KR101980453B1 - Composition For Promoting Production of Stem Cell-derived Exosomes - Google Patents

Abstract

The present invention relates to a composition for promoting the production of stem cell-derived exosomes including one or more substances selected from the group consisting of pioglitazone, metformin, and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR); and a method for producing mesenchymal stem cell-derived exosomes using the composition. In the case of using the composition for promoting the production of stem cell-derived exosomes for stem cell culture, since the production amount of stem cell-derived exosomes and the content of protein and RNA in exosomes are increased, it is possible to mass-produce high-quality exosomes more efficiently than conventionally known methods. Therefore, the present invention can be useful for related R&D and commercialization.

Description

Composition for Promoting Production of Stem Cell-derived Exosomes

The present invention is a composition for promoting stem cell-derived exosomes production composition comprising one or more substances selected from the group consisting of pioglitazone (pioglitazone), metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) and stem cells derived from the same It relates to a method for producing exosomes.

Extracellular Vesicles are vesicles composed of spherical lipid-bilayers of 30-1000 nm in size, including Microvesicles, Exosomes, and the like. The lipid bilayer of exosomes has the same phospholipid bilayer structure as cells of origin (donor cells), and is a construct of substances secreted by the cells extracellularly, and performs a functional role such as cell-cell communication and cellular immune mediation. It is known. Exosomes contain cell-specific components that reflect the biological function peculiar to the cells of origin (donor cells), and include various soluble proteins, exogenous proteins, and transmembrane protein components in addition to phospholipids, mRNAs and miRNAs. The marker proteins of exosomes are well known, such as CD63 and CD81, mainly including receptors on cell surfaces such as EGFR, molecules involved in signal transduction, proteins involved in cell adhesion, and heat shock proteins. And Alix, which are involved in endoplasmic reticulum formation. These exosomes are released from all animal cells such as mast cells, lymphocytes, astrocytes, platelets, nerve cells, endothelial cells, epithelial cells, and found in various body fluids such as blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid. do. Exosomes can also pass through the blood-brain barrier (BBB), and have high selective permeability to allow cell membrane penetration of epidermal and endothelial cells, and thus, a drug delivery system (DDS), a nanocarrier for certain drugs. It is also used for development.

Exosomes and microvesicles secreted from mesenchymal stem cells are known to be involved in cell-to-cell communication and show regenerative medical efficacy of stem cells. It is known to have a trophic effect on paracrine factors secreted by cells without prolonged survival after transplanting stem cells into the body, and these factors include growth factors and chemokines. Low molecules such as cytokines are secreted by extracellular vesicles such as exosomes, which are derived from stem cells. Therefore, exosomes are used to characterize stem cells and evaluate their therapeutic efficacy.

Recently, studies on the therapeutic effects of various diseases using exosomes secreted by mesenchymal stem cells, rather than mesenchymal stem cells themselves, are being actively conducted. It is expected to be a new alternative to overcome this problem. Commercial use of these exosomes requires large amounts of high quality exosomes. However, the amount of exosomes currently available from stem cells is very small, and the development of a substance capable of increasing the production of stem cell-derived exosomes is still insufficient. Accordingly, there is a need for the development of new methods and materials for promoting the production of stem cell-derived exosomes.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Republic of Korea Patent Publication No. 10-1643825

The present inventors made diligent research efforts to develop methods and materials for promoting the production of stem cell-derived exosomes. As a result, when pre-treatment of pioglitazone, metformin, and AICAR to stem cells, not only the number of stem cell-derived exosomes, but also a significant increase in the content of proteins and RNA in the exosomes was completed.

Accordingly, an object of the present invention is to provide a composition for promoting stem cell-derived exosome generation comprising at least one substance selected from the group consisting of pioglitazone, piformlitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). It is.

Another object of the present invention is to provide 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).

Still another object of the present invention is to provide a stem cell therapeutic agent comprising the composition for promoting stem cell-derived exosome generation.

Still another object of the present invention is to provide a stem cell therapeutic agent comprising an exosome derived from the stem cells.

Another object of the present invention is the step of culturing the stem cells in a cell culture medium containing one or more substances selected from the group consisting of pioglitazone (pioglitazone), metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) It provides a method for producing a stem cell-derived exosomes comprising.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention promotes the generation 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) It provides a composition for.

