KR102199640B1 - Composition For Promoting Production of Stem Cell-derived Exosomes and Increasing Stemness comprising Lanifibranor - Google Patents

Abstract

The present invention relates to a composition for promoting production of stem cell-derived exosomes and enhancing stemness, comprising lanifibranor. When the composition of the present invention is used for stem cell culture, the stemness of stem cells and the production of stem cell-derived exosomes are increased, and thus high-quality stem cells and stem cell-derived exosomes can be more efficiently produced. Therefore, the composition can be usefully used for related R&D and commercialization.

Description

Composition For Promoting Production of Stem Cell-derived Exosomes and Increasing Stemness comprising Lanifibranor}

The present invention relates to a composition for promoting the generation of stem cell-derived exosomes including Lanifibranor and enhancing the function of stem cells, and more particularly, to a composition for stem cell cultivation of stem cells ( stemness) to increase the stem cell proliferation rate, stem cell ability, and the production of stem cell-derived exosomes by promoting the production of stem cell-derived exosomes including ranifibranor and enhancing stem cell ability.

Extracellular Vesicles are vesicles composed of a 30-1000 nm-sized spherical lipid-bilayer including microvesicles and exosomes.

The lipid bilayer of exosomes has the same phospholipid double membrane structure as the cells of origin (donor cells), and is a composition of substances secreted by cells extracellularly, and plays a functional role such as cell-cell communication and cellular immunity mediation. It is known.

Exosomes contain cell-specific components that reflect the unique biological functions of the cell of origin (donor cell), and include various soluble proteins, exogenous proteins, and transmembrane protein components in addition to phospholipids, mRNA, and miRNA.

These exosomes are excreted from all animal cells, such as mast cells, lymphocytes, astrocytes, platelets, nerve cells, endothelial cells, and epithelial cells, and are 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 penetration of epidermal cells and endothelial cells into the cell membrane, so the drug delivery system (DDS) is a nanocarrier for specific drugs. It is also being used for development.

It is known that exosomes and microvesicles secreted from mesenchymal stem cells are involved in cell-to-cell communication and show regenerative medical treatment efficacy of stem cells.

It is known to have a trophic effect on paracrine factors secreted from cells without long-term survival after stem cells are transplanted into the body, and these factors include growth factors and chemokines. ), small molecules such as cytokines are secreted by extracellular vesicles such as exosomes, and these exosomes are derived from stem cells. Therefore, exosomes are used to characterize stem cells and evaluate their therapeutic efficacy.

Recently, research on the therapeutic effect of various diseases is being actively conducted using exosomes secreted by mesenchymal stem cells rather than using the mesenchymal stem cells themselves, and through this, the limitations of conventional stem cell therapies in academia and industry. It is expected that it will be a new alternative to overcome the problem.

In order to use these exosomes commercially, a large amount of and high quality exosomes are required. However, currently, the amount of exosomes that can be obtained from stem cells is only a very small amount, and development of a substance capable of increasing the production of stem cell-derived exosomes is still insufficient.

On the other hand, it is known that the mesenchymal stem cells have a prolonged cultivation period and, at the same time, decrease proliferative capacity and differentiation capacity into various lineages through multiple passages. In addition, it is known that the ability to form colonies is significantly reduced in mesenchymal stem cells in the later passages compared to mesenchymal stem cells in the early passage. Aging of mesenchymal stem cells is associated with weakened proliferative capacity over long culture periods rather than age of bone marrow donors, and appears to be associated with a decrease in telomerase activity.

As such, the amount of mesenchymal stem cells that can be obtained from humans is currently limited, and problems such as a decrease in differentiation capacity due to subculture are inevitable to induce differentiation of the obtained cells into a specific lineage. These shortcomings are unrealistic in clinical application requiring accurate induction of differentiation into target cells and securing a large amount of appropriate stem cells for the same. Accordingly, there is a need for a method capable of maintaining the proliferation rate for a long period of time and maintaining the ability to differentiate into various lines.

Accordingly, there has been a need for a new method and material development capable of not only promoting the production of stem cell-derived exosomes, but also maintaining the proliferation rate and differentiation capacity of stem cells for a long period of time.

