WO2010107287A2 - Composition pour la croissance pileuse/capillaire utilisant des cellules souches mésenchymateuses foetales du liquide amniotique - Google Patents

Composition pour la croissance pileuse/capillaire utilisant des cellules souches mésenchymateuses foetales du liquide amniotique Download PDF

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WO2010107287A2
WO2010107287A2 PCT/KR2010/001730 KR2010001730W WO2010107287A2 WO 2010107287 A2 WO2010107287 A2 WO 2010107287A2 KR 2010001730 W KR2010001730 W KR 2010001730W WO 2010107287 A2 WO2010107287 A2 WO 2010107287A2
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composition
stem cells
mesenchymal stem
amniotic fluid
cells
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WO2010107287A3 (fr
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유승권
윤병선
정혜연
전은경
문재희
김종건
이중한
박을순
맹이삭
김준성
이장호
이황희
이종원
조경식
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주식회사 스템메디언스
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth

Definitions

  • 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 isolated from early embryos
  • Three types of embryonic germ 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.
  • 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.
  • recent studies have shown that cells isolated from adipose or other tissues have similar characteristics to bone marrow-derived mesenchymal stem cells.
  • isolation and the acquisition of large amounts of cells are difficult, so there is an urgent need for other alternative sources.
  • the present inventors focused on the amniotic fluid that can be easily separated without harming the mother or the fetus.
  • 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.
  • 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.
  • amniotic cells have the advantage of easily obtaining a large amount of cells compared to other adult stem cells that have been studied previously.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the composition of the present invention is preferably transferrin, alpha-2-HS-glycoprotein, collagenase and matrix metalloproteinases (MMPs). It includes.
  • 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.
  • 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.
  • the amniotic fluid-derived mesenchymal stem cells of the present invention have the ability to differentiate into adipocytes, osteoblasts and chondrocytes.
  • 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.
  • the differentiation medium into osteoblasts may be made of high glucose DMEM, 100 mM dexamethasone, 10 mM ⁇ -glycerophosphate, 0.2 mM ascorbate and 10% FBS. have.
  • 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].
  • 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.
  • 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.
  • composition of the present invention is effective in promoting hair growth or preventing hair loss.
  • 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.
  • 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.
  • the cosmetic composition may further include a skin absorption promoting material to enhance the effect.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the present invention may be prepared as pharmaceutical compositions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CCMM cell culture minimum medium
  • 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, ⁇ Minimal Essential Medium (GMEM) (Glasgow's Minimal essential Medium) and IMEM (Iscove's Modified Dulbecco's Medium), but are not limited thereto.
  • the medium may include antibiotics such as penicillin, streptomycin, or gentamicin.
  • 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.
  • the low glucose DMEM medium may further comprise 4ng / ml bFGF solution with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin.
  • 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.
  • 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.
  • 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.
  • a 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.
  • 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).
  • MMPs matrix metalloprotein Matrix metalloproteinases
  • 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. .
  • an oxidative source such as L-glutamine
  • an energy metabolite such as sodium pyruvate
  • 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.
  • 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.
  • 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).
  • 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.
  • Example 1 Confirmation of allogeneic mesenchymal stem cell shape using culture and culture conditions of amniotic fetal derived cell line obtained from mother
  • 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%.
  • Example 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.
  • chromosomes are analyzed by stopping cell division with colcemid for 1 to 2 hours and observing by G-banding staining.
  • 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).
  • 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 (PPAR ⁇ ), 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.
  • the conditions for production of conditioned media by cell number from established amniotic fetal derived mesenchymal stem cells were established.
  • 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 ⁇ l 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.
  • 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).
  • Example 7 Confirm the effect of conditioned media on in vivo
  • conditioned medium affects hair growth.
  • bald patients were stimulated with microneedle after mesotherapy of conditioned medium once a week.
  • miniaturization was improved, hair density was increased, and thickness was also increased (FIG. 9A).
  • FIG. 9B 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.

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Abstract

La présente invention concerne un milieu relatif aux cellules souches mésenchymateuses foetales du liquide amniotique. Plus spécifiquement, la présente invention concerne une composition pour la croissance pileuse/capillaire ou la prévention de la dépilation, ces compositions comprenant le milieu relatif aux cellules souches mésenchymateuses foetales comme principe actif. De plus, la présente invention concerne un procédé de préparation de cette composition qui consiste à incuber des cellules souches mésenchymateuses foetales provenant du liquide amniotique et à récupérer le milieu.
PCT/KR2010/001730 2009-03-20 2010-03-19 Composition pour la croissance pileuse/capillaire utilisant des cellules souches mésenchymateuses foetales du liquide amniotique WO2010107287A2 (fr)

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KR10-2009-0024056 2009-03-20
KR1020090024056A KR101218101B1 (ko) 2009-03-20 2009-03-20 양수 내 태아 유래 중간엽줄기세포를 이용한 발모촉진용 조성물

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WO2010107287A3 WO2010107287A3 (fr) 2011-01-20

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CN110191714A (zh) * 2017-01-11 2019-08-30 思特科技公司 制备用于毛发生长的组合物的方法，该组合物包含从引入了nanog的羊水中胎儿衍生间充质干细胞获得的外泌体

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KR101218101B1 (ko) 2013-01-03
KR20100105167A (ko) 2010-09-29

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