Pioglitazone, which is one of the components of the composition of the present invention, is a diabetic drug having a hypoglycemic effect of thiazolidinedione (TZD) family. It has a hypoglycemic effect, but the effect on cardiovascular disease has not been reported statistically significant.

Metformin, another component of the composition of the present invention, is a biguanides oral diabetes treatment. Metformin also improves blood sugar, but there is still limited evidence to prevent cardiovascular disease. One of the mechanisms of action is the activation of AMP-activated protein kinase (AMPK) in the liver, which prevents glucose biosynthesis, promotes glucose uptake into cells, and inhibits metabolic syndrome. The mechanism of action of metformin has not been accurately determined, but 1) it inhibits cell respiration in mitochondria, 2) activates AMPK, and 3) prevents the increase of glucagon-induced concentration of cAMP (Cyclic adenosine monophosphate). It is known to inhibit the activity of protein kinase A and to affect gut flora in the gut.

Another component of the composition of the present invention AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is an intermediate in the production of inosine monophosphate (inosine monophosphate). AICAR is known to stimulate AMP-dependent protein kinase (AMPK) as an analog of AMP and has been used clinically for the treatment and protection of cardiac ischemic injury.

The composition according to the present invention may further include a known exosome production promoting material in addition to the above-mentioned components.

As used herein, the term “Exosome” is a cell-derived endoplasmic reticulum, which is present in the body fluids of almost all eukaryotic organisms. The diameter of the exosomes is on the order of 30-100 nm, which is larger than the LDL protein but much smaller than the red blood cells. Exosomes are released from cells when the multivesicular bodies are fused with the cell membrane, or directly from the cell membrane. It is well known that exosomes perform important and specialized functions such as coagulation and intercellular signaling.

As used herein, the term “stem cell” refers to a cell that has the ability to differentiate into two or more different types of cells while having self-replicating ability as an undifferentiated cell. Stem cells of the present invention may be autologous or allogeneic stem cells, may be from any type of animal, including humans and non-human mammals, and is not limited to whether the stem cells are derived from adults or embryos Do not. Stem cells of the present invention include embryonic stem cells, induced pluripotent stem cells or adult stem cells, specifically, induced pluripotent stem cells or adult stem cells.

As used herein, the term 'embryonic stem cell' is a cultured in vitro by extracting the inner cell mass from the blastocyst embryo just before the fertilized egg implants in the mother's womb, and will differentiate into cells of all tissues of the individual. It refers to a cell having a pluripotent or pluripotent or self-renewal capable of being pluripotent, and in a broad sense, an embryoid derived from embryonic stem cells. bodies). Embryonic stem cells of the present invention include embryonic stem cells of all derived from humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, etc., preferably human embryonic stem cells.

As used herein, the term 'induced pluripotent stem cells' refers to cells induced to have pluripotent differentiation ability through artificial dedifferentiation from differentiated cells and is also referred to as induced pluripotent stem cells (iPSC). . Artificial dedifferentiation process is performed by the use of virus-mediated or non-viral vectors with retroviruses and lentiviruses, introduction of non-virus-mediated dedifferentiation factors using protein and cell extracts, or by stem cell extracts, compounds, etc. Includes reverse differentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have similar cell morphology, similar gene and protein expression patterns, and have pluripotency in vitro and in vivo. When inserted into the blastocyst of the mouse, chimera mice are formed, and germline transmission of the gene is possible. Induced pluripotent stem cells of the present invention include all induced pluripotent stem cells derived from humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, etc., but preferably induced pluripotent stems derived from humans It is a cell.

The adult stem cells are stem cells of various tissues of human or animal origin (for example, hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells, testicular stem cells, etc.) and human Or mesenchymal stromal cells derived from animal tissues, mesenchymal stem cells derived from induced pluripotent stem cells of various tissue origins of humans or animals, multipotent stem cells, and the like, specifically, derived from human or animal tissues. Mesenchymal stem cells derived from mesenchymal stem cells or induced pluripotent stem cells, but are not limited thereto. The mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane or placenta, and the like.

In the present specification, "promoting production" refers to an increase in production, production, release, etc. of a specific substance compared to the control within the same time. Specifically, the present invention means that the amount of exosomes produced from stem cells, and the amount of protein, RNA, etc. in the exosomes is increased.