Domestic registered patent No. 10-1439074

Accordingly, the present inventors have developed a method and material capable of not only promoting the production of stem cell-derived exosomes, but also maintaining the proliferation rate and differentiation ability of stem cells for a long period of time, and pretreatment of Lanifibranor to stem cells. In this case, it was confirmed that the effect of increasing the number of stem cell-derived exosomes and the content of proteins and RNAs in the exosomes as well as increasing the stemness and proliferation of the stem cells was remarkably excellent.

Accordingly, an object of the present invention is to provide a composition for promoting the generation of stem cells-derived exosomes comprising ranifibranore.

Another object of the present invention is to provide a stem cell therapeutic agent for promoting the generation of stem cells-derived exosomes comprising ranifibranore.

Another object of the present invention is to provide a composition for enhancing stem cell ability (stemness) of stem cells comprising rani fibra nor.

Another object of the present invention is to provide a stem cell therapeutic agent for enhancing stem cell ability of stem cells including ranifibranore.

Another object of the present invention is to provide a stem cell-derived exosome that has been pretreated with ranifibranor.

Another object of the present invention is to provide a stem cell therapeutic agent comprising, as an active ingredient, stem cell-derived exosomes pretreated with ranifibranor.

Another object of the present invention is to provide a method for producing stem cell-derived exosomes comprising a pretreatment step of culturing stem cells in a cell culture medium containing ranifibranor.

Another object of the present invention is to provide a method for enhancing stem cell capacity of stem cells, including a pretreatment step of culturing stem cells in a cell culture medium containing ranifibranor.

The present invention relates to a composition for promoting the generation of stem cell-derived exosomes, including Lanifibranor, and for enhancing the stemness of stem cells, including the Ranifibranor according to the present invention. Stem cells cultured through the composition promote exosome production and enhance the function of stem cells.

The inventors of the present invention have thus determined that the composition for promoting the generation of stem cells-derived exosomes and enhancing stem cell capacity, including ranifibranor, not only increases the stem cell capacity and proliferation capacity (Proliferation) of stem cells when pre-treatment in stem cell culture, It was confirmed that the number of stem cell-derived exosomes and the content of protein and RNA in the exosomes were significantly increased.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a composition for promoting the generation of exosomes derived from stem cells comprising ranifibranore.

Ranifibranor of the present invention is a peroxisome proliferator-activated receptors (PPAR) agonist, which activates three PPAR isoforms known as PPARα, PPARδ and PPARγ, respectively, to induce antifibrotic, anti-inflammatory and beneficial metabolic changes in the body. It is a known small molecule. PPAR is a ligand-activated transcription factor belonging to the family of nuclear hormone receptors that regulate the expression of genes. PPAR plays an essential role in the regulation of cell differentiation, development and tumor formation. It is known to reduce abnormal growth of connective tissue by activating PPAR, which regulates fibrosis. In addition, ranifibranor has been used clinically as a therapeutic agent for systemic sclerosis and idiopathic pulmonary fibrosis.

In one embodiment of the present invention, the composition for promoting the generation of exosomes derived from stem cells including ranifibranor may further include a known exosome generation promoting substance other than ranifibranor.

As used herein, the term "exosome" refers to a cell-derived endoplasmic reticulum, which is present in almost all eukaryotic body fluids, and refers to an endoplasmic reticulum having a diameter of about 30-100 nm, which is larger than LDL protein, but much smaller than red blood cells. do. It is well known that exosomes are released from cells when multivesicular bodies are fused with cell membranes or can be released directly from cell membranes, and perform important and specialized functions such as coagulation and intercellular signaling.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more different types of cells while having self-replicating ability as undifferentiated cells.

In one embodiment of the present invention, the stem cells may be autologous or allogeneic stem cells, may be derived from any type of animal including humans and non-human mammals, may be stem cells derived from adults, and derived from embryos. It may be a stem cell. For example, the stem cells may be selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem cells (iPSCs), but are not limited thereto.

In one embodiment of the present invention, the adult stem cells are derived from adult stem cells derived from human or animal tissues, mesenchymal stromal cells derived from human or animal tissues, and induced pluripotent stem cells derived from human or animal tissues. It may be selected from the group consisting of mesenchymal stem cells, but is not limited thereto.

