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WO2019107939A1 - Composition pour favoriser la production d'un exosome dérivé d'une cellule souche - Google Patents

Composition pour favoriser la production d'un exosome dérivé d'une cellule souche Download PDF

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WO2019107939A1
WO2019107939A1 PCT/KR2018/014873 KR2018014873W WO2019107939A1 WO 2019107939 A1 WO2019107939 A1 WO 2019107939A1 KR 2018014873 W KR2018014873 W KR 2018014873W WO 2019107939 A1 WO2019107939 A1 WO 2019107939A1
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stem cells
derived
exosome
cell
stem cell
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김수
오연목
이슬기
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Asan Foundation
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention was made by the task number HI16C2187 under the support of the Ministry of Health and Welfare of the Republic of Korea.
  • the research institute of the above-mentioned task is the Health Industry Promotion Agency; the research project name is “Advanced Medical Technology Development”; the research title is “ Development of a therapeutic agent for chronic obstructive pulmonary disease ", and” Asan Medical Center in Seoul “.
  • the research period is 2016.11.08-2021.07.31.
  • the present invention is also made by the task No. NRF-2015K1A4A3046807 under the support of the Ministry of Science, Technology & Information and Communications of the Republic of Korea.
  • the research institute of the above subject is Korea Research Foundation, the name of the project is "overseas excellent research institute attracting business” Asan-Minnesota Implantation and Innovation Center ", the host institution is” Seoul Asan Hospital “and the period of research is 2015.09.01-2021.08.31.
  • the present invention relates to a composition for promoting the production of stem cell-derived exosomes comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide)
  • the present invention relates to a method for producing exosomes.
  • Extracellular vesicles are vesicles composed of a lipid-bilayer of 30-1000 nm size including microvesicles, exosome and the like.
  • the lipid bilayer of exosomes is a phospholipid bilayer structure like the origin cells (donor cells), and is a constituent of a cell secreted outside the cell, and functions as a cell-cell communication and a cellular immune intervention .
  • Exosomes contain cell-specific components that reflect the biological functions peculiar to the origin cells (donor cells) and include various water-soluble proteins, extrinsic proteins, and membrane-penetrating protein components in addition to phospholipids, mRNAs, and miRNAs.
  • markers for exosomes CD63 and CD81 are well known.
  • Exosomes can also penetrate the blood-brain barrier (BBB) and are highly selective for the permeability of epidermal and endothelial cell membranes, resulting in a drug delivery system (DDS), a nanocarrier of certain drugs, It is also used for development.
  • BBB blood-brain barrier
  • DDS drug delivery system
  • exosomes and microvesicles secreted from mesenchymal stem cells are involved in cell-to-cell communication and are known to exhibit regenerative medical therapeutic effects of stem cells. It has been known that after transplantation of stem cells into the body, trophic effect is exerted on paracrine factors secreted from cells without long-term survival. These factors include growth factors, chemokines ) And cytokines are secreted by extracellular vesicles such as exosomes, and these exosomes originate from stem cells. Thus, exosomes have been used to characterize stem cells and to assess their therapeutic efficacy.
  • the present inventors have sought to develop methods and materials for promoting the production of stem cell-derived exosomes. As a result, it was confirmed that when pre-treating the stem cells with pioglitazone, metformin, and AICAR, the content of protein and RNA in the exosome as well as the number of stem cell-derived exosomes was greatly increased, thereby completing the present invention.
  • an object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) .
  • the present invention provides a method for promoting the production of stem cell-derived exosomes comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) ≪ / RTI >
  • Pioglitazone which is one of the components of the composition of the present invention, is a diabetes remedy having a hypoglycemic effect based on thiazolidinedione (TZD). Although there is a blood glucose lowering effect, the effect on cardiovascular disease is not statistically significant.