According to one embodiment of the present invention, the composition of the present invention can be used in culture of stem cells as a medium composition, and administered in parallel with the stem cells as a pharmaceutical composition for promoting in vivo exosome production Prior or subsequent administration can be used to enhance the in vivo efficacy of stem cells. Enhancing the in vivo efficacy of the stem cells, on the premise that the efficacy of stem cells having a therapeutic purpose (in vivo efficacy) for specific diseases such as arthritis or neuropathy is mediated by the exosomes released by the stem cells, When the composition is administered with stem cell administration, it means that the production of exosomes from the stem cells is promoted, thereby enhancing the therapeutic effect (in vivo efficacy) of stem cells.

When the composition of the present invention is a pharmaceutical composition, pharmaceutically acceptable carriers that may be included in the pharmaceutical composition of the present invention are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, Starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, Magnesium stearate, mineral oil, and the like, but is not limited thereto. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be administered orally and parenterally, such as intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, pulmonary, rectal, intradural, ocular. , Dermal administration and transdermal administration.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a specific embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-1000 mg / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation 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 extracts, powders, suppositories, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

In the present invention, "medium composition" refers to a composition containing essential components necessary for the growth, survival and proliferation of cells in vitro, and includes all of the stem cell culture medium commonly used in the art. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / Commercial such as F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMOD (Isocove's Modified Dulbecco's Medium), KnockOut DMEM Media prepared by or artificially synthesized may be used, but 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 an amino acid, an antibiotic, and the like.

The medium composition of the present invention can be prepared by adding the components of the present invention (pioglitazone, metformin, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide)) in a conventional stem cell culture medium. .

According to a specific embodiment of the present invention, the pioglitazone may be added at a concentration of 0.01 to 100 μM, 0.01 to 10 μM, 0.01 to 5 μM, 0.1 to 50 μM, 0.1 to 25 μM, 0.1 to 10 μM in the medium. For example, the concentration may be 0.1-10 μM, 1-10 μM, 1-8 μM, 1-5 μM, 3-5 μM, and most specifically 4 μM, but is not limited thereto.

According to another specific embodiment of the present invention, the metformin is metformin at a concentration of 0.01-100 mM, 0.01-10 mM, 0.01-5 mM, 0.1-50 mM, 0.1-25 mM, 0.1-10 mM. It may be added, more specifically, it may be added in a concentration of 0.1 ~ 10 mM, 1 ~ 10 mM, 1 ~ 8 mM, 1 ~ 5 mM, 3 ~ 5 mM, most specifically 4 mM, but is not limited thereto. It doesn't happen.

According to another specific embodiment of the present invention, the AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is 1 ~ 1000 μM, 1 ~ 800 μM, 1 ~ 700 μM, 1 ~ 600 μM, 1 ~ 500 μM in the medium , 1-400 μM, 1-300 μM, 1-200 μM, 1-100 μM, 10-1000 μM, 10-800 μM, 10-700 μM, 10-600 μM, 10-500 μM, 10-400 μM , 10-300 μM, 10-200 μM, 10-100 μM, 50-500 μM, 50-400 μM, 50-300 μM, 50-200 μM, 50-100 μM, can be added at the most specific 100-1000 μM, 1-800 μM, 1-700 μM, 1-600 μM, 1-500 μM, 1-400 μM, 1-300 μM, 1-200 μM, 1-100 μM It may be added, but is not limited thereto.

According to one aspect of the invention, the invention is exosomes 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) To provide.

According to another aspect of the present invention, the present invention provides a method for producing stem cell-derived exosomes comprising the following steps:

(a) culturing the 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).

According to one embodiment of the invention, the production method may further comprise the following steps: (b) washing the cultured stem cells and then further culturing in cell culture medium and (c) the cell culture medium Separating the exosomes from the.

In the step (a), the stem cells are pretreated with one or more substances selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) to stimulate the stem cells. By this process, stem cells produce a greater number of exosomes than stem cells not pretreated with the substance, and the content of proteins and RNA in exosomes is also increased.

In a specific embodiment of the present invention, the cell culture medium of step (b) further comprises fetal bovine serum from which exosomes have been removed. The reason for using FBS from which exosomes are removed in the cell culture medium is that exosomes derived from FBS, in addition to exosomes secreted by stem cells, are generally used because FBS contains a large amount of exosomes derived from bovine serum. This is to prevent mixing.

