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WO2025121569A1 - Composition pour induire la différenciation de cellules souches dérivées du tissu adipeux en cellules de papille dermique à l'aide d'un composite de 6bio, moroniside et gomme de mastic solubilisée - Google Patents

Composition pour induire la différenciation de cellules souches dérivées du tissu adipeux en cellules de papille dermique à l'aide d'un composite de 6bio, moroniside et gomme de mastic solubilisée Download PDF

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WO2025121569A1
WO2025121569A1 PCT/KR2024/007206 KR2024007206W WO2025121569A1 WO 2025121569 A1 WO2025121569 A1 WO 2025121569A1 KR 2024007206 W KR2024007206 W KR 2024007206W WO 2025121569 A1 WO2025121569 A1 WO 2025121569A1
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cells
adipose
stem cells
derived stem
composition
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Korean (ko)
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심태진
김지훈
홍인기
임영철
서재아
김종필
이혜숙
김미진
김다솔
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Frombio Co Ltd
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Frombio Co Ltd
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    • 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
    • A61K35/33—Fibroblasts
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a composition for inducing differentiation of adipose-derived stem cells into hair papilla cells using a complex of 6BIO, moroniside, and water-soluble mastic gum.
  • Hair loss is broadly classified into natural hair loss, in which hair in the growth phase naturally falls out from the hair follicle through a normal growth cycle, and abnormal hair loss, which is caused by genetic factors, hormonal abnormalities, mental stress, disease, and drug side effects.
  • the hair follicle contains the hair bulb, dermal papilla, hair matrix, melanocytes, sebaceous glands, and hair follicle muscles.
  • Dermal papilla cells are a type of dermal fibroblast cells located in the dermal papilla at the bottom of the hair follicle. Dermal papilla cells have capillaries distributed throughout them, which supply oxygen and nutrients to the hair follicle.
  • IGF-1 insulinlike growth factor-1
  • KGF keratinocyte growth factor
  • ⁇ -FGF ⁇ -fibroblast growth factor
  • HGF hepatocyte growth factor
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • hair growth progresses as the epithelial cells surrounding the hair papilla divide to form a hair shaft.
  • This epithelial cell division is regulated by the hair papilla cells, and in male pattern baldness, the part where male hormones act on the hair follicle is also the hair papilla, and the hair papilla cells play a very important role in hair growth.
  • hair loss treatments approved by the U.S. Food and Drug Administration include minoxidil, finasteride, and baricitinib.
  • finasteride has side effects such as decreased sexual function and birth defects, and is limited to use on male patients.
  • Minoxidil has side effects such as allergic dermatitis, itching, and hair loss recurrence when use is discontinued.
  • Baricitinib has cardiovascular side effects, and its use is limited to severe alopecia areata.
  • Surgical procedures such as injection of the stromal vascular fraction of adipose tissue containing adipose-derived stem cells (ADSCs) into the scalp or transplantation of hair tissue suspension have been attempted and reported to be effective.
  • ADSCs adipose-derived stem cells
  • surgical procedures have the problems of being accompanied by pain and being expensive (Stem Cell Res. Ther. 12 (1) (2021) 486).
  • the present invention discloses a technique for differentiating adipose-derived mesenchymal stem cells into papilla cells using a complex of 6BIO, moroniside, and water-soluble mastic gum.
  • the purpose of the present invention is to provide a composition for inducing differentiation of adipose-derived stem cells into hair papilla cells using a complex of 6BIO, moroniside, and water-soluble mastic gum.
  • Another object of the present invention is to provide a method for differentiating adipose-derived stem cells into mammary papilla cells using the composition.
  • the present invention was completed by confirming in the examples below that a complex of 6BIO, moroniside, and water-soluble mastic gum induces differentiation of human adipose-derived stem cells into dermal papilla cells when treated with factors such as BMP2 (Bone morphogenetic protein 2).
  • the present invention is provided based on these experimental results, and in one aspect, the present invention can be understood as a composition for inducing differentiation of adipose-derived (i.e., isolated from adipose tissue) stem cells into hair papilla cells, which comprises a complex of 6BIO, moroniside, and water-soluble mastic gum as active ingredients.
  • adipose-derived i.e., isolated from adipose tissue
  • hair papilla cells which comprises a complex of 6BIO, moroniside, and water-soluble mastic gum as active ingredients.
