetal mesenchymal stem cells derived from the amniotic fluid. The medium used for passage is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source, and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (GMEM) (Glasgow's Minimal essential Medium) and IMEM (Iscove's Modified Dulbecco's Medium), but are not limited thereto. In addition, the medium may include antibiotics such as penicillin, streptomycin, or gentamicin.

In the present invention, fetal mesenchymal stem cells derived from amniotic fluid can be obtained by culturing cells isolated from amniotic fluid in basal medium containing FBS and bFGF, preferably in low glucose DMEM medium containing 10% FBS. It can be obtained by incubating by adding 4ng / ml bFGF. In a preferred embodiment of the present invention, the low glucose DMEM medium may further comprise 4ng / ml bFGF solution with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin.

In step (c), the obtained amniotic fluid-derived mesenchymal stem cells are cultured in serum-free medium for 1 to 10 days to prepare a conditioned medium.

As used herein, the term "conditioned medium" refers to a cell growth suspension in which the cells are subjected to liquid suspension culture, and then, when the apoptotic growth phase is reached, the dividing cells are removed by centrifugation or filtration, and only the culture medium is collected and mixed with the culture substrate. Say one badge. This uses an unknown growth factor that is extracted in the medium from the dividing cells, and is widely used for low density cell plating or protoplast culture.

For the purposes of the present invention, the conditioned medium composition of the present invention is a composition comprising a solution from which fetal-derived mesenchymal stem cells are removed from a medium in which fetal-derived mesenchymal stem cells are cultured, and derived from fetal-derived mesenchymal stem cells. It refers to a composition containing abundant substances such as growth factors, preferably transferrin, alpha-2-HS-glycoprotein, collagenase and matrix metalloprotein Matrix metalloproteinases (MMPs).

In the present invention, in order to obtain a conditioned medium of fetal-derived mesenchymal stem cells, the mesenchymal stem cells obtained from amniotic fluid containing Ham's F-12 nutrient mixture containing amino acids or their analogues and vitamins or their analogs It is preferable to use a serum culture medium. Serum-free medium containing Ham's F-12 nutrient mixture according to the present invention is based on DMEM without a pH indicator such as phenol red and Ham's F-12 nutrient mixture is added at a ratio of approximately 1: 0.5-2. . At this time, it is possible to add an oxidative source such as L-glutamine, an energy metabolite such as sodium pyruvate, and a carbon regulator such as sodium bicarbonate. This mixed solution is used to help maintain the growth and homeostasis of cells and to secrete various minerals and amino acids that are involved in improving cell stability and maintenance in subculture after initial culture of mesenchymal stem cells. Vitamin-based nutrients and other factors, which can promote higher production of the factor, are formed in a proportion.

In a specific embodiment of the present invention, the embryo-derived cells isolated from the amniotic fluid obtained from the mother are passaged in a medium containing FBS and bFGF to obtain the fetal-derived mesenchymal stem cells in the amniotic fluid, followed by DMEM / F-12 serum-free medium. Alternatively, the conditioned medium was prepared by centrifugation and filtration of culture medium obtained by culturing for 3 days with different numbers of amniotic cells in DMEM / F12-ITS (Insulin-Transferrin-Selenite) complex medium (FIG. 5).

In the step (d), the conditioned medium culture is collected to prepare a composition for improving the skin condition desired in the present invention. Collection of the conditioned medium culture may be performed by a method known in the art, and may be collected by centrifugation or filtration.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, the scope of the present invention is not limited by these examples.

실시예 1: 산모로부터 얻은 양수 내 태아유래 세포주의 배양 및 배양 조건을 이용한 동종의 중간엽줄기세포 모양을 확인Example 1: Confirmation of allogeneic mesenchymal stem cell shape using culture and culture conditions of amniotic fetal derived cell line obtained from mother

There are many floats in the amniotic fluid obtained from mothers. To separate it, the amniotic fluid was put in T-flask and incubated at 37 ° C. The next day, the rest were removed except for the cells on the floor. Cells adhered to the bottom were separated at the bottom using trypsin and collected by centrifugation, and then 4ng / ml with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin in low glucose DMEM. Cells were seeded in 100 mm cell culture dishes by resuspension in basal medium consisting of bFGF. After 12 to 24 hours, the new medium was replaced. Amniotic fetal-derived cell lines had a variety of shapes (heterogenous populations), and after two or three passages, only one fibroblast was found. In the form as shown in Figure 1 the medium was changed every 2 to 3 days and the passage was cultured when the cell density of 80 ~ 90%.

실시예 2: 핵형 분석을 통해 양수 내 태아유래 세포라는 것을 확인Example 2: Identification of fetal-derived cells in amniotic fluid through karyotyping

Karyotyping was performed to determine whether the amniotic fluid was derived from amniotic fluid.

