WO2016126122A2 - Composition pour induction de différenciation de chondrocyte ou régérération de tissu cartilagineux à base d'exosomes extraits de cellules souches se différenciant en chondrocytes - Google Patents

Composition pour induction de différenciation de chondrocyte ou régérération de tissu cartilagineux à base d'exosomes extraits de cellules souches se différenciant en chondrocytes Download PDF

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WO2016126122A2
WO2016126122A2 PCT/KR2016/001230 KR2016001230W WO2016126122A2 WO 2016126122 A2 WO2016126122 A2 WO 2016126122A2 KR 2016001230 W KR2016001230 W KR 2016001230W WO 2016126122 A2 WO2016126122 A2 WO 2016126122A2
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stem cells
composition
chondrocytes
cartilage
exosomes
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Korean (ko)
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WO2016126122A3 (fr
Inventor
조용우
최지숙
우창희
최영찬
조동규
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Industry University Cooperation Foundation IUCF HYU
Industry University Cooperation Foundation of Sogang University
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Industry University Cooperation Foundation IUCF HYU
Industry University Cooperation Foundation of Sogang University
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Priority claimed from KR1020160013293A external-priority patent/KR101706642B1/ko
Application filed by Industry University Cooperation Foundation IUCF HYU, Industry University Cooperation Foundation of Sogang University filed Critical Industry University Cooperation Foundation IUCF HYU
Priority to EP16746863.6A priority Critical patent/EP3235500B1/fr
Priority to JP2017538397A priority patent/JP6577038B2/ja
Priority to CN201680008522.3A priority patent/CN107223153B/zh
Publication of WO2016126122A2 publication Critical patent/WO2016126122A2/fr
Publication of WO2016126122A3 publication Critical patent/WO2016126122A3/fr
Priority to US15/642,156 priority patent/US20170296590A1/en
Anticipated expiration legal-status Critical
Priority to US17/894,936 priority patent/US20230047434A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • 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
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • 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
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin

Definitions

  • the present invention comprises a composition for inducing chondrocyte differentiation or cartilage tissue regeneration comprising exosomes extracted from stem cells differentiated into chondrocytes as an active ingredient, a medium composition for inducing chondrocyte differentiation comprising the composition, for chondrocyte regeneration It relates to an injection, a pharmaceutical composition for treating cartilage disease, and a method for treating cartilage disease.
  • a pharmaceutical preparation for treating osteoarthritis including Clodronic Acid and Hyaluronic Acid (Korean Patent Publication No. 2008-0082657) and the like have been developed.
  • the method may provide temporary pain reduction, but there is a lack in inducing regeneration into cartilage tissues, and thus, effective factors to help the differentiation of cartilage tissues are required.
  • stem cell therapy using stem cells such as cord blood, adipose tissue, synovial membrane, and muscle stem is generally performed.
  • stem cells such as cord blood, adipose tissue, synovial membrane, and muscle stem.
  • cartilage cells or adult stem cells obtained from patients were cultured in vitro, dispersed in hydrogels, and then transplanted into defective areas.
  • Direct cell injection allows regeneration of tissues close to normal cartilage, but inevitable surgical procedures for obtaining cells, difficulty in in vitro culture, limitation of cell number depending on the size of damaged tissue, and low differentiation rate I have a problem.
  • a single administration can reduce temporary pain and increase joint mobility, ultimately, regeneration of damaged cartilage tissue was difficult.
  • the present inventors extracted only exosomes from stem cells differentiated into chondrocytes, and completed the present invention by confirming the cartilage regeneration effect of the composition including the extracted exosomes.
  • Patent Document 1 Korean Unexamined Patent Publication No. 2013-0028012
  • Patent Document 2 Korean Patent Publication No. 1279812
  • Patent Document 3 Korean Unexamined Patent Publication No. 2008-0082657
  • Patent Document 4 Korean Unexamined Patent Publication No. 2013-0072983
  • Patent Document 5 Korean Unexamined Patent Publication No. 2013-0009651
  • An object of the present invention is to provide a composition for inducing chondrocyte differentiation or cartilage tissue regeneration comprising an exosome extracted from stem cells differentiated into chondrocytes as an active ingredient.