  • the complex of 6BIO, moroniside and water-soluble mastic gum acts as an active ingredient that induces differentiation of human adipose-derived stem cells into dermal papilla cells, either alone or together with a carrier component that is inactive per se.
  • 6BIO (6-Bromoindirubin-3'-oxime) is a known substance with a CAS number of 667463-62-9, a semi-synthetic derivative of indirubin found in edible molluscs or plants, and is an inhibitor of Glycogen synthase kinase 3 ⁇ (Gsk-3 ⁇ ), which is known to play an important role in inducing osteogenic differentiation of bone marrow mesenchymal stem cells, osteogenic differentiation of periodontal ligament stem cells, and bone regeneration (Int J Mol Sci. 2022 Aug 4;23(15):8676; An Acad Bras Cienc. 2019 Mar 21;91(1):e20180459; ACS Biomater. Sci. Eng. 2021 Jan 11;7(1):232-231).
  • the IUPAC name of moroniside is ⁇ methyl (1S,3R,4aS,8S,8aS)-3-hydroxy-1-methyl-8-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-1,3,4,4a,8,8a-hexahydropyrano[3,4-c]pyran-5-carboxylate ⁇ , a known compound, and its CAS number is 25406-64-8. It is a substance known to regulate hair growth through the Wnt/ ⁇ -catenin signaling pathway (Scientific Reports, (2016) 8:13785).
  • water-soluble mastic gum refers to a suspension (i.e., a suspension in which mastic gum is pulverized into microparticles (having an average particle diameter of 1 to 999 ⁇ m) or nanoparticles (having an average particle diameter of 1 to 999 nm) and suspended in water as a solvent (i.e., a state in which it is dispersed without coagulation or precipitation in water as a solvent) .
  • a suspension i.e., a suspension in which mastic gum is pulverized into microparticles (having an average particle diameter of 1 to 999 ⁇ m) or nanoparticles (having an average particle diameter of 1 to 999 nm) and suspended in water as a solvent (i.e., a state in which it is dispersed without coagulation or precipitation in water as a solvent) .
  • Such water-soluble mastic gum can be obtained by pulverizing mastic gum into microparticles or nanoparticles, adding water thereto, stirring, and mixing, and in particular, it may be preferable to obtain mastic gum by pulverizing mastic gum into microparticles, adding water thereto, mixing, and pulverizing the mixture (or mixed solution) to thereby pulverize mastic gum particles in the mixture into nanoparticles.
  • a suspending agent may be added in an appropriate amount before and after mixing the mastic gum in the microparticle or nanoparticle form with water and before and after pulverization.
  • suspending agent known in the art can be used, for example, water-soluble polymers such as gum arabic, gum tragacanth, agar, karaya gum, locust bean gum, guar gum, xanthan gum, ghatti gum, and pectin; cyclodextrin, glycerin or its fatty acid ester derivatives; modified cellulose such as methyl cellulose or hydroxymethyl cellulose; various surfactants; etc.
  • These suspending agents can be used alone or in mixtures of two or more, and can be used in an appropriate amount for suspension, specifically in a range of 20 to 200 parts by weight per 100 parts by weight of mastic gum.
  • mastic gum refers to a resin obtained from a mastic tree ( Pistacia lentiscus ).
  • composition of the present invention may additionally contain BMP2 (Bone morphogenetic protein 2), BMP4, etc., in addition to the active ingredient, water-soluble mastic gum, in order to supplement or increase the differentiation-inducing activity of water-soluble mastic gum.
  • BMP2 Breast morphogenetic protein 2
  • BMP4 BMP4
  • These factors may be of human origin (isolated from human blood, etc., or produced by a genetic recombination method, and having the same amino acid sequence as that of a human).
  • composition of the present invention may contain a complex of active ingredients, 6BIO, moroniside and mastic gum, in an amount necessary to sufficiently or to a desired degree differentiate adipose-derived stem cells into dermal papilla cells.
  • 6BIO is contained in an amount of 2 to 10 ⁇ M
  • moroniside in an amount of 5 to 15 ⁇ M
  • water-soluble mastic gum in an amount of 50 to 1,000 ppm, particularly in an amount of 100 to 300 ppm.