Step (b) is a process of inducing secretion or production of exosomes from stem cells by washing the stem cells pretreated with the material according to the present invention to remove the pretreatment material and culturing in a new cell culture medium. The stem cell pretreatment material (pioglitazone, metformin, AICAR) of step (a) is removed by the washing process of step (b) and does not remain in the exosomes or culture medium produced from the stem cells, stem cells after washing. Do not. Therefore, in step (a), exosomes and culture media produced from stem cells and stem cells treated with the stem cell pretreatment material of the present invention (pioglitazone, metformin, AICAR) are not subsequently affected by the residue of the pretreatment material. It can be usefully used for research or treatment of diseases.

According to a specific embodiment of the present invention, the further culture of step (b) is 12 hours to 120 hours, 24 hours to 96 hours, 48 hours to 96 hours, or 60 hours to 84 hours, most specifically 72 hours It may be cultured, but is not limited thereto.

In addition, in another embodiment of the present invention, the step of separating the exosomes from the cell culture medium of the step (c) is the culture medium of the stem cells further cultured in the step (b) from 5 to 5 to 200-400 xg After centrifugation for 20 minutes to remove the remaining cells and cell residues, the supernatant was taken and centrifuged at 9,000-12,000 xg for 60-80 minutes, then the supernatant was again centrifuged at 90,000-120,000 xg for 80-100 minutes. The exosomes remaining in the lower layer can be obtained by separating and removing the supernatant. According to a specific embodiment of the present invention, the mesenchymal stem cell culture medium is collected and centrifuged at 300 xg for 10 minutes to remove remaining cells and cell residues, and the supernatant is taken and filtered using a 0.22 μm filter. , Centrifuge for 70 minutes at 10,000 xg, 4 ℃ using a high speed centrifuge. The supernatant was centrifuged again and centrifuged at 100,000 x g, 4 ° C. for 90 minutes using an ultracentrifuge to separate the exosomes remaining in the lower layer.

As demonstrated in the following examples, the pretreatment of the stem cells with the pretreatment material according to the present invention increases the amount of protein and RNA in the exosomes as well as the amount of exosomes produced in comparison to the stem cells not pretreated. . Therefore, exosomes derived from stem cells pretreated with the composition for promoting exosome production of the present invention, unlike the exosomes derived from conventional stem cells or stem cells not pretreated with the composition for promoting exosome production of the present invention. Substantially different in that the protein content and RNA content are significantly improved. Stem cell-derived exosome production promoting composition of the present invention will require additional research, but not only increase the number of exosomes produced by stem cells, but also increases the content of proteins and RNA present in the exosomes excellent functionality It is estimated that they can produce a large amount of exosomes.

In addition, according to an aspect of the present invention, the present invention is a pioglitazone (pioglitazone), metformin (metformin) and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) at least one selected from the group consisting of or a composition comprising the same and It provides a stem cell therapy comprising stem cells.

In the present invention, the 'cell therapeutic agent' refers to the biological characteristics of cells by proliferating, selecting, or otherwise releasing autologous, allogenic, and xenogenic cells in vitro to restore the function of cells and tissues. Drugs used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing. These cell therapies can be broadly classified into two categories. The first is a "stem cell therapy" for tissue regeneration, long-term function recovery or immune cell function control. It is an “immune cell therapy” for the regulation of immune response such as hyperactivity. Since the composition of the present invention and its components promote the production of stem cell-derived exosomes, thereby having an effect of enhancing the therapeutic efficacy of stem cells, within the present specification, "cell therapeutic agent" is "stem cell therapeutic agent" Means.

The stem cell therapeutic agents include cardiovascular diseases such as myocardial infarction and heart failure, liver cancer, gastric cancer, colon cancer, prostate cancer, bladder cancer, lung cancer and other cancer diseases, atopic dermatitis, arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, colitis and multiple It may be used for the treatment of inflammatory diseases such as sclerosis, or diseases to be treated such as autoimmune diseases, but is not limited thereto.

Since the stem cell therapeutic agent has the composition and components for promoting stem cell-derived exosomes of the present invention described above in common, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

In addition, according to another aspect of the present invention, the present invention is one or more substances selected from the group consisting of pioglitazone (pioglitazone), metformin (metformin) and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) (or stem cell derived) The present invention provides a stem cell therapeutic agent comprising an exosome derived from stem cells pretreated with an exosome generation promoting composition) as an active ingredient.