  • Metformin another of the components of the composition of the present invention, is a treatment for diabetes mellitus for biguanides. Metformin has also been shown to improve blood sugar, but the evidence for the prevention of cardiovascular disease is still limited. One mechanism of action is to activate the AMP-activated protein kinase (AMPK) in the liver, thereby inhibiting the biosynthesis of the grape, promoting glucose uptake into cells, and inhibiting the metabolic syndrome.
  • AMPK AMP-activated protein kinase
  • metformin Although the mechanism of action of metformin is not clearly known, it has been suggested that 1) it suppresses cell respiration in mitochondria, 2) activates AMPK, 3) prevents the increase of glucagon-induced cAMP (cyclic adenosine monophosphate) (Protein kinase A), and 4) it affects the gut flora in the intestines.
  • AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), another component of the composition of the present invention, is an intermediate for the production of inosine monophosphate.
  • AICAR is known to stimulate AMP-dependent protein kinase (AMPK) as an analog of AMP and has been used clinically as an agent for treating and protecting cardiac ischemic injury.
  • AMPK AMP-dependent protein kinase
  • composition according to the present invention may further comprise a known substance for promoting exosome formation in addition to the above-mentioned components.
  • exosome is a cell-derived vesicle and is present in body fluids of almost all eukaryotes. Generally, the diameter of the exosomes is about 30-100 nm, which is larger than the LDL protein, but much smaller than the red blood cells. Although exosomes are distinguished from " microemboli " having diameters of about 100-1000 nm, the term " exosome " is used herein to encompass " microembossies " Is used. Exosomes are released from or released directly from the cell when the multivesicular bodies are fused with the cell membrane. It is well known that exosomes perform important and specialized functions such as coagulation and intercellular signaling.
  • stem cell refers to a cell having the ability to self-replicate as an undifferentiated cell and capable of differentiating into two or more different kinds of cells.
  • the stem cells of the present invention may be autologous or allogeneic stem cells, and may be derived from any type of animal, including human and non-human mammals, whether stem cells derived from an adult or stem cells derived from an embryo Do not.
  • the stem cells of the present invention include embryonic stem cells, inducible pluripotent stem cells, or adult stem cells, specifically, inducible pluripotent stem cells or adult stem cells.
  • embryonic stem cell refers to a cell obtained by extracting an inner cell mass from a blastocyst embryo immediately before fertilization of the embryo into the uterus of a mother and culturing the same in vitro, Refers to a cell having pluripotent or totipotent self-renewal capability, and in a broad sense embryonic stem cells derived from embryonic stem cells, bodies.
  • the embryonic stem cells of the present invention include all embryonic stem cells derived from human, monkey, pig, horse, cattle, sheep, dog, cat, mouse, rabbit and the like, but are preferably human-derived embryonic stem cells.
  • the term 'inducible pluripotent stem cells' refers to cells induced to multipotential differentiation through an artificial reprogramming process from differentiated cells, which is also referred to as iPSC (induced pluripotent stem cells) .
  • An artificial reprogramming process may be performed by introduction of a non-viral-mediated reprogramming factor using virus-mediated or non-viral vector utilization, retroviruses and lentiviruses, proteins and cell extracts, or by stem cell extracts, And includes a de-differentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells.
  • the inducible pluripotent stem cells of the present invention include all the inducible pluripotent stem cells derived from human, monkey, pig, horse, cattle, sheep, dog, cat, mouse, rabbit and the like, but preferably human pluripotent stem It is a cell.
  • the adult stem cells may be stem cells derived from various tissues of human or animal (such as hematopoietic stem cells, mammary stem cells, intestinal stem cells, endothelial stem cells, neural stem cells, olfactory neural stem cells, testicular stem cells, etc.) Or mesenchymal stromal cells derived from animal tissues, mesenchymal stem cells derived from pluripotent stem cells derived from various tissue origins of human or animal, pluripotent stem cells, and the like, Mesenchymal stem cells derived from mesenchymal stem cells, or mesenchymal stem cells derived from induced pluripotent stem cells.
  • the mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion or placenta, but are not limited thereto.
  • the term " promoting production " means that the production, production and release of a specific substance are increased within the same time period as compared with the control.