The term "embryonic stem cell" in the present specification is a cell extracted during the development of an embryo, and the inner cell mass is extracted from the blastocyst embryo, just before the fertilized egg implants in the mother's uterus, and cultured in vitro. Means one thing.

Embryonic stem cells refer to cells that are pluripotent or totipotent that can differentiate into cells of all tissues of an individual, and have self-renewal capabilities. Means to include embryoid bodies derived from embryonic stem cells.

In one embodiment of the present invention, the stem cells may include embryonic stem cells derived from all of humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., but are not limited thereto.

The term "adult stem cell" as used herein refers to a cell extracted from umbilical cord blood, bone marrow, blood, etc. of a mature adult, and refers to a cell immediately before differentiation into a specific organ cell, and develops into a body tissue when necessary. It refers to cells in an undifferentiated state that have the ability to do so.

In one embodiment of the present invention, the adult stem cells are adult stem cells from various tissues of humans or animals, mesenchymal stromal cells derived from human or animal tissues, and induced pluripotent stems from various tissues of humans or animals. Mesenchymal stem cells and multipotent stem cells derived from cells may be included, but are not limited thereto.

In one embodiment of the present invention, the human or animal or animal tissue may be selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, but is not limited thereto.

In one embodiment of the present invention, the stem cells of various tissues of human or animal origin are 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 testis stem cells. It may be selected from, but is not limited thereto.

The term "induced pluripotent stem cell (iPSC)" as used herein refers to cells induced to have pluripotent differentiation from differentiated cells through an artificial dedifferentiation process, and "redifferentiated stem cells" and It can be used in the same sense.

The artificial dedifferentiation process is performed by introduction of a non-viral-mediated dedifferentiation factor using a virus-mediated or non-viral vector using retrovirus, lentivirus and Sendai virus, protein and cell extract, etc., or stem cell extract, It may include a dedifferentiation process by a compound or the like.

Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, they have a similar cell shape, are similar in gene and protein expression, have pluripotency in vitro and in vivo, and form teratoma. And, when inserted into the blastocyst of a mouse, chimera mice are formed, and germline transmission of genes is possible.

In one embodiment of the present invention, the induced pluripotent stem cells may include induced pluripotent stem cells derived from all of humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, etc., but are limited thereto. It is not.

As used herein, the term "promoting production" means that the production, production, release, etc. of a specific substance is increased within the same time as compared to the control group, and specifically, the amount of exosomes produced from stem cells, and exosomes It means that the content of protein, RNA, etc. is increased.

Another aspect of the present invention is a composition for enhancing stem cell ability of stem cells comprising ranifibranore.

The term "stem cell ability" as used herein refers to pluripotency capable of producing all cells and self-renewal capable of producing cells that resemble magnetism indefinitely, for example For example, increasing the proliferation of stem cells, increasing telomerase activity, increasing the expression of stemness acting signals, or increasing the cell migration activity while maintaining the undifferentiated cells in the undifferentiated state. Refers to increasing, and may include the appearance of one or more of these features.

In one embodiment of the present invention, the composition of the present invention may be used for culturing stem cells as a medium composition, and as a pharmaceutical composition for promoting exosome production in vivo, it is administered in parallel with stem cells in vivo, or It can be administered before or after to enhance the in vivo efficacy of stem cells.

The term "strengthening in vivo efficacy of stem cells" in the present specification presupposes that the efficacy of stem cells with therapeutic purposes (in vivo efficacy) for specific diseases such as arthritis or neuropathy is mediated by exosomes released by stem cells. As a result, when the composition of the present invention is administered together with the administration of stem cells, the generation of exosomes from stem cells is promoted, and as a result, it refers to the use of enhancing the disease treatment effect (in vivo efficacy) of stem cells.

In one embodiment of the present invention, the medium composition refers to a composition containing essential components necessary for cell growth, survival, and proliferation in vitro, and is a medium for culturing stem cells commonly used in the art. Includes all, 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/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), IMDM (Isocove's Modified Dulbecco's Medium), KnockOut DMEM , E8 (Essential 8 Medium) or the like, a commercially prepared medium or an artificially synthesized medium may be used, but is not limited thereto.

In one embodiment of the present invention, the medium composition generally includes a carbon source, a nitrogen source, and trace element components, and may further include amino acids, antibiotics, and the like.