  • Another object of the present invention is to provide a chondrocyte differentiation inducing medium composition comprising the chondrocyte differentiation inducing or chondrogenic regeneration composition.
  • Still another object of the present invention is to provide an injection for cartilage tissue regeneration comprising the composition for inducing chondrocyte differentiation or cartilage tissue regeneration.
  • Another object of the present invention is to provide a pharmaceutical composition for treating cartilage disease comprising the composition for inducing chondrocyte differentiation or cartilage tissue regeneration.
  • Another object of the present invention is to provide a method for treating cartilage disease comprising administering to the mammal a therapeutically effective amount of the composition for inducing chondrocyte differentiation or cartilage tissue regeneration.
  • one embodiment of the present invention provides a composition for inducing chondrocyte differentiation or cartilage tissue regeneration comprising exosomes extracted from stem cells differentiated into chondrocytes as an active ingredient.
  • stem cells that are differentiated into chondrocytes refers to stem cells that are in the process of differentiating from chondrocyte stem cells to chondrocytes as shown in FIG. 1. From this, exosomes containing the genetic information, proteins, and growth factors of chondrocytes can be extracted.
  • exosome refers to a membrane vesicle that is secreted from various cell types, and binds to other cells and tissues to deliver membrane components, proteins, and RNA. It is known.
  • Exosomes extracted from stem cells that are differentiated into chondrocytes may have basic characteristics of stem cells, and may contain important growth factors, various bioactive proteins and genetic information, etc. in chondrocyte differentiation.
  • exosomes can be prepared using exosome extraction methods known in the art, for example
  • the "induced chondrocyte differentiation” may mean to induce stem cells to differentiate into chondrocytes.
  • the "cartilage tissue regeneration” may mean the regeneration of cartilage tissue by restoring damaged cartilage tissue or inducing the generation of scarce cartilage tissue.
  • Stem cells that are differentiated into chondrocytes may be adult stem cells capable of differentiating into chondrocytes.
  • Adult-derived stem cells capable of differentiating into chondrocytes may be bone marrow stem cells, umbilical cord blood stem cells or adipose derived stem cells, for example, adipose derived stem cells.
  • the bone marrow stem cells, umbilical cord blood stem cells or adipose derived stem cells may be stem cells derived from a human body, an animal or a plant.
  • the composition for cartilage regeneration according to the present invention is different from the conventional techniques in that it uses exosomes extracted from stem cells that differentiate into chondrocytes as an effective substance for effective cartilage regeneration.
  • Various growth factors related to the proliferation and differentiation of the cells supported on the extracted and purified exosomes effectively regenerate sustainable cartilage tissues, and problems of in vitro cell culture of existing autologous chondrocytes and adult stem cells, and fibrocartilage. Problems such as furnace regeneration or tissue calcification due to cell death can be solved.
  • Stem cell-derived exosomes extracted during differentiation into chondrocytes according to the present invention effectively deliver not only stem cell characteristics but also active factors related to differentiation into chondrocytes, and minimize the side effects while treating with existing stem cells. The same effect can be expected.
  • Stem cell derived exosomes extracted during differentiation into chondrocytes according to the present invention are biological membrane vesicles secreted from the cells.
  • it since it has a lipid structure similar to that of the cell membrane, when absorbed into the body, it has an excellent absorption rate to neighboring cells, and thus, it is possible to induce effective regeneration of cartilage tissue by fast delivery of agonist substance.
  • Another embodiment of the present invention provides a composition for inducing chondrocyte differentiation comprising the composition for inducing chondrocyte differentiation or chondrocyte regeneration.
  • the medium composition may include, but is not limited to, an exosome in a concentration of 1 to 100 ⁇ g / mL, specifically, 1 to 60 ⁇ g / mL, for example, 50 ⁇ g / mL.