  • the present invention means that the amount of exosome produced from stem cells and the content of protein, RNA and the like in the exosome are increased.
  • the composition of the present invention can be used as a culture medium for stem cell culture and can be administered in vivo together with stem cells as a pharmaceutical composition for promoting exosome production in vivo , ≪ / RTI > may be administered in a prophylactic or post-operative manner to enhance the in vivo efficacy of stem cells.
  • Strengthening in vivo efficacy of the stem cells refers to the fact that the efficacy of stem cells having a therapeutic purpose (in vivo efficacy) for certain diseases such as arthritis or neuropathy is mediated by exosomes releasing stem cells,
  • the use of the composition in combination with stem cell administration promotes the production of exosome from stem cells, thereby enhancing the therapeutic effect (in vivo efficacy) of stem cells.
  • composition of the present invention is a pharmaceutical composition
  • pharmaceutically acceptable carriers that can be contained in the pharmaceutical composition of the present invention include those conventionally used in the present invention, such as lactose, dextrose, sucrose, sorbitol, mannitol, Starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, Magnesium stearate, mineral oil, and the like.
  • the pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, kaolin, sorbiol, sorbitol, etc.
  • 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 and parenterally, and can be administered orally or parenterally, for example, by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, intrathecal administration, , Skin administration, transdermal administration, and the like.
  • the appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
  • the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg / kg.
  • the pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.
  • the formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, suppositories, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
  • culture medium composition in the present invention means a composition containing essential components necessary for cell growth, survival and proliferation in vitro, and includes all the culture medium for stem cell culture commonly used in the art (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 Such as F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), ⁇ -MEM ( ⁇ -Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), Isocove's Modified Dulbecco's Medium Or an artificially synthesized medium may be used, but the present invention is not limited thereto.
  • the medium composition of the present invention generally includes a carbon source, a nitrogen source, and a trace element component, and may further include amino acids, antibiotics, and the like.
  • the above-mentioned culture composition of the present invention can be produced by adding the constituents of the present invention (pioglitazone, metformin, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide)) into conventional culture medium for stem cell culture .
  • the pioglitazone may be added to the medium at a concentration of 0.01 to 100 ⁇ M, 0.01 to 10 ⁇ M, 0.01 to 5 ⁇ M, 0.1 to 50 ⁇ M, 0.1 to 25 ⁇ M, and 0.1 to 10 ⁇ M , More specifically 0.1 to 10 ⁇ M, 1 to 10 ⁇ M, 1 to 8 ⁇ M, 1 to 5 ⁇ M, 3 to 5 ⁇ M, and most specifically 4 ⁇ M.
  • the metformin is present in the medium at a concentration of 0.01 to 100 mM, 0.01 to 10 mM, 0.01 to 5 mM, 0.1 to 50 mM, 0.1 to 25 mM, 0.1 to 10 mM May be added, more specifically, at a concentration of 0.1 to 10 mM, 1 to 10 mM, 1 to 8 mM, 1 to 5 mM, 3 to 5 mM, and most specifically 4 mM, It is not.
  • the AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is contained in the medium at a concentration of 1 to 1000 ⁇ M, 1 to 800 ⁇ M, 1 to 700 ⁇ M, 1 to 600 ⁇ M, , 1 to 400 ⁇ M, 1 to 300 ⁇ M, 1 to 200 ⁇ M, 1 to 100 ⁇ M, 10 to 1000 ⁇ M, 10 to 800 ⁇ M, 10 to 700 ⁇ M, 10 to 600 ⁇ M, 10 to 500 ⁇ M and 10 to 400 ⁇ M , 10 to 300 ⁇ M, 10 to 200 ⁇ M, 10 to 100 ⁇ M, 50 to 500 ⁇ M, 50 to 400 ⁇ M, 50 to 300 ⁇ M, 50 to 200 ⁇ M and 50 to 100 ⁇ M, and the most specific At a concentration of 100 1 to 1000 ⁇ M, 1 to 800 ⁇ M, 1 to 700 ⁇ M, 1 to 600 ⁇ M, 1 to 500 ⁇ M, 1 to 400 ⁇ M,
  • the present invention provides an exosome derived from stem cells pretreated with at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) .