In one embodiment of the present invention, the medium composition may be prepared by adding the constituent of the present invention ranifibranor into a conventional stem cell culture medium.

In one embodiment of the present invention, Lanifibranor is 1 to 1000 μM, 1 to 500 μM, 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM in the medium, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 5 to 1000 μM, 5 to 500 μM, 5 to 100 μM, 5 to 90 μM, 5 to 80 μM, 5 to 70 μM, 5 to 60 μM, 5 to 50 μM, 5 to 40 μM, 5 to 30 μM, 5 to 20 μM may be added, for example, it may be added at a concentration of 10 μM, but is not limited thereto.

In one embodiment of the present invention, the composition for enhancing stem cell ability of stem cells including ranifibranor may be a pharmaceutical composition.

In one embodiment of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate , Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. However, it is not limited thereto.

In one embodiment of the present invention, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, 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).

In one embodiment of the present invention, the pharmaceutical composition may be administered orally and parenterally, such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, rectal administration, dura mater It may be administered by intramuscular administration, ocular administration, skin administration, and transdermal administration, but is not limited thereto.

In one embodiment of the present invention, the pharmaceutical composition is variously depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient. The dosage may be determined, and may be determined or prescribed as a dosage effective for the desired treatment or prophylaxis. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg/kg.

In one embodiment of the present invention, the pharmaceutical composition is formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs. It can be manufactured in unit dosage form or can be manufactured by putting it inside a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer, but is limited thereto. It is not.

Another aspect of the present invention is a stem cell-derived exosome pretreated with ranifibranor.

Description of the common content between the composition according to the present invention and the stem cell-derived exosomes will be omitted to avoid excessive complexity of the present specification.

Another aspect of the present invention is a stem cell therapeutic agent for promoting the generation of stem cells-derived exosomes, including ranifibranore.

Another aspect of the present invention is a stem cell therapeutic agent for enhancing stem cell stem cell ability, including ranifibranor.

Another aspect of the present invention is a stem cell therapeutic agent comprising, as an active ingredient, stem cell-derived exosomes pretreated with ranifibranor.

As used herein, the term "cell therapy" refers to the biological properties of cells by proliferating and selecting in vitro or other methods of living autologous, allogenic, and xenogenic cells to restore the function of cells and tissues. It refers to medicines used for treatment, diagnosis, and prevention purposes through a series of actions such as changing.

These cell therapy drugs can be classified into two broad categories, the first being "stem cell therapy" for tissue regeneration, organ function recovery, or immune cell function regulation, and the second, suppressing or immune response in vivo. It is a "immune cell therapy" for regulating immune responses such as boosting of

The composition according to the present invention increases the stem cell ability of stem cells and promotes the generation of stem cell-derived exosomes, thereby enhancing the therapeutic efficacy of stem cells, and therefore, "cell therapy" within the present specification. Means "stem cell therapeutic agent".

In one embodiment of the present invention, the stem cell therapeutic agent is cardiovascular disease such as myocardial infarction and heart failure, liver cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cancer diseases such as lung cancer, atopic dermatitis, arthritis, autoimmune encephalomyelitis, systemic It may be used to treat inflammatory diseases such as lupus erythematosus, colitis, and multiple sclerosis, or diseases to be treated such as autoimmune diseases, but is not limited thereto.

Since the stem cell therapeutic agent according to the present invention has the above-described compositions and components of the present invention in common, descriptions of the contents in common between the two are omitted in order to avoid excessive complexity of the present specification.

Another aspect of the present invention is a method for producing stem cell-derived exosomes comprising the following steps:

A culture step of culturing stem cells in a cell culture medium containing ranifibranore.

In one embodiment of the present invention, the method for producing stem cell-derived exosomes may further include the following steps:

An additional culturing step of further culturing in a cell culture medium after washing the cultured stem cells; And

Separation step to separate exosomes.

Hereinafter, a method for producing stem cell-derived exosomes of the present invention will be described in detail.

The culturing step of the method for producing stem cell-derived exosomes according to the present invention is a process in which stem cells are pretreated with ranifibranor to stimulate stem cells. By this process, stem cells produce a greater number of exosomes than stem cells that have not been pretreated with the above substances, and the content of proteins and RNA in the exosomes is also increased.