  • the media composition for inducing chondrocyte differentiation is dexamethasone, insulin, ascorbate, cartilage growth factor IGF (Insulin-like Growth Factor) and TGF- ⁇ 1 to differentiate stem cells into chondrocytes. It may further include a differentiation inducer such as (Transforming Growth Factor ⁇ 1), but is not limited thereto.
  • Another embodiment of the present invention provides an injection for cartilage tissue regeneration comprising the composition for inducing chondrocyte differentiation or cartilage tissue regeneration.
  • the injection may further comprise phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the injection may be used to carry a composition for inducing chondrocyte differentiation or cartilage tissue regeneration in phosphate buffered saline.
  • the injection may comprise a hydrogel instead of phosphate buffered saline.
  • the hydrogel may be any one or more selected from the group consisting of hyaluronic acid, gelatin, alginate, chitosan, fibrin, elastin, collagen, and methyl cellulose, and specifically, may be hyaluronic acid hydrogel, but is not limited thereto.
  • the injection may include an exosome in a concentration of 1 to 1000 ⁇ g / mL, specifically 10 to 900 ⁇ g / mL, more specifically 10 to 800 ⁇ g / mL, for example, 500 ⁇ g / mL, but is not limited thereto. It doesn't work.
  • the injection may be administered by injection into a site of damage such as cartilage of a mammal such as a mouse, a mouse, a livestock, or a human.
  • the composition for cartilage regeneration according to the present invention is economical in terms of operation time and cost because it is easy to inject into the body as an injection composition, thereby reducing the pain, sequelae and economic burden of the patient.
  • the stem cells are differentiated into chondrocytes, the extracted exosomes contain extracellular matrix derivatives and various growth factors related to the proliferation and differentiation of cells, thereby inducing effective regeneration of damaged cartilage tissue. Therefore, long-term effects can be expected with a single procedure to overcome the existing problems that had to be performed periodically to obtain a lasting effect.
  • Another embodiment of the present invention provides a pharmaceutical composition for treating cartilage disease comprising the composition for inducing chondrocyte differentiation or cartilage tissue regeneration.
  • cartilage disease may be a cartilage disease resulting from damage to the cartilage tissue, specifically osteoarthritis, deformable arthrosis, chondrogenic dysplasia, degenerative arthritis, rheumatoid arthritis, osteomalacia, fibrous osteoarthritis and aplasia It may be selected from the group consisting of bone diseases, but is not limited thereto.
  • treatment of cartilage disease may mean that damage to cartilage tissue is treated by regenerating damaged cartilage by intra-articular injection of a composition for treating cartilage disease.
  • the pharmaceutical composition according to the above embodiment may be various oral or parenteral formulations.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin.
  • lubricants such as magnesium stearate, talc and the like are also used.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the pharmaceutical composition according to the above embodiment may be used as a non-aqueous solvent, a suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.
  • a suspension solvent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.
  • injectable esters such as ethyl oleate, and the like.
  • the dosage forms of the pharmaceutical compositions according to the above embodiments may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
  • the salt is not particularly limited as long as it is pharmaceutically acceptable.
  • hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid and the like can be used.
  • the pharmaceutical composition according to the above embodiment may be parenterally or orally administered as desired, and may be administered in one to several times so as to be administered in an amount of 0.1 to 500 mg and 1 to 100 mg per kg of body weight per day. Can be. Dosage for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of disease, and the like.
  • compositions according to the above embodiments may be powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, ointments, creams, external preparations, suppositories, and sterile injectable solutions, etc., according to conventional methods. It may be used in formulated in any form suitable for pharmaceutical formulations.
  • the pharmaceutical composition according to the above embodiment may be administered to mammals such as rats, mice, livestock, humans by various routes such as parenteral, oral, and all modes of administration may be expected, but preferably oral, rectal Or by intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
  • the pharmaceutical composition according to the embodiment is dexamethasone (insame), insulin (asulin), ascorbate, cartilage growth factor IGF (Insulin-like Growth Factor) and TGF- ⁇ 1 to differentiate stem cells into chondrocytes. It may further include a differentiation inducer such as (Transforming Growth Factor ⁇ 1), but is not limited thereto.