The stem cell therapeutic agent comprises exosomes and components 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 content in common between the two is omitted in order to avoid excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(A) The present invention is a composition for promoting stem cell-derived exosomes production comprising at least one substance selected from the group consisting of pioglitazone (pioglitazone), metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) and using the same It relates to a method for promoting the production of mesenchymal stem cell-derived exosomes.

(b) When the composition for promoting stem cell-derived exosomes production according to the present invention is used for stem cell culture, the production of stem cell-derived exosomes and the content of protein and RNA isolated from exosomes is increased compared to the conventionally known methods More efficient mass production of high quality exosomes, which can be useful for the relevant research and development and commercialization.

1 is a diagram showing an electron microscope image of mesenchymal stem cell-derived exosomes (microvesicles) treated with pioglitazone, AICAR, and metformin, which are components of the present invention.
2 is a diagram showing the size and distribution of mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, and metformin, which are components of the present invention.
3 is a diagram confirming protein markers of mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, and metformin (Metformin), which are components of the present invention.
Figure 4 shows the number of mesenchymal stem cell-derived exosomes (microvesicles) treated with pioglitazone, AICAR, and metformin, which are the constituents of the present invention, of the mesenchymal stem cell-derived exosomes without treatment of the material. It is a figure compared with the number.
Figure 5 is a mesenchymal stem untreated with the amount of protein isolated in the mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, metformin (Metformin) as a component of the present invention Figure shows the amount of protein in cell-derived exosomes.
Figure 6 is a mesenchymal stem untreated with the substance content of RNA isolated in mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, metformin (Metformin) as a component of the present invention Figure shows the content of RNA in the cell-derived exosomes.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  One. Pioglitazone ( pioglitazone ), Metformin ( metformin ), AICAR (5- Aminoimidazole -4- carboxamide  stem cell derivation following ribonucleotide treatment Exosomes  detach

Culture medium containing 4 μM pioglitazone, 4 mM metformin, or 100 μM AICAR [high glucose DMEM (Gibco, Cat no.11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Umbilical cord tissue-derived mesenchymal stem cells were cultured for 1 week in Non-Essential Amino Acids Solution (100X) (Gibco, Cat no. 11140-050). Umbilical cord stem-derived mesenchymal stem cells are umbilical cord stem-derived mesenchymal stem cells established by separating the fetal umbilical cord tissues from the Asan Hospital Stem Cell Center. After the incubation was completed, the mesenchymal stem cells pretreated with the respective pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM) were washed with fetal bovine serum (Fetal Bovine). Serum, FBS) was incubated for an additional 72 hours in a culture medium added 10%. The reason for using FBS with exosomes removed is to prevent the incorporation of FBS-derived exosomes in addition to the exosomes secreted by cells because the commonly used FBS contains a large amount of exosomes derived from bovine serum. . After 72 hours of incubation, 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 collected, filtered using a 0.22 μm filter, and then centrifuged at 10,000 × g, 4 ° C. for 70 minutes using a high speed centrifuge. The supernatant was centrifuged again and centrifuged at 100,000 x g, 4 ° C for 90 minutes using an ultracentrifuge to remove the supernatant. Exosome remaining in the lower layer was diluted in PBS (phosphate buffered salin) was used in the following experiment.

Example  2. Pioglitazone ( pioglitazone ), Metformin ( metformin ), AICAR (5- Aminoimidazole -4- carboxamide  stem cell derivation following ribonucleotide treatment Exosomes  Confirm

Exosomes were isolated from mesenchymal stem cell cultures treated with pioglitazone 4 μM, metformin 4 mM or AICAR 100 μM as in Example 1, respectively, through nanoparticle tracking assay (NanoSight NS300, Malvern). The size of the exosomes was confirmed (Fig. 2). The morphology of the exosomes was confirmed using an electron microscope (FIG. 1), and the expression of CD9 and CD63 was confirmed using an antibody against CD9 and CD63, which are exosome-specific antigens, by Western blot (FIG. 3).

