KR20220033403A - Composition for treating inflammatory diseases comprising exosomes derived from human epidural adipose tissue derived stem cell - Google Patents

Abstract

The present invention relates to a composition for treating, alleviating or preventing inflammatory diseases, including exosomes derived from human epidural adipose tissue-derived stem cells. The exosomes have increased expression of anti-inflammatory factor cytokines IL-4, IL-10, and IL-13, and when the exosome treatment, the expression of inflammatory cytokines TNF-α and IL-6 is decreased, and the expression of the anti-inflammatory cytokine IL-10 is increased, so that the composition can be usefully used for preventing, alleviating or treating inflammatory diseases.

Description

Composition for treating inflammatory diseases comprising exosomes derived from human epidural adipose tissue derived stem cell

The present invention relates to a composition for treating, improving or preventing inflammatory diseases, including exosomes derived from human epidural adipose tissue-derived stem cells.

Inflammation is one of the defense mechanisms of living tissues against tissue damage, external stimuli, or various infectious agents. It is an immune response that occurs locally to restore the damaged area to its original state. That is, the inflammatory response is necessary to protect the living body and remove the products generated by tissue damage. However, when such an inflammatory reaction occurs over a certain level or occurs chronically, it progresses to a disease state such as chronic inflammation, resulting in serious abnormalities.

Currently, the most potent anti-inflammatory drugs are steroids. However, most steroid preparations are chemically synthesized substances, and when used for a long period of time, side effects such as adrenal suppression, retention of body fluids, and cataracts are often accompanied. Therefore, there is a need for research on substances capable of inhibiting various kinds of inflammation while having few side effects.

Republic of Korea Patent Publication No. 10-1766341

The inventors of the present invention confirmed that the exosomes derived from human epidural adipose tissue-derived stem cells had lower levels of pro-inflammatory cytokines and chemokines compared to exosomes derived from human skin epithelial cells, whereas the expression of anti-inflammatory cytokines was increased. Thus, the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the treatment or prevention of inflammatory diseases, comprising exosomes derived from human epidural adipose tissue-derived stem cells as an active ingredient.

Another object of the present invention is to provide a food composition for improving or preventing inflammatory diseases, comprising exosomes derived from human epidural adipose tissue-derived stem cells as an active ingredient.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases, comprising exosomes derived from human epidural adipose tissue-derived stem cells as an active ingredient.

In addition, the present invention provides a food composition for improving or preventing inflammatory diseases, comprising exosomes derived from human epidural adipose tissue-derived stem cells as an active ingredient.

In an embodiment of the present invention, the exosome may include anti-inflammatory cytokines.

In one embodiment of the present invention, the anti-inflammatory cytokine may be at least one selected from the group consisting of IL-4, IL-10 and IL-13.

The present invention relates to a composition for the treatment, improvement or prevention of inflammatory diseases, including exosomes derived from human epidural adipose tissue-derived stem cells, wherein the exosomes are anti-inflammatory factor cytokines IL-4, IL-10 And IL-13 expression is increased, and the expression of inflammatory cytokines TNF-α and IL-6 is decreased when the exosome treatment is performed, and the expression of IL-10, an anti-inflammatory cytokine, is increased, so prevention of inflammatory diseases , can be usefully used for improvement or treatment.

Figure 1a is an observation of the culture results of human skin epithelial cells and human epidural adipose stem cells.
Figure 1b shows the surface-specific antigen expression pattern of human epidural adipose stem cells (negative expression (CD14, CD34, CD45) and positive expression (CD73, CD90, CD105)).
Figure 2a confirms the morphology of the human skin epithelial cell-derived exosome and human epidural adipose stem cell-derived exosome.
Figure 2b confirms the positive expression of CD63 and CD81 in exosomes derived from human skin epithelial cells and human epidural adipose stem cells.
Figure 2c shows the size distribution, average diameter, and concentration measurement results of exosomes derived from human skin epithelial cells and human epidural adipose stem cells.
3 is a comparative analysis of the cytokine and chemokine expression patterns of human epidural adipose stem cell-derived exosomes compared to human skin epithelial cell exosomes (*p-value < 0.05, ** ≤ 0.001).
Figure 4a shows the results of human mononuclear THP-1 cells differentiated into macrophages, treated with LPS and/or EV, and then observed under a microscope.
Figure