  • composition of the present invention includes BMP2 and BMP4, it is preferable to include BMP2 and BMP4 in a range of 0.3 to 3 ng/ml in order to sufficiently or to the intended degree differentiate into mammary papilla cells.
  • composition of the present invention may contain a solvent in addition to a complex such as the active ingredient 6BIO.
  • a solvent may be a cell culture medium, a buffer, an isotonic solution, or other appropriate solvents such as purified water or DMSO (dimethyl sulfoxide).
  • the cell culture medium is not particularly limited and any basic medium used in the art for culturing mammalian cells can be used.
  • the basic medium is for cell growth, proliferation and/or expansion, and basically contains sugars, amino acids, and inorganic salts, and may optionally contain vitamins, trace elements, antimicrobial agents, growth factors, hormones, buffers, isotonic agents, etc.
  • Monosaccharides, disaccharides, etc. can be used as the sugar, and specifically, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, or a mixture of one or more of these can be used.
  • the amino acids contained in the basic medium include aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, norvaline, tryptophan, phenylalanine, isoleucine, leucine, lysine, hydroxyproline, sarcosine and/or proline.
  • the amino acid is preferably a synthetic amino acid, and such synthetic amino acid may be in the form of a dipeptide or tripeptide. Such dipeptide or tripeptide can be converted into the free amino acid in a cell culture containing the cells.
  • the basic medium may additionally contain inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate, which help maintain osmotic balance and regulate membrane potential by providing sodium, potassium, and calcium ions.
  • inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate, which help maintain osmotic balance and regulate membrane potential by providing sodium, potassium, and calcium ions.
  • the basic medium may optionally include vitamins.
  • vitamins are essential for cell growth and proliferation and cannot be synthesized in sufficient amounts by cells, so they need to be sufficiently supplemented in the cell culture medium.
  • Vitamins such as vitamin A, vitamin B group, vitamin C, and vitamin E may be included in the basic medium.
  • vitamin B group such as thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, and nicotinamide are preferably added to promote cell growth.
  • the basic medium may optionally contain, in addition to sugars, amino acids, and vitamins, trace elements, antibiotics, growth factors, hormones, buffers, and tonics.
  • Trace elements may be added to the basal medium to ensure proper cell growth and maintain enzyme function, and examples of such trace elements include copper, zinc, selenium, and tricarboxylic acid intermediates.
  • Antimicrobial agents may be added to the basic medium to prevent contamination by external microorganisms, and specifically, antibiotics such as penicillin, streptomycin, and fungizone, antifungal agents such as amphotericin B, and mycoplasma inhibitors such as gentamicin, ciprofloxacin, azithromycin, and tylosin may be used.
  • antibiotics such as penicillin, streptomycin, and fungizone
  • antifungal agents such as amphotericin B
  • mycoplasma inhibitors such as gentamicin, ciprofloxacin, azithromycin, and tylosin
  • Growth factors can be added to the basal medium for cell proliferation, and examples of such growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (bFGF), and activin A.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • IGF insulin-like growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TGF transforming growth factor
  • VEGF vascular endothelial growth factor
  • bFGF fibroblast growth factor-2
  • activin A activin A.
  • Hormones that can be added to the basic medium include insulin, hydrocortisone, triiodothyronine, estrogen, androgen, progesterone, prolactin, follicle-stimulating hormone, gastrin-releasing peptide, dexamethasone, estradiol, and glucagon.
  • the basic medium may contain buffers such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris, or isotonic agents such as sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose, and glucose.
  • buffers such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris
  • isotonic agents such as sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arab
  • the basic medium may contain cell adhesion factors such as type I or type II collagen, gelatin, fibronectin, laminin, poly-L-lysine, poly-D-lysine, albumin that binds salts, free fatty acids, hormones, and vitamins and transports them between tissues and cells, and plays a role in regulating pH and osmotic pressure, and transferrin that plays an important role in iron transport.