  • the present invention provides a method for producing a stem cell-derived exosome comprising the steps of:
  • culturing stem cells in a cell culture medium comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide).
  • the production method may further comprise the steps of: (b) washing the cultured stem cells and further culturing them in a cell culture medium; and (c) culturing the cell culture medium Lt; / RTI >
  • the step (a) is a step of stimulating stem cells by pretreating stem cells with at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide).
  • stem cells produce a greater number of exosomes than stem cells that have not been pretreated with the material, and the content of proteins and RNA in the exosome is also increased.
  • the cell culture medium of step (b) further comprises fetal bovine serum from which exosome has been removed.
  • the reason for using FBS from which the exosome has been removed from the cell culture medium is that the FBS commonly used contains a large amount of exosome derived from bovine serum. Therefore, besides the exosome secreted by stem cells, the FBS- In order to prevent them from being incorporated.
  • the step (b) is a process for inducing the secretion or production of exosome from stem cells by washing the stem cells pretreated with the material according to the present invention, removing the pretreatment material, and culturing in a new cell culture medium.
  • the stem cell pretreatment material prioglitazone, metformin, AICAR
  • step (a) is removed by the washing process of step (b), and the stem cell after the washing, the exosome produced in the stem cell, Do not.
  • the stem cells treated with the stem cell pre-treatment material (pioglitazone, metformin, AICAR) of the present invention and the exosome produced in the stem cells and the culture medium can be cultured in the subsequent Can be usefully used for research or for the treatment of diseases.
  • the stem cell pre-treatment material prioglitazone, metformin, AICAR
  • step (b) the further incubation of step (b) is carried out for 12 to 120 hours, 24 hours to 96 hours, 48 hours to 96 hours, or 60 hours to 84 hours, most particularly 72 hours But is not limited thereto.
  • the step of separating the exosome from the cell culture medium of step (c) comprises culturing the culture medium of the stem cells further cultured in step (b) After centrifugation for 20 minutes, the supernatant was removed and the supernatant was centrifuged at 9,000-12,000 xg for 60-80 minutes. The supernatant was then centrifuged and centrifuged at 90,000-120,000 x g for 80-100 minutes The exosome remaining in the lower layer can be obtained by separating and removing the supernatant.
  • the mesenchymal stem cell culture medium is collected and centrifuged at 300 xg for 10 minutes to remove the remaining cells and cell debris.
  • the supernatant is collected and filtered using a 0.22 ⁇ m filter , And centrifuged at 10,000 xg and 4 ° C for 70 minutes using a high-speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 10 minutes at 4 ° C using an ultracentrifuge to remove the supernatant, thereby separating the remaining exosomes from the lower layer.
  • composition of the present invention for promoting the production of exosomes derived from stem cells needs further study, but not only increases the number of exosomes generated by stem cells, but also increases the content of protein and RNA present in exosome, It is presumed that a large amount of exosome can be produced.
  • the present invention also provides a composition comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide)
  • the present invention provides a stem cell treatment agent containing stem cells.
  • the 'cell therapeutic agent' refers to a cell therapeutic agent that is used to regenerate autologous, allogenic, xenogenic cells in vitro or in vitro to restore cell and tissue function, And the like, which are used for the purpose of treatment, diagnosis and prevention.
  • These cell treatments can be roughly divided into two categories. First, they are “stem cell treatment agents” for tissue regeneration, regeneration of long-term function, or function of immune cells. Second, the inhibition of in vivo immune response or immune response It is an "immune cell therapeutic agent" for controlling the immune response such as hyperactivity. Since the composition of the present invention and its components promote the production of stem cell-derived exosomes and thereby enhance the therapeutic effect of stem cells, the term " cell therapy agent " .