In the additional culturing step of the method for producing stem cell-derived exosomes according to the present invention, the stem cells pretreated with Ranifibranor according to the present invention are washed to remove the pretreatment material, and then cultured in a new cell culture medium from stem cells. It is a process that induces the secretion or production of exosomes. The stem cell pretreatment material (Ranifibranor) in the culture step is removed by the washing process in the additional culture step, and does not remain in the stem cells after washing, exosomes produced from stem cells, or the culture medium. Therefore, the stem cells treated with the stem cell pretreatment material (Ranifibranor) of the present invention and exosomes and culture medium produced from the stem cells in the cultivation stage are used for subsequent studies or treatment of diseases without the effect of the residual of the pretreatment material. It can be usefully used.

In one embodiment of the present invention, the cell culture medium in the additional culture step may additionally contain fetal bovine serum (FBS) from which exosomes have been removed.

The reason for using FBS with exosomes removed in the cell culture medium is that the FBS generally used contains a lot of exosomes derived from bovine serum, so in addition to exosomes secreted by stem cells, exosomes derived from FBS are mixed. It is to prevent it from becoming.

In one embodiment of the present invention, the additional cultivation of the additional culturing step is performed for 12 hours to 120 hours, 24 hours to 96 hours, 48 hours to 96 hours, or 60 hours to 84 hours, for example, 72 hours. It may be cultured, but is not limited thereto.

In the separation step of the method for producing stem cell-derived exosomes according to the present invention, the culture medium of the stem cells additionally cultured in the additional culture step is centrifuged at 200-400xg for 5 to 20 minutes to remove remaining cells and cell residues. After that, take the supernatant and perform high-speed centrifugation at 9,000-12,000xg for 60-80 minutes, then take the supernatant again and centrifuge at 90,000-120,000xg for 80-100 minutes and remove the supernatant to remove the exosomes remaining in the lower layer. Can be obtained.

For example, the mesenchymal stem cell culture medium is collected and centrifuged at 300xg for 10 minutes to remove remaining cells and cell residues, and the supernatant is collected and filtered using a 0.22 μm filter, and then a high-speed centrifuge (high speed centrifuge) at 10,000xg and 4℃ for 70 minutes, and then take the centrifuged supernatant again and centrifuge at 100,000xg at 4℃ for 90 minutes using an ultracentrifuge to remove the supernatant. By doing so, it is possible to separate the exosomes remaining in the lower layer, but is not limited thereto.

Another aspect of the present invention is a method of enhancing stem cell ability of stem cells, comprising the following steps:

A culture step of culturing stem cells in a cell culture medium containing ranifibranore.

Since the method of enhancing the stem cell function of stem cells is the same as the method for producing the stem cell-derived exosomes of the present invention described above and each step, the contents in common between the two are described in order to avoid excessive complexity of the present specification. Is omitted.

The present invention relates to a composition for promoting the production of stem cell-derived exosomes including Lanifibranor and enhancing the function of stem cells. When the composition of the present invention is used for stem cell culture, the Since the production of stem cell ability and stem cell-derived exosomes is increased, it is possible to more efficiently produce high-quality stem cells and stem cell-derived exosomes, and this can be usefully used for related research and development and commercialization.

1 is a graph confirming an increase in stem cell proliferation according to treatment with a stem cell pretreatment material (Lanifibranor) according to an embodiment of the present invention.
2A and 2B are diagrams illustrating an increase in stem cell capacity of stem cells according to treatment with a stem cell pretreatment material (Lanifibranor) according to an embodiment of the present invention.
3 is a diagram illustrating the distribution of exosomes according to the size of the stem cell pretreatment material (Lanifibranor) treatment according to an embodiment of the present invention.
4 is a diagram illustrating an increase in the number of exosomes according to treatment with a stem cell pretreatment material (Lanifibranor) according to an embodiment of the present invention.
5 is a diagram illustrating an increase in the amount of exosome-derived protein according to the treatment of a stem cell pre-treatment material (Lanifibranor) according to an embodiment of the present invention.
6 is a diagram illustrating an increase in the amount of exosome-derived RNA according to the treatment of a stem cell pretreatment material (Lanifibranor) according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example: Isolation of stem cells-derived exosomes according to Lanifibranor treatment

Lanifibranor pretreatment

LANIFIBRANOR 10 μM-containing culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100X) (Gibco, Cat no.11140-050)] from umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) was cultured for 1 week.