  • Another embodiment of the present invention provides a method for treating cartilage disease comprising administering to a mammal a therapeutically effective amount of the composition for inducing chondrocyte differentiation or cartilage tissue regeneration.
  • the composition may be a pharmaceutical composition for treating cartilage disease comprising the composition for inducing chondrocyte differentiation or cartilage tissue regeneration, or may be a cartilage regeneration injection including the composition for chondrocyte differentiation or cartilage tissue regeneration. Therefore, the contents described about the pharmaceutical composition for treating cartilage disease or cartilage regenerative injection may be equally applied to the treatment method.
  • the mammal may be a mammal suffering from cartilage disease, and may be, for example, a rat, a mouse, a livestock, a human, and the like, having a cartilage disease.
  • the “therapeutically effective amount” means an amount of the composition sufficient to exhibit a cartilage disease therapeutic response in a mammal.
  • Such dosage forms include parenteral and oral administration, but may be administered by injection into a site of injury such as cartilage in a mammal, for example.
  • exosomes (Adipose tissue-derived stem cell; ASC-EXO) extracted from proliferating stem cells and exosomes (Chongenic differentiating stem cell-derived exosome; Chondro; -EXO) confirming the size, it was confirmed that the average size of each 88.17 nm, 83.6 nm (Fig. 3).
  • composition comprising exosomes (Chondro-EXO) extracted from stem cells differentiated into chondrocytes according to the present invention was confirmed that the cartilage tissue regeneration effect is excellent when injected in vivo ( 7 and 8).
  • the composition for inducing chondrocyte differentiation or cartilage regeneration contains exosomes extracted from stem cells that differentiate into chondrocytes as effective factors. Exosomes secreted during the differentiation of stem cells contain growth factors related to chondrocyte differentiation as well as genes and proteins related to cell proliferation and regeneration of stem cells. In addition, it is possible to deliver agonists into cells stably and rapidly as cell-derived carriers while minimizing side effects on existing cell therapy agents. Therefore, as an injection composition for the prevention and treatment of cartilage diseases, it is possible to reduce the pain of patients with simple procedures and to treat cartilage diseases continuously and effectively after the procedure.
  • FIG. 1 is a schematic diagram of exosomes extracted from stem cells that are differentiated into chondrocytes and their applications.
  • Figure 2 shows the timing of extraction of exosomes from stem cells that are differentiated into chondrocytes and observed the shape change of stem cells differentiated into chondrocytes and cartilage specific substrate synthesis through alcian blue staining. It is confirmed figure.
  • Figure 3 is a diagram of the characteristics of exosomes (Chondro-Exo) extracted from stem cells differentiated into chondrocytes and exosomes (ASC-Exo) extracted from proliferating stem cells:
  • FIG. 4 shows Exo-CheckTM exosome antibody arrays of exosomes extracted from stem cells differentiated into chondrocytes.
  • GM stem cell growth medium
  • ASC-EXO exosomes extracted from proliferating stem cells
  • Chondro-EXO exosomes extracted from stem cells differentiated into chondrocytes
  • DM chondrocyte differentiation medium (differentiation medium)
  • dotted line The part where condensation has taken place between cells.
  • Figure 6 is the result of analysis of human adipose derived stem cells into chondrocytes 21 days after induction of differentiation; A: Confirmation of synthesis of acid mucosubstance and acid mucin, cartilage specific substrates by alcian blue staining, B: Cartilage specificity through safranin-o staining Confirmation of synthesis of proteoglycans, GM: stem cell growth medium, Chondro-EXO: exosomes derived from stem cells differentiated into chondrocytes, DM: chondrocyte differentiation medium .
  • FIG. 7 shows exosomes (Chondro-EXO) extracted from stem cells differentiated into chondrocytes in osteoarthritis model mice and cartilage tissue after injection of phosphate-buffered saline (PBS) as a control. Safranin-o staining was performed to confirm the regeneration of the (knee joint, 100x); T: tibia, F: femur.