Karyotyping is the number, size, and shape of chromosomes, called karyotypes, and chromosome mutations and sex can be determined by examining the chromosomes of the fetus. In order to perform karyotyping, chromosomes are analyzed by stopping cell division with colcemid for 1 to 2 hours and observing by G-banding staining. As a result of karyotype analysis, the fetal-derived cells in the amniotic fluid have normal chromosomes and the sex chromosomes are XY, indicating that the cells are derived from the fetus rather than the mother (FIG. 2).

실시예 3: 양수 내 태아유래 세포가 중간엽줄기세포와의 유사성 확인Example 3: Confirmation of Amniotic Fetal-Derived Cells with Mesenchymal Stem Cells

We confirmed whether fetal-derived cells in amniotic fluid have characteristics of multipotent mesenchymal stem cells.

First, after analyzing the immunological phenotype through flow cytometry, it was confirmed that the fetal-derived cells in the amniotic fluid were expressed with CD13, CD29, and CD44 when compared with human bone marrow-derived mesenchymal stem cells (FIG. 3). Although the expression of these surfaces is a specific marker for mesenchymal stem cells, different characteristics may be present in each cell, so we examined the differentiation ability to bone, fat, and chondrocytes, which are representative characteristics of mesenchymal stem cells.

Amniotic fetal-derived cells are differentiated like human bone marrow-derived mesenchymal stem cells, and amniotic fetal-derived cells are differentiated like human bone marrow-derived mesenchymal stem cells. The genes expressed are osteopontin and osteocalcin, and fats are lipoprotein lipase (LPL), fatty acid binding protein (aP2) Peroxysome proliferator-activated receptor gamma (PPARγ), in which cartilage forms as cartilage forms and is associated with typical gene type II collagen and type I collagen. It was confirmed by reverse transcription chain reaction (RT-PCR) that the differentiation into agrican (aggrecan). Alizarin red S staining is used to confirm the formation of calcium salts when differentiating into bones. Lipids are formed when differentiating into fats and oil red O staining to stain them. Staining) and cartilage was confirmed by the specific staining for each differentiation using Alcian blue staining (Alcian blue staining) in order to stain the cartilage specific secretion substrate (Fig. 4 ). Therefore, in the present invention, it was confirmed that the fetal-derived cells in the amniotic fluid are cells having characteristics similar to those of the bone marrow-derived mesenchymal stem cells.

실시예 4: 양수 내 태아유래 중간엽줄기세포에서 조건별 컨디션드 배지 제조Example 4 Preparation of Conditioned Medium in Amniotic Fluid-Derived Mesenchymal Stem Cells

The conditions for production of conditioned media by cell number from established amniotic fetal derived mesenchymal stem cells were established.

First, the established mesenchymal stem cells were trypsinized in a 100 mm cell culture dish and separated from the bottom. Collected cells were collected in a 15ml tube and centrifuged. The collected cells were released in 5 ~ 10ml medium and mixed well. 20 μl of the cell suspension in the tube was measured using a hematocytometer, and then, low glucose DMEM was composed of 10% FBS, 1% L-glutamine, 1% penicillin-streptomycin and 4 ng / ml bFGF. the ^{^{^{5x10 4, 1x10 5, 2.5x10 5}}} , 5x10 5 cells in the basal medium were seeded in 100mm cell culture dish. After 12 hours, the medium was changed to low glucose DMEM serum-free medium and DMEM / F12-ITS bFGF medium and incubated for 72 hours. After 72 hours, each aliquot of each culture was transferred to a tube, followed by centrifugation, and a 0.20 syringe filter to produce conditioned medium (FIG. 5).

실시예 5: 양수 내 태아유래 중간엽줄기세포에서 조건별 컨디션드 배지 성분 확인Example 5 Identification of Conditioned Medium Components by Amniotic Fluid in Fetal Derived Mesenchymal Stem Cells

In order to analyze the composition of the conditioned medium obtained by using fetal-derived mesenchymal stem cells in amniotic fluid, two-dimensional electrophoresis was used to determine what proteins were expressed and the amount of change was measured. After electrophoresis, multiple protein spots were identified on gels by colloidal coomassie blue staining or silver staining. Analysis of the components of the conditioned medium revealed that transferrin, a protein that carries iron to the bone marrow, alpha-2-HS-glycoprotein, which accelerates cell regeneration, collagenase, Matrix metalloproteinases (MMPs) and many growth factor factors were analyzed to contain (Figure 6).