Further culture

After the culture was completed, the mesenchymal stem cells pretreated with LANIFIBRANOR were washed and cultured for an additional 72 hours in a culture medium containing 10% fetal bovine serum (FBS) from which exosomes were removed. The reason for using FBS from which exosomes have been removed is to prevent the incorporation of FBS-derived exosomes in addition to exosomes secreted by cells, since the commonly used FBS contains a lot of exosomes derived from bovine serum. .

Exosome separation

After 72 hours of incubation, the mesenchymal stem cell culture medium treated with the pre-treatment material was collected and centrifuged at 300xg for 10 minutes to remove the remaining cells and cell residues. The supernatant was taken and filtered using a 0.22 μm filter, and then centrifuged at 10,000xg and 4°C for 70 minutes using a high speed centrifuge. The centrifuged supernatant was retaken and centrifuged at 100,000xg for 90 minutes at 4℃ using an ultracentrifuge to remove the supernatant. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered salin) and used in the following experiments.

Experimental Example 1: Enhancement of stem cell function according to Lanifibranor treatment

1-1. Stem cell proliferation increase by lanifibranor treatment

100uL culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100X) (Gibco, Cat no. 11140-050)]] Mesenchymal stem cells and mesenchymal stem cells pretreated with Lanifibranor of the above example were seeded at a rate of 3000 cells per well, respectively, in a 96-well plate containing, and then cultured in a CO 2 incubator for 24 hours. Then, after adding a CCK solution (10 uL/well) and incubating for 4 hours in a CO2 incubator, the proliferation rate was confirmed by measuring absorbance at a wavelength of 450nm (420~480nm).

As can be seen in FIG. 1, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pre-treatment material [Lanifibranor] increased by about 181% from 1 to 1.81 compared to the mesenchymal stem cells not treated with any material.

1-2. Increased stemness of stem cells by lanifibranor treatment

10mL culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100X) (Gibco, Cat no. 11140-050)] Mesenchymal stem cells and mesenchymal stem cells pretreated with Lanifibranor of the above Example were inoculated at 1000 cells per dish and cultured for 21 days in a 100 mm cell culture dish containing. The dish to which the cultured cells were attached was washed twice with PBS and fixed with 95% methanol for about 2 minutes at room temperature. After washing the fixed cells 3 times with PBS, 0.5% crystal violet solution [crystal violet (sigma, C-3886, USA) 5g, methanol 100ml] was added, stained for 5 minutes, washed with water, and dried at room temperature. . The number of colonies composed of 50 or more cells in each dish was counted and compared.

As can be seen in FIGS. 2A and 2B, CFU-F of mesenchymal stem cells (56.7 cells) pretreated with a stem cell pretreatment material [Lanifibranor] compared to mesenchymal stem cells (40 cells) not treated with any material. The number of colonies increased by about 142%.

Experimental Example 2. Increased exosome production efficiency according to Lanifibranor treatment

2-1. Increased number of exosomes following lanifibranor treatment

Through a nanoparticle tracking assay (NanoSight NS300, Malvern), the number of exosomes isolated in the above Example was confirmed. At this time, for comparison, the number of separated exosomes was confirmed in the same manner as in Example except that no pretreatment material was treated. The results are shown in Table 1, FIGS. 3 and 4 below.

Yield(per 10 ⁶ Cells) MSC-Exo Lani-MSC-Exo No. of exosome particles(10 ⁹ ) 2.30 11.4

(MSC: untreated mesenchymal stem cells, Lani-MSC-Exo: Lanifibranor-treated mesenchymal stem cells)

As can be seen in Tables 1 and 4, about 4.95 times as many exosomes were produced in mesenchymal stem cells pretreated with a stem cell pre-treatment material (Lanifibranor) compared to mesenchymal stem cells not treated with any material. .

From the above results, it can be seen that the number of exosomes produced by the mesenchymal stem cells increases when the stem cell pretreatment material according to the present invention is treated on the mesenchymal stem cells.