  • cell adhesion factors such as type I or type II collagen, gelatin, fibronectin, laminin, poly-L-lysine, poly-D-lysine, albumin that binds salts, free fatty acids, hormones, and vitamins and transports them between tissues and cells, and plays a role in regulating pH and osmotic pressure, and transferrin that plays an important role in iron transport.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Base Medium Eagle
  • RPMI 1640 F-10, F-12, DMEM/F12
  • MEM- ⁇ Minimal Essential Medium- ⁇
  • G-MEM Glasgow's Minimal Essential Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • MacCoy's 5A medium AmnioMax complete medium, AminoMaxII complete medium
  • EBM Endothelial Basal Medium
  • Chang's Medium MesenCult-XF
  • DMEM/HG Dulbecco's Modified Eagle's Medium high glucose
  • MCDB+DMEM/LG MCDB +Dulbecco's Modified Eagle's Medium low glucose
  • the solvent included in the composition of the present invention may be a buffer solution
  • the buffer solution may be a saline solution containing citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, Tris, etc. as a buffer.
  • it may be phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HEPES buffered saline), DPBS (Dulbecco's phosphate-buffered saline), etc.
  • the solvent included in the composition of the present invention may also be an isotonic solution containing sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene, glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol trihalose, glucose, etc. as isotonic agents.
  • isotonic solutions may be Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, 5% glucose aqueous solution, and may be manufactured and used directly or purchased and used as commercially available ones.
  • the adipose-derived stem cell (ADSC) of the present invention has self-renewal ability and can differentiate into various lineages of cells such as skin, cartilage, and bone, and is easy to collect and can be mass-cultivated compared to bone marrow-derived stem cells or umbilical cord blood-derived stem cells, and has a large stem cell content, so it has high utilization in cell therapy.
  • the adipose-derived stem cell has self-renewal ability and differentiation ability, its origin is not particularly limited and may be derived from a human, monkey, pig, horse, cow, sheep, dog, cat, mouse, or rabbit. It is preferably derived from a human.
  • the present invention relates to a method for producing mammary papilla cells differentiated from adipose-derived stem cells, comprising the step of treating and culturing a culture medium for adipose-derived stem cells with the composition for inducing differentiation of the present invention as described above.
  • the culture temperature may be in the range of 25 to 40°C, preferably 35 ⁇ 2°C.
  • the culturing is performed until differentiation into mammary papilla cells is sufficient or as intended.
  • the culturing can be performed for a period of 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or more.
  • the culture may be performed in an amount of carbon dioxide (CO 2 ) of 10% to 1% (v/v), preferably 8% to 2% (v/v), and especially 5% (v/v) so that differentiation into mammary gland cells can occur smoothly.
  • CO 2 carbon dioxide
  • the culturing can be performed in a closed incubator, particularly in a closed incubator maintained in a sterile state.
  • Incubators suitable for the present invention can be GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising mammary papilla cells differentiated from adipose-derived stem cells obtained according to the manufacturing method described above.
  • the pharmaceutical composition of the present invention can be prepared as an oral or parenteral formulation by a conventional method known in the art, depending on the route of administration, including a pharmaceutically acceptable carrier or excipient.
  • Such pharmaceutically acceptable carriers or excipients which do not have special toxicity to the human body and do not inhibit the activity or properties of the drug, may be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline solution and sterile water), syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, Ringer's solution, buffers, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof.
  • suitable carriers or excipients include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc., and one or more components may be used alone or in combination. If necessary, other conventional pharmaceutical additives such as antioxidants, buffers, and bacteriostatic agents may be added and used.
  • the pharmaceutical composition of the present invention when formulated as an oral administration agent, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and when formulated as a parenteral administration agent, particularly an injection, it can be manufactured in the form of a unit dosage ampoule or multiple dosage forms.
  • the pharmaceutical composition of the present invention can also be manufactured in the form of a solution, suspension, tablet, pill, capsule, sustained-release preparation, etc.
  • the pharmaceutical composition of the present invention is formulated in the form of a unit dosage form suitable for administration into a patient's body according to a method conventional in the pharmaceutical field, and may be administered by an oral administration route or a parenteral administration route such as a skin, intralesional, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal, or rectal route using an administration method conventionally used in the art.
  • an oral administration route or a parenteral administration route such as a skin, intralesional, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal, or rectal route using an administration method conventionally used in the art.
  • the dosage (effective amount) of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and a person skilled in the art can appropriately determine the dosage by considering these factors.
  • the pharmaceutical composition of the present invention is prepared as an injection in the form of a unit dose, and when prepared as an injection in the form of a unit dose, the amount of mast cells contained per unit dose of the pharmaceutical composition of the present invention can be in the range of 10 2 -10 7 cells/ml.