  • PBS phosphate-buffered saline
  • FIG. 8 is a graph showing a Mankin score showing the degree of induction of osteoarthritis based on Table 1; PBS: Phosphate-buffered saline (negative control), Chondro-EXO: Exosomes extracted from stem cells differentiated into chondrocytes, Femoral condyle: Femoral joints, Tibial plateau: Tibial plateau.
  • human adipose derived stem cells were cultured in a general culture medium (Dulbecco Modified Eagle Medium high glucose (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin).
  • DMEM Dulbecco Modified Eagle Medium high glucose
  • Differentiation medium 5% fetal bovine serum, 1% penicillin / streptomycin, 100 nM dexamethasone, 0.15 mM ascorbic acid, 1X ITS (Insulin-Transferrin-Sodium selenite), 10 ng / mL TGF- Differentiation was induced into chondrocytes by replacing with Dulbecco Modified Eagle Medium high glucose (DMEM) containing ⁇ 1 (Transforming Growth Factor ⁇ 1) and incubating for a total of 5 weeks.
  • DMEM Dulbecco Modified Eagle Medium high glucose
  • the stem cells were replaced with DMEM medium without phenol red while maintaining serum for 24 hours before extracting the exosomes, and cell culture supernatants were recovered.
  • the supernatant obtained after concentration was mixed with exosome isolation reagent in a 1: 0.5 ratio and stored at 4 ° C. for one day.
  • exosome precipitate was obtained by centrifugation at 10,000 ⁇ g for 60 minutes, filtered through a 0.22 ⁇ m filter (exosome spin column), and washed with phosphate-buffered saline (PBS). The washed exosome precipitate was centrifuged at 10,000 xg for 60 minutes and then resuspended in PBS. After the supernatant was recovered, the differentiation medium was added to induce chondrocyte differentiation of stem cells, and this process was repeated for 5 weeks. After 2 weeks of differentiation medium replacement, the intercellular condensation observed when differentiating into chondrocytes began to appear and after 5 weeks colonies were produced (FIG. 2). Therefore, the exosomes were extracted from the supernatant recovered during the period from 2 weeks to 5 weeks after the differentiation induction which clearly shows the change in cell shape.
  • PBS phosphate-buffered saline
  • exosomes (Adipose tissue-derived stem cell (ASC-EXO)) were extracted from proliferating human adipose derived stem cells and used as a comparison group. Specifically, exosomes were extracted from the proliferating human adipose derived stem cells by following the same method as Example 1 except that no differentiation medium was used.
  • ASC-EXO Adipose tissue-derived stem cell
  • exosome-specific markers known as exosome membrane surface markers (CD63, CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5 and TSG101) was confirmed by antibody reaction using exosome antibody arrays ( 4).
  • exosomes extracted from proliferating stem cells ACS-EXO
  • exosomes extracted from stem cells differentiated into chondrocytes Chondro
  • the medium composition was used in addition to the concentration of exosome 10 ⁇ g / mL to the stem cell culture medium (Dulbecco Modified Eagle Medium high glucose (DMEM) containing 5% fetal bovine serum, 1% penicillin / streptomycin).
  • DMEM Dulbecco Modified Eagle Medium high glucose
  • DMEM Dulbecco Modified Eagle Medium high glucose
  • Cartilage differentiation medium 5% fetal bovine serum, 1% penicillin / streptomycin, 100 nM dexamethasone, 0.15 mM ascorbic acid, 1X ITS (Insulin-Transferrin-Sodium selenite), 10 ng / mL TGF- ⁇ 1 (Transforming Growth Factor) Dulbecco Modified Eagle Medium high glucose (DMEM) containing ⁇ 1) was used.
  • the medium composition was replaced every 35 days for 3 days, and the change in cell morphology was confirmed using a microscope for stem cells induced differentiation into chondrocytes.