2-2. Increased amount of exosome-derived protein by lanifibranor treatment

The protein was isolated from the exosomes isolated in the above example using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595nm through Bradford analysis to determine the protein. Quantified. At this time, for comparison, proteins were separated and quantified from exosomes separated in the same manner as in Examples, except that no pretreatment material was treated. The results are shown in Table 2 and FIG. 5 below.

Yield(per 10 ⁶ Cells) MSC-Exo Lani-MSC-Exo Exosomal protein(ug) 2.0 9.2

(MSC: untreated mesenchymal stem cells, Lani-MSC-Exo: Lanifibranor-treated mesenchymal stem cells)

As can be seen in Tables 2 and 5, compared to the mesenchymal stem cells not treated with any material, about 4.6 times more exosome-derived protein in the mesenchymal stem cells pretreated with the stem cell pre-treatment material (Lanifibranor) Was produced.

From the above results, it can be seen that when the stem cell pretreatment material according to the present invention is treated on mesenchymal stem cells, not only the number of exosomes but also the content of exosome-derived proteins is increased.

2-3. Increased amount of exosome-derived RNA by lanifibranor treatment

Total exosome RNA was isolated from the exosomes isolated in the above example using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the concentration of RNA was measured using a nanodrop. At this time, for comparison, total exosome RNA was isolated from the isolated exosomes in the same manner as in Example, except that no pretreatment material was treated, and the concentration was measured. The results are shown in Table 3 and FIG. 6 below.

Yield(per 10 ⁶ Cells) MSC-Exo Lani-MSC-Exo Exosomal RNA (ng) 27.00 110.34

(MSC: untreated mesenchymal stem cells, Lani-MSC-Exo: Lanifibranor-treated mesenchymal stem cells)

As can be seen in Table 3 and FIG. 6, about 4.1 times more exosome-derived RNA in mesenchymal stem cells pretreated with stem cell pre-treatment material (Lanifibranor) compared to mesenchymal stem cells not treated with any material. Was produced.

From the above results, it can be seen that when the stem cell pretreatment material according to the present invention is treated on mesenchymal stem cells, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) increases.

Sintering

It can be seen that when the stem cell pretreatment material (Lanifibranor) according to the present invention is treated on mesenchymal stem cells, the stem cell ability (Stemness) of the stem cells is increased. In addition, when the stem cell pretreatment material (Lanifibranor) according to the present invention is treated on mesenchymal stem cells, not only the number of exosomes but also the production of exosome-derived proteins increases, and the amount of exosome-derived RNA (exosomal RNA) is increased. It can be seen that the content also increases.

Therefore, the stem cell pretreatment material (Lanifibranor) according to the present invention not only increases the stem cell ability of stem cells, but also promotes the production of stem cell-derived exosomes, so that the production of stem cells with excellent functionality and mass production of exosomes It is expected to be used for purposes.

Claims (14)

A composition for promoting the generation of stem cells-derived exosomes, including Ranifibranor. The composition of claim 1, wherein the stem cells are selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem cells (iPSCs). The method of claim 2, wherein the adult stem cells are intermediates derived from adult stem cells derived from human or animal tissues, mesenchymal stromal cells derived from human or animal tissues, and induced pluripotent stem cells derived from human or animal tissues. The composition is selected from the group consisting of mesenchymal stem cells. The composition of claim 3, wherein the human or animal tissue is selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, 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 testis stem cells. The composition. delete A composition for enhancing stem cell ability (stemness) of stem cells, including Ranifibranor. delete Stem cell-derived exosomes pretreated with Ranifibranor. Stem cell treatment containing exosomes derived from stem cells pretreated with Lanifibranor as an active ingredient. Stem cell-derived exosome production method comprising the following steps:
A culture step of culturing stem cells in a cell culture medium containing Lanifibranor. The method of claim 11, wherein the method further comprises the following steps:
An additional culturing step of further culturing in a cell culture medium after washing the cultured stem cells; And
Separation step to separate exosomes. The method of claim 11, wherein the cell culture medium contains fetal bovine serum from which exosomes have been removed. A method for enhancing the stemness of stem cells, including the following steps:
A culture step of culturing stem cells in a cell culture medium containing Lanifibranor.

Images