  • the stem cell treatment agent is useful as a therapeutic agent for cardiovascular diseases such as myocardial infarction and heart failure, cancer diseases such as liver cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer and lung cancer, atopic dermatitis, arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, But are not limited to, inflammatory diseases such as sclerosis, or autoimmune diseases.
  • cardiovascular diseases such as myocardial infarction and heart failure
  • cancer diseases such as liver cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer and lung cancer, atopic dermatitis, arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, but are not limited to, inflammatory diseases such as sclerosis, or autoimmune diseases.
  • stem cell therapeutic agent and the composition for promoting exosome production derived from stem cells of the present invention are common to each other, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.
  • the present invention provides a pharmaceutical composition comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (or 5-aminoimidazole-4-carboxamide ribonucleotide) A composition for accelerating the production of exosomes) as an active ingredient.
  • a pharmaceutical composition comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (or 5-aminoimidazole-4-carboxamide ribonucleotide)
  • a composition for accelerating the production of exosomes as an active ingredient.
  • the stem cell treatment agent may be selected from the group consisting of exosomes derived from stem cells pretreated with one or more substances selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) Because they are common, the description common to both is omitted in order to avoid the excessive complexity of the present disclosure.
  • the present invention relates to a composition for promoting the production of stem cell-derived exosomes comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) And to a method for promoting the production of exosomes derived from mesenchymal stem cells.
  • a composition for promoting the production of stem cell-derived exosomes comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide)
  • AICAR 5-Aminoimidazole-4-carboxamide ribonucleotide
  • stem cell-derived composition for promoting exosome formation (b) When the stem cell-derived composition for promoting exosome formation according to the present invention is used for stem cell culture, the production amount of stem cell-derived exosomes and the content of proteins and RNA isolated from exosomes are increased, It is possible to mass-produce high quality exosome more efficiently and can be usefully used for related research and development and commercialization.
  • FIG. 1 is an electron micrograph of an exosomes (microembossed bodies) derived from mesenchymal stem cells treated with Pioglitazone, AICAR and Metformin as constituents of the present invention.
  • FIG. 2 is a diagram showing the size and distribution of exosomes (microembodents) derived from mesenchymal stem cells treated with Pioglitazone, AICAR and Metformin, which are constituents of the present invention.
  • FIG. 3 is a view showing protein markers of exosomes (microembossed bodies) derived from mesenchymal stem cells treated with Pioglitazone, AICAR and Metformin as constituents of the present invention.
  • Fig. 4 is a graph showing the number of exosomes (microvesicles) derived from mesenchymal stem cells treated with Pioglitazone, AICAR, and Metformin, which are constituents of the present invention, Fig.
  • FIG. 5 is a graph showing the relationship between the amount of protein isolated in mesenchymal stem cell-derived exosomes (micro-endoplasmic reticulum) treated with Pioglitazone, AICAR, and Metformin as constituents of the present invention, As compared to the amount of protein in the cell-derived exosome.
  • FIG. 6 is a graph showing the relationship between the amount of RNA isolated in mesophyll stem cell-derived exosomes (microemboli) treated with Pioglitazone, AICAR, and Metformin, which are components of the present invention, As compared to the content of RNA in the cell-derived exosome.
  • Pioglitazone pioglitazone
  • Metformin metformin
  • AICAR 5-Aminoimidazole-4-carboxamide ⁇ ucleotide
  • the mesenchymal stem cells derived from umbilical cord tissue were cultured for 1 week in Non-Essential Amino Acids Solution (100X) (Gibco, Cat no.11140-050).
  • the mesenchymal stem cells derived from the umbilical cord tissue are mesenchymal stem cells derived from the umbilical cord tissue, which were established by separating the embryonic tissue of the embryo at the Asan Medical Center.
  • the mesenchymal stem cells pretreated with the respective pretreatment substances were washed and the fetal bovine serum (Fetal Bovine Serum, FBS) supplemented with 10% FBS was further cultured for 72 hours.
  • FBS Fetal Bovine Serum
  • the mesenchymal stem cell culture medium treated with each pretreatment material was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris.