  • the cells treated with exosomes (Chondro-EXO) extracted from stem cells differentiated into chondrocytes showed a precartilage condensation between cells at the level similar to the positive control group from the 21st day.
  • GM negative control group
  • ASC-EXO exosomes
  • a medium composition containing exosomes (Chondro-EXO) extracted from stem cells differentiated into chondrocytes was used.
  • the media composition is Dulbecco Modified Eagle containing 5, 10, 30, 50 ⁇ g / mL exosomes extracted from stem cells differentiated into chondrocytes (5% fetal bovine serum, 1% penicillin / streptomycin).
  • Medium high glucose (DMEM) was used in addition.
  • Stem cell culture medium (Dulbecco Modified Eagle Medium high glucose (DMEM) containing 5% fetal bovine serum, 1% penicillin / streptomycin) was used as a negative control, and cartilage differentiation medium (5% fetal bovine serum, Dulbecco Modified Eagle Medium high glucose containing 1% penicillin / streptomycin, 100 nM dexamethasone, 0.15 mM ascorbic acid, 1X ITS (Insulin-Transferrin-Sodium selenite), 10 ng / mL TGF- ⁇ 1 (Transforming Growth Factor ⁇ 1) (DMEM)) was used.
  • DMEM Transforming Growth Factor ⁇ 1
  • the medium composition was changed every 3 days for 21 days, and the stem cells differentiated into chondrocytes were alkian blue staining and safranin-o staining. The differentiation of cells was analyzed.
  • the exosomes extracted from stem cells differentiated into chondrocytes have an excellent effect of inducing differentiation from stem cells to chondrocytes.
  • the knee joint was cut about 1 cm along the side of the patella using a surgical tool. After excision by exposing the joint capsule, the medial meniscotibial ligament connected to the medial meniscus is cut out, the joint capsule and the skin are closed in order, and finished with a suture thread. It was.
  • the exosome composition was prepared by supporting exosomes extracted from stem cells differentiated into chondrocytes of Example 1 in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the exosome composition has a final concentration of exosome 500 ⁇ g / mL, and injected 6 ⁇ L (3 ⁇ g / 6 ⁇ L per mouse) in the joint cavity of the mouse.
  • Phosphate-buffered saline (PBS) was used as a negative control. After 11 weeks, the regeneration of cartilage tissue was confirmed using safranin-o staining.
  • cartilage-specific substrates were synthesized when the composition containing exosomes extracted from stem cells differentiated into chondrocytes was synthesized compared to the negative control group and regenerated similarly to natural cartilage without damage to the superficial zone. Confirmed. The regenerated cartilage was stained red. Therefore, it was confirmed that the composition containing the exosomes extracted from stem cells differentiated into chondrocytes has excellent cartilage regeneration effect (FIG. 7).

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Abstract

La présente invention se rapporte à : une composition pour induction de différenciation de chondrocyte ou régénération de tissu cartilagineux, contenant, en tant qu'ingrédients actifs, des exosomes extraits de cellules souches se différenciant en chondrocytes ; une composition de milieu pour induction de différenciation de chondrocyte, une injection pour régénération de tissu cartilagineux, et une composition pharmaceutique pour le traitement de troubles des cartilages, dont toutes contiennent la composition ; et un procédé pour le traitement de troubles des cartilages. Selon un mode de réalisation spécifique de la présente invention, la composition pour induction de différenciation de chondrocyte ou régénération du cartilage, contient, en tant que facteurs actifs, des exosomes extrait de cellules souches se différenciant en chondrocytes. Les exosomes à sécréter pendant la différenciation de cellules souches contiennent des facteurs de croissance associés à la différenciation de chondrocyte, et des gènes et des protéines associés à la prolifération et à la régénération de cellules souches, et ainsi l'induction de régénération de tissu cartilagineux endommagé est excellente. En outre, un matériau actif peut être distribué de manière stable et rapide en tant que véhicule dérivé de cellules dans des cellules alors que les effets secondaires sur un agent thérapeutique cellulaire classique sont réduits au minimum. Par conséquent, la douleur d'un patient peut être réduite par une simple opération au moyen d'une composition d'injection pour prévenir et traiter les troubles des cartilages, et les troubles des cartilages peuvent être traités en continu et efficacement après une opération.