  • the present invention relates to a method for producing a pharmaceutical composition, comprising the steps of treating and culturing human adipose-derived stem cells with a composition for inducing differentiation of adipose-derived stem cells (particularly human adipose-derived stem cells) into dermal papilla cells, which comprises a complex such as 6BIO as an active ingredient, to differentiate the human adipose-derived stem cells into dermal papilla cells, obtaining differentiated dermal papilla cells, and mixing the obtained dermal papilla cells with the pharmaceutically acceptable carrier or excipient described above to produce a pharmaceutical composition.
  • a composition for inducing differentiation of adipose-derived stem cells particularly human adipose-derived stem cells
  • dermal papilla cells which comprises a complex such as 6BIO as an active ingredient
  • the composition for inducing differentiation of the present invention may additionally contain BMP2 (Bone morphogenetic protein 2), BMP4, etc., in addition to the complex such as 6BIO, which is an active ingredient, in order to supplement or increase the differentiation inducing activity of the complex.
  • BMP2 Bis morphogenetic protein 2
  • BMP4 BMP4
  • 6BIO 6BIO
  • a composition for inducing differentiation of adipose-derived stem cells into mammary papilla cells using a complex such as 6BIO can be provided.
  • composition for inducing differentiation of the present invention is a technology capable of differentiating hair papilla cells from adipose-derived stem cells, and such differentiated hair papilla cells can be usefully used as a cell therapy agent for hair loss treatment.
  • Figure 1 is a schematic diagram showing the process of differentiating mammary papilla cells from human adipose-derived stem cells according to the present invention.
  • Figures 2 to 4 show the results of confirming the degree of differentiation of human adipose-derived stem cells into papilla cells and the degree of expression of papilla cell-specific factors LEF-1, Wnt5 ⁇ , and BMP4 using FACS (fluorescence activated cell sorter) or RT-PCR (reverse transcription polymerase chain reaction).
  • FACS fluorescence activated cell sorter
  • RT-PCR reverse transcription polymerase chain reaction
  • Human adipose tissue used in the present invention was purchased from Goma Biotech Co., Ltd. (Seoul, Korea), and stem cells were isolated from the adipose tissue according to the method of Zuk et al. (Mol Biol Cell. 2022 Dec; 13(12): 4279-4295).
  • stem cells from adipose tissue After isolating stem cells from adipose tissue, they were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml), and 10% heat-inactivated serum under 95% air, 5% CO2, and 37°C conditions. When the cells grew by attaching to the culture dish, they were collected using 0.05% trypsin/10 mM EDTA and maintained in DMEM supplemented with 10% (w/v) Fetal Bovine Serum (FBS).
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • penicillin 100 U/ml
  • streptomycin 100 ug/ml
  • heat-inactivated serum under 95% air, 5% CO2, and 37°C conditions.
  • FBS Fetal Bovine Serum
  • a new composition of a differentiation-inducing medium was prepared in order to differentiate human adipose-derived stem cells into dermal papilla cells.
  • the medium was prepared by using a medium (DMEM containing 10% (w/v) of FBS) used for culturing human adipose-derived stem cells as in Example 1, and a substance for inducing differentiation into dermal papilla cells, which is known as a differentiation-inducing substance, namely, a glycogen synthase kinase 3a/b inhibitor (6-Bromoindirubin-3'-oxime; 6BIO), morroniside (MOR), soluble mastic gum, bone morphogenetic protein 2 (BMP2), and/or bone morphogenetic protein 4 (BMP4), to prepare five types of differentiation-inducing culture media.
  • concentrations of the differentiation-inducing substances used in the media and the compositions of the five types of differentiation-inducing media are as shown in Tables 1 and 2 below.
  • the differentiation-inducing substances used in this experiment 6BIO, BMP2, and BMP4, were purchased from Sigma-Aldrich (USA), and moroniside was purchased from BIOFRON (USA).
  • 20% of the selected mastic gum powder weight, 75.7% of purified water weight, 4% of cyclodextrin weight, and 0.3% of additive (gatti gum) weight were added and dispersed using a homomixer (KNS Company, Ltd.) at 30 to 50°C and 2,000 to 3,000 rpm for 30 to 60 minutes to a particle size of approximately 20 to 50 ⁇ m.