  • the supernatant was filtered through a 0.22 ⁇ m filter and centrifuged at 10,000 ⁇ g, 4 ° C. for 70 minutes using a high-speed centrifuge.
  • the centrifuged supernatant was again taken and centrifuged at 100,000 ⁇ g for 90 minutes at 4 ° C. using an ultracentrifuge to remove the supernatant.
  • the remaining exosomes were diluted in PBS (phosphate buffered saline) and used in the following experiments.
  • Exosomes were isolated from mesenchymal stem cell culture medium treated with 4 ⁇ M of pioglitazone, 4 mM of metformin or 100 ⁇ M of AICAR as in Example 1, and cultured in a nanoparticle tracking assay (NanoSight NS300, Malvern) (Fig. 2).
  • the morphology of the exosomes was confirmed using an electron microscope (Fig. 1), and expression of CD9 and CD63 was confirmed using antibodies against CD9 and CD63, which are exosome-specific antigens, by Western blot (Fig. 3).
  • Pioglitazone pioglitazone
  • Metformin metformin
  • AICAR 5-Aminoimidazole-4-carboxamide ⁇ ucleotide
  • Exosomes were isolated from the mesenchymal stem cell culture medium treated with stem cell pre-treatment material (pioglitazone 4 ⁇ M, metformin 4 mM, AICAR 100 ⁇ M), and then subjected to nanoparticle tracking assay (NanoSight NS300, Malvern) The number of exosomes derived from mesenchymal stem cells was compared.
  • mesenchymal stem cells pretreated with stem cell pretreatment material (pioglitazone 4 [mu] M, metformin 4 mM, AICAR 100 [mu] And about 22% to about 61% of the exosomes. From the above results, it can be seen that the number of exosomes produced by mesenchymal stem cells increases when mesenchymal stem cells are treated with the stem cell pre-treatment material according to the present invention.
  • mesenchymal stem cells pretreated with stem cell pretreatment materials compared to mesenchymal stem cells not treated with any substance Derived protein from about 3.4 to about 4.6 times the amount of exosome-derived protein. From this, it can be seen that when the mesenchymal stem cells are treated with the stem cell pre-treatment material, the content of exosomal protein as well as the number of exosomes increases.
  • a total exosome RNA and protein isolation kit was prepared from the exosomes isolated from mesenchymal stem cell culture medium treated with stem cell pre-treatment material [pioglitazone 4 ⁇ M, metformin 4 mM, AICAR 100 ⁇ M] Invitrogen) was used to isolate total exosome RNAs, and RNA concentrations were measured using nanodrops.
  • mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 ⁇ M, metformin 4 mM, AICAR 100 ⁇ M) compared to mesenchymal stem cells not treated with any substance , which is about 3.2 times to about 4.3 times larger than that of the wild type, was produced.
  • MSC untreated mesenchymal stem cells
  • Pio-MSC pioglitazone-treated mesenchymal stem cells
  • AICAR-MSC AICAR-treated mesenchymal stem cells
  • Met-MSC metformin-
  • the mesenchymal stem cells were treated with the stem cell pre-treatment material (pioglitazone, metformin, AICAR) according to the present invention
  • the production of exosomal protein as well as the number of exosomes increased
  • the content of exosomal RNA is also increased. Therefore, the composition for promoting the production of stem cell-derived exosomes according to the present invention needs to be further studied, but not only increases the number of exosomes produced by stem cells but also increases the content of proteins and RNA present in exosomes It is presumed that a large amount of functional exosome can be produced.

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Abstract

La présente invention concerne une composition comprenant au moins un matériau choisi dans le groupe constitué par la pioglitazone, la metformine et le 5-aminoimidazole-4-carboxamide ribonucléotide (AICAR) pour favoriser la production d'exosomes dérivés de cellules souches et un procédé de production d'exosomes dérivés de cellules souches mésenchymateuses l'utilisant. Quand elle est utilisée pour cultiver des cellules, la composition pour favoriser la production d'exosomes dérivés de cellules souches selon la présente invention augmente la génération d'exosomes dérivés de cellules souches et les teneurs en protéines et ARN dans les exosomes, ce qui permet ainsi de produire en masse des exosomes de qualité plus efficacement que par les procédés classiques connus. Par conséquent, la composition peut être efficacement appliquée à la recherche et au développement pertinents et à la commercialisation.