PCT/KR2016/001230 2015-02-04 2016-02-04 Composition pour induction de différenciation de chondrocyte ou régérération de tissu cartilagineux à base d'exosomes extraits de cellules souches se différenciant en chondrocytes Ceased WO2016126122A2 (fr)

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Application Number Priority Date Filing Date Title
EP16746863.6A EP3235500B1 (fr) 2015-02-04 2016-02-04 Composition pour induction de différenciation de chondrocyte ou régérération de tissu cartilagineux à base d'exosomes extraits de cellules souches se différenciant en chondrocytes
JP2017538397A JP6577038B2 (ja) 2015-02-04 2016-02-04 軟骨細胞に分化されている幹細胞由来のエキソソームを含む軟骨細胞への分化誘導または軟骨組織再生用組成物
CN201680008522.3A CN107223153B (zh) 2015-02-04 2016-02-04 包含源于正分化成软骨细胞之干细胞的外排体的用于软骨细胞分化诱导或软骨组织再生的组合物
US15/642,156 US20170296590A1 (en) 2015-02-04 2017-07-05 Composition for inducing chondrocyte differentiation and regenerating cartilage tissue
US17/894,936 US20230047434A1 (en) 2015-02-04 2022-08-24 Composition for inducing chondrocyte differentiation and regenerating cartilage tissue

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KR20150017349 2015-02-04
KR10-2015-0017349 2015-02-04
KR1020160013293A KR101706642B1 (ko) 2015-02-04 2016-02-03 연골세포로 분화되고 있는 줄기세포로부터 추출된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물
KR10-2016-0013293 2016-02-03

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CN112601533A (zh) * 2017-12-06 2021-04-02 设计细胞股份有限公司 将含有源自干细胞的外泌体的培养液作为有效成分包含的关节炎预防或治疗用组合物
CN113166725A (zh) * 2018-09-19 2021-07-23 耶迪特普大学 用于生成软骨组织的分离的中隔软骨外来体
CN115487208A (zh) * 2022-10-24 2022-12-20 格莱康美生物医学技术(北京)有限公司 一种用于治疗骨关节炎/软骨损伤的组合物及其制备方法

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EP2399990B1 (fr) * 2003-06-27 2015-07-22 DePuy Synthes Products, Inc. Cellules dérivées de cordon ombilical post-partum pour l'utilisation dans le traitement de maladies du coeur et du système circulatoire
CA2711218C (fr) * 2008-01-04 2019-08-06 Lydac Neuroscience Limited Procede de production d'une population de cellules differenciees
CA2879322A1 (fr) * 2012-07-19 2014-01-23 Reneuron Limited Microparticules de cellules souches
CN103767985A (zh) * 2012-10-22 2014-05-07 吉林省霍普金斯药物研究院有限责任公司 人源血液或间充质干细胞分泌exosome的制备与应用

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CN112601533A (zh) * 2017-12-06 2021-04-02 设计细胞股份有限公司 将含有源自干细胞的外泌体的培养液作为有效成分包含的关节炎预防或治疗用组合物
CN112601533B (zh) * 2017-12-06 2024-02-06 设计细胞股份有限公司 将含有源自干细胞的外泌体的培养液作为有效成分包含的关节炎预防或治疗用组合物
CN113166725A (zh) * 2018-09-19 2021-07-23 耶迪特普大学 用于生成软骨组织的分离的中隔软骨外来体
EP3852875A4 (fr) * 2018-09-19 2022-06-22 Yeditepe Universitesi Exosome de cartilage septal isolé utilisé pour générer un tissu cartilagineux
CN115487208A (zh) * 2022-10-24 2022-12-20 格莱康美生物医学技术(北京)有限公司 一种用于治疗骨关节炎/软骨损伤的组合物及其制备方法

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