실시예 6: 인비트로(in vitro) 상에서 컨디션드 배지 효과 확인Example 6 Confirmation of Conditioned Medium Effects in Vitro

Count the number of cells using fetal derived mesenchymal stem cells in amniotic fluid 5x10⁴, 1x10⁵, 2.5x10⁵,5 x 10⁵The dogs were changed to each low glucose DMEM serum-free medium and DMEM / F12-ITS bFGF medium, and cultured for 3 days to make a conditioned medium to examine the effect of fibroblast growth. 1x10 of the fibroblasts in 24 wells⁴After 12 hours of seeding, the medium was changed to conditioned medium and experimented for 3 days. At the end of the experiment, remove the conditioned medium from each of the 24 wells, wash with PBS twice, fix the cells with 10% formalin, and stain with crystal violet. ). After removing the dye solution in running water and drying for about 24 hours, the value was measured by measuring the absorbance through destaining with 10% acetic acid. 40-60% of low glucose DMEM serum-free conditioned medium and 20% of DMEM / F12 ITS-bFGF conditioned medium compared to cells grown in low glucose DMEM serum-free medium and DMEM / F12 ITS-bFGF medium, but not in Conditioned medium. It was confirmed that the growth of about 30% was promoted (FIG. 7).

실시예 7: 인비보(in vivo) 상에서 컨디션드 배지가 미치는 효과를 확인Example 7: Confirm the effect of conditioned media on in vivo

Count the number of cells using fetal-derived mesenchymal stem cells in amniotic fluid and change to 5x10 ⁵ low-glucose DMEM-bFGF medium and incubate for 3 days to create a conditioned medium. I looked at it. Experiments were conducted in mice (C57BL6) and rats (SD), respectively. The dorsal lower limbs of the two types of animals were removed in a rectangular shape on both sides of the spine and completely removed using a remover to remove the roots. The control group was applied to normal low glucose DMEM serum-free medium, not to the conditioned medium, and the treated group was applied to the site where the hair was removed once daily using a brush. After 12 days, as a result of checking the hair growth state of the control and the treatment, it was confirmed that the hair growth of the treatment treated with the conditioned medium was increased compared to the normal medium rather than the conditioned medium. In particular, in the case of the mouse (C57BL6), melanocytes are formed in the skin when the hair is generated, and the skin becomes black. In the treatment group coated with the conditioned medium, the change in the color of the skin darker than the control group coated with the normal medium, not the conditioned medium. It could be confirmed (Fig. 8).

실시예 8: 임상에서 컨디션드 배지가 미치는 효과를 확인Example 8 Confirmation of the Effect of Conditioned Medium in the Clinic

Count the number of cells using fetal-derived mesenchymal stem cells in amniotic fluid and change to 5x10 ⁵ with low-glucose DMEM-bFGF medium and incubate for 3 days to produce conditioned medium and compare with normal medium, not conditioned medium. We examined whether conditioned medium affects hair growth. In clinical trials, bald patients were stimulated with microneedle after mesotherapy of conditioned medium once a week. When observed after 8 to 15 weeks, miniaturization was improved, hair density was increased, and thickness was also increased (FIG. 9A). By stimulating and treating in the same manner, it was confirmed that the hair density was increased, the hair was thickened, and the hair loss site was reduced (FIG. 9B).

Claims

A composition for promoting hair growth or preventing hair loss comprising a culture solution of fetal mesenchymal stem cells in the amniotic fluid as an active ingredient.
The composition of claim 1, wherein the amniotic fluid-derived mesenchymal stem cells have the following characteristics. :

(a) all show positive immunological characteristics for CD13, CD29 and CD44;

(b) attached to and grown on a cell culture dish and exhibited spin-shape morphological properties; And

(c) has the ability to differentiate into mesodermal derived cells.
The culture of the amniotic fluid-derived mesenchymal stem cells according to claim 1, wherein the culture medium is transferrin, alpha-2-HS-glycoprotein, collagenase and matrix metalloprotein. Composition comprising the metals (matrix metalloproteinases, MMPs).
The composition of claim 1, wherein the composition is a cosmetic composition.
The group of claim 4 wherein the composition consists of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray A composition characterized by having a formulation selected from.
The composition of claim 1, wherein the composition is a pharmaceutical composition.
Culturing the fetal derived mesenchymal stem cells in amniotic fluid; And collecting the culture solution.
(a) isolating fetal derived cells in amniotic fluid obtained from the mother;

(b) passaging in FBS and bFGF-containing medium to obtain fetal mesenchymal stem cells in amniotic fluid;

(c) culturing the obtained amniotic fluid-derived mesenchymal stem cells in serum-free medium or DMEM / F12-ITS (Insulin-Transferrin-Selenite) complex medium for 1 to 10 days to prepare a conditioned medium; And

(d) collecting the conditioned medium culture, wherein the composition of claim 1 is prepared.