PCT/KR2018/014873 2017-11-29 2018-11-28 Composition pour favoriser la production d'un exosome dérivé d'une cellule souche Ceased WO2019107939A1 (fr)

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CN111154648A (zh) * 2019-12-27 2020-05-15 新乡医学院三全学院 一种干细胞源性外泌体培养装置
CN111394303A (zh) * 2020-03-23 2020-07-10 天津百恩生物科技有限公司 含有干细胞激活剂的培养基及间充质干细胞的培养方法
CN113862220A (zh) * 2021-11-05 2021-12-31 杭州隽和生物医药有限公司 间充质干细胞外泌体的制备及其应用
WO2023017539A1 (fr) * 2021-08-11 2023-02-16 Pandorum Technologies Private Limited Procédés de culture de cellules souches mésenchymateuses, compositions et mises en oeuvre de celles-ci
JP2023528083A (ja) * 2020-07-29 2023-07-03 プレクソジェン インコーポレイテッド 幹細胞由来エクソソームを含む組成物及びその製造方法
RU2799432C1 (ru) * 2020-05-25 2023-07-05 Ск-Эксоджин Ко., Лтд. Способ получения экзосом, происходящих из мезенхимальных стволовых клеток, и культурального раствора, продуцированного из них
CN117143812A (zh) * 2023-10-31 2023-12-01 中国人民解放军军事科学院军事医学研究院 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用

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KR20220085036A (ko) * 2020-12-14 2022-06-21 서울대학교산학협력단 소포체 스트레스 유발 물질로 처리된 세포 유래 엑소좀 및 이의 용도

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CN111154648A (zh) * 2019-12-27 2020-05-15 新乡医学院三全学院 一种干细胞源性外泌体培养装置
CN111154648B (zh) * 2019-12-27 2022-10-25 新乡医学院三全学院 一种干细胞源性外泌体培养装置
CN111394303A (zh) * 2020-03-23 2020-07-10 天津百恩生物科技有限公司 含有干细胞激活剂的培养基及间充质干细胞的培养方法
CN111394303B (zh) * 2020-03-23 2022-12-09 天津百恩生物科技有限公司 含有干细胞激活剂的培养基及间充质干细胞的培养方法
RU2799432C1 (ru) * 2020-05-25 2023-07-05 Ск-Эксоджин Ко., Лтд. Способ получения экзосом, происходящих из мезенхимальных стволовых клеток, и культурального раствора, продуцированного из них
JP2023528083A (ja) * 2020-07-29 2023-07-03 プレクソジェン インコーポレイテッド 幹細胞由来エクソソームを含む組成物及びその製造方法
JP7568239B2 (ja) 2020-07-29 2024-10-16 プレクソジェン インコーポレイテッド 幹細胞由来エクソソームを含む組成物及びその製造方法
WO2023017539A1 (fr) * 2021-08-11 2023-02-16 Pandorum Technologies Private Limited Procédés de culture de cellules souches mésenchymateuses, compositions et mises en oeuvre de celles-ci
CN113862220A (zh) * 2021-11-05 2021-12-31 杭州隽和生物医药有限公司 间充质干细胞外泌体的制备及其应用
CN113862220B (zh) * 2021-11-05 2024-02-13 杭州隽和生物医药有限公司 间充质干细胞外泌体的制备及其应用
CN117143812A (zh) * 2023-10-31 2023-12-01 中国人民解放军军事科学院军事医学研究院 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用
CN117143812B (zh) * 2023-10-31 2024-01-26 中国人民解放军军事科学院军事医学研究院 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用

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