Example  3. Pioglitazone ( pioglitazone ), Metformin ( metformin ), AICAR (5- Aminoimidazole -4- carboxamide  ribonucleotide treatment Exosomes  production Increase efficiency

3-1. Pioglitazone ( pioglitazone ), Metformin ( metformin ), AICAR (5- Aminoimidazole -4- carboxamide  ribonucleotide treatment Exosomes  Number increase comparison

Exosomes were isolated from the mesenchymal stem cell cultures treated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM), and the same were obtained through nanoparticle tracking assay (NanoSight NS300, Malvern). The number of mesenchymal stem cell-derived exosomes was compared.

The results are shown in FIG.

As shown in Figure 4, compared with mesenchymal stem cells not treated with any substance, mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM) In as little as 22% to as much as 61% produced a large number of exosomes. From the above results, it can be seen that when the stem cell pretreatment material according to the present invention is treated to mesenchymal stem cells, the number of exosomes produced by mesenchymal stem cells increases.

3-2. Pioglitazone ( pioglitazone ), Metformin ( metformin ), AICAR (5- Aminoimidazole -4- carboxamide  ribonucleotide treatment Exosomes  Derived Protein Content Comparison

Total exosome RNA and protein isolation kit from exosomes isolated from mesenchymal stem cell culture treated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM) , Invitrogen) was used to isolate proteins and quantify proteins by measuring absorbance at 595 nm through bradford analysis.

The results are shown in FIG.

5, mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM) compared to mesenchymal stem cells treated with no substance. It was confirmed that in about 3.4 times produced about 4.6 times as much amount of exo-derived protein. From this, it can be seen that when the stem cell pretreatment material is treated on mesenchymal stem cells, the content of exosome-derived protein (exosomal protein) is increased as well as the number of exosomes.

3-3. Pioglitazone ( pioglitazone ), Metformin ( metformin ), AICAR (5- Aminoimidazole -4- carboxamide  ribonucleotide) According to treatment Exosomes  Derived RNA Content Comparison

Kit for RNA separation from exosomes isolated from mesenchymal stem cell culture treated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM) Invitrogen) was used to isolate total exosome RNA and to compare and measure the concentration of RNA using nanodrop.

The results are shown in FIG.

6, mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM, AICAR 100 μM) compared to mesenchymal stem cells treated with no substance. In about 3.2 times to about 4.3 times as much amount of the exo-derived RNA was produced.

3-4. Sintered

The results of Examples 3-1 to 3-3 are shown in Table 1 below.

(MSC: untreated mesenchymal stem cells, Pio-MSC: pioglitazone-treated mesenchymal stem cells, AICAR-MSC: AICAR-treated mesenchymal stem cells, Met-MSC: metformin-treated mesenchymal stem cells)

As shown in Table 1 above, when the stem cell pretreatment material (pioglitazone, metformin, AICAR) according to the present invention is treated to mesenchymal stem cells, the number of exosomes as well as the production of exosomal protein are increased. It can be seen that the content of exosome-derived RNA (exosomal RNA) also increases. Therefore, the composition for promoting stem cell-derived exosomes production of the present invention may require additional research, but not simply increase the number of exosomes produced by stem cells, but also increase the content of proteins and RNA present in the exosomes. It is estimated that a large amount of exosomes can be produced.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (12)

Composition for promoting stem cell-derived exosomes production comprising pioglitazone (pioglitazone).
The composition of claim 1, wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPS).
The method of claim 2, wherein the adult stem cells are adult stem cells of human or animal tissue origin, mesenchymal stromal cells derived from human or animal tissue, or derived from induced pluripotent stem cells of human or animal tissue origin The mesenchymal stem cells, characterized in that the composition.
The composition of claim 3, wherein the mesenchymal stem cells are derived from a tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, and placenta.
The method of claim 3, wherein the adult stem cells of human or animal tissue origin are selected from the group consisting of hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells and testicular stem cells The composition characterized in that.
The composition of claim 1, wherein the composition increases the number of exosomes, exosome-derived protein, and exosome-derived RNA.
delete delete delete Method for producing stem cell-derived exosomes comprising the following steps:
(a) culturing the stem cells in a cell culture medium containing pioglitazone.
The method of claim 10, further comprising: (b) further culturing the cultured stem cells in cell culture medium; And (c) separating the exosomes from the cell culture medium.
The method of claim 11, wherein the cell culture medium is characterized in that the fetal bovine serum (Fetal bovine serum) is removed from the exosomes.

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