  • the above mastic gum dispersion was carried out using a bead mill (Dientech Co., Ltd.) at 20 to 40°C for 3 to 6 hours until the final dispersed particles had an average of 600 to 1,000 nm. Thereafter, the dispersion was filtered through a 40 to 60 ⁇ m housing filter to obtain a mastic gum aqueous solution in a suspended state.
  • the obtained water-soluble mastic gum was stored in a refrigerator at 4°C, and when the original solution was used, it was left at 40°C for 30 minutes and then shaken sufficiently to use the homogenized solution.
  • Example 3 Induction of differentiation from human adipose-derived stem cells into mammary papilla cells
  • human adipose-derived stem cells In order to induce differentiation of human adipose-derived stem cells into dermal papilla cells, they were cultured for 24 hours at a concentration of 1 ⁇ 10 5 cells/well in a 6-well plate under the culture conditions used in Example 1. When the cells attached to the bottom, they were washed with the solvent DPBS (Dulbecco's Phosphate-buffered saline), and differentiation was induced for 7 days using each differentiation medium prepared in Example 2. At this time, the differentiation medium was replaced every day, and the existing differentiation medium was removed and replaced with 2 mL of new differentiation medium to induce differentiation.
  • Fig. 1 A schematic diagram of the differentiation process of human adipose-derived stem cells into dermal papilla cells of the present invention is shown in Fig. 1.
  • LEF-1 and Wnt5 ⁇ which are specific factors of dermal papilla cells, were identified using FACS (fluorescence activated cell sorter) for human adipose-derived stem cells differentiated according to the above method (Annals of Dermatology 2019;31(2):164-174; Int J Med Sci. 2013; 10(6):738-746; Int J Mol Sci. 2023 Feb; 24(4): 3961).
  • Human adipose-derived stem cells differentiated according to each condition were treated with 0.05% trypsin/EDTA to detach the cells, and then the cells were fixed and stained using a Fixation/Permeabilization solution kit (BDcytofix/Cytoperm TM , BD).
  • the cells were fixed for 20 minutes at 4°C with the BD Cytofix/Cytoper solution in the kit, and washed three times with BD Perm/Wash TM buffer. Afterwards, a solution of LEF-1 antibody (Anti-LEF-1 antibody phycoerythrin, PE-A; Santacruz) or Wnt5a antibody (Anti-Wnt5a antibody phycoerythrin, PE-A; Santacruz) diluted in BD Perm/Wash TM buffer was added and stained at 4°C for 30 minutes. After staining, the cells were washed three times using BD Perm/Wash TM buffer and finally suspended in PBS and analyzed using a flow cytometer (FACSLyric, BD Biosciences).
  • LEF-1 antibody Anti-LEF-1 antibody phycoerythrin, PE-A; Santacruz
  • Wnt5a antibody Anti-Wnt5a antibody phycoerythrin, PE-A; Santacruz
  • the proportion of cells positive for LEF-1 did not exceed 25% when treated with 6BIO and BMP2, when treated with MOR and BMP2, and when treated with 6BIO, MOR, and BMP2, but the proportion of cells positive for LEF-1 increased significantly to 66.4% and 72.93% when treated with 6BIO, mastic gum, and BMP2, and when treated with 6BIO, MOR, mastic gum, and BMP2, respectively.
  • human adipose-derived stem cells were prepared at 1x105 cells/well in a 6-well plate and cultured for 24 hours under the conditions of a 37°C, 5% CO2 incubator. After 24 hours of culture, the culture medium was removed, washed with DPBS, and differentiation was induced for 7 days using each differentiation medium prepared through Example 2 above. After differentiation was completed, total RNA was isolated from the cells attached to the bottom using Trizol Reagent (Invtrogen).
  • RNA was quantified using NanoDrop TM , and cDNA was synthesized using 1 ug of each total RNA sample and AccuPower CycleScript RT PreMix (dN6) Kit (Bioneer, Korea). 80 ⁇ l of RNase-water was added to 20 ⁇ l of the synthesized cDNA and used as a cDNA template.
  • Each sample which was mixed with 1 ⁇ l of cDNA template, 10 ⁇ l of SYBRTM Green Master Mix, 1 ⁇ l each of BMP4 and LEF-1 gene primers, and 7 ⁇ l of RNase free water, was subjected to qPCR using a QuantStudio 3 Real-Time PCR instrument, and the primer information is shown in Table 3.
  • the CT values measured through qPCR were compared by calculating the relative expression rate using the 2 - ⁇ CT method.
  • Primer gene Primer sequence BMP4-Forward CGGGCCAGGAAGAAGAATAAG (SEQ ID NO: 1) BMP4-Reverse CCAGTCATTCCAGCCCACAT (SEQ ID NO: 2) LEF-1-Forward ACAGATCACCCCACCTCTTG (SEQ ID NO: 3) LEF-1-Reverse ATAGCTGGATGAGGGATGCC (SEQ ID NO: 4)
  • Fig. 4 The results are shown in Fig. 4. Referring to Fig. 4, there was not much difference in the expression level of the BMP4 gene, a breast papilla cell-specific factor, compared to the control ADSCs when treated with MOR and mastic gum, whereas when treated with 6BIO, the gene expression level of BMP4 increased, and when 6BIO and mastic gum were treated together and when 6BIO, MOR, and mastic gum were treated together, the gene expression of BMP4 increased even more (Fig. 4 (A)). This trend was also observed in the gene expression level of LEF-1. BMP4 is a specific factor expressed in hair papilla cells and is used as an indicator factor showing the degree of hair papilla cell differentiation (Genes Dev.

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Abstract

La présente invention concerne une composition pour induire la différenciation de cellules souches dérivées du tissu adipeux en cellules de papille dermique à l'aide d'un composite de 6BIO, moroniside et gomme de mastic solubilisée. La composition d'induction de la différenciation selon la présente invention est une technique capable de différencier les cellules de papille dermique à partir de cellules souches dérivées du tissu adipeux par l'utilisation d'un composite de 6BIO, de moroniside et de gomme de mastic solubilisée, et ainsi les cellules de papille dermique différenciées peuvent être utiles comme agent thérapeutique cellulaire pour traiter la perte de cheveux.
PCT/KR2024/007206 2023-12-05 2024-05-28 Composition pour induire la différenciation de cellules souches dérivées du tissu adipeux en cellules de papille dermique à l'aide d'un composite de 6bio, moroniside et gomme de mastic solubilisée Pending WO2025121569A1 (fr)

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KR1020230174478A KR102697520B1 (ko) 2023-12-05 2023-12-05 6bio와 모로니사이드 및 수용화 매스틱 검의 복합물을 이용한 지방 유래 줄기세포의 모유두세포로의 분화 유도용 조성물
KR10-2023-0174478 2023-12-05

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120003175A1 (en) * 2009-03-04 2012-01-05 Zadik Hazan Therapeutic uses of mastic gum fractions
KR20190009716A (ko) * 2017-07-19 2019-01-29 서울대학교산학협력단 인간 유래 만능줄기세포에서 모유두전구세포로의 분화방법 및 이의 용도
KR102328824B1 (ko) * 2021-07-12 2021-11-23 주식회사 프롬바이오 수용화 매스틱 검과 감태나무 추출물을 이용한 발모 촉진 또는 탈모 억제용 조성물
KR102543431B1 (ko) * 2022-10-05 2023-06-15 주식회사 프롬바이오 지방 유래 줄기세포의 모유두세포로의 분화 유도용 조성물 및 그 조성물을 이용한 분화 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120003175A1 (en) * 2009-03-04 2012-01-05 Zadik Hazan Therapeutic uses of mastic gum fractions
KR20190009716A (ko) * 2017-07-19 2019-01-29 서울대학교산학협력단 인간 유래 만능줄기세포에서 모유두전구세포로의 분화방법 및 이의 용도
KR102328824B1 (ko) * 2021-07-12 2021-11-23 주식회사 프롬바이오 수용화 매스틱 검과 감태나무 추출물을 이용한 발모 촉진 또는 탈모 억제용 조성물
KR102543431B1 (ko) * 2022-10-05 2023-06-15 주식회사 프롬바이오 지방 유래 줄기세포의 모유두세포로의 분화 유도용 조성물 및 그 조성물을 이용한 분화 방법
KR102588893B1 (ko) * 2022-10-05 2023-10-13 주식회사 프롬바이오 지방 유래 줄기세포의 모유두세포로의 분화 유도용 조성물 및 그 조성물을 이용한 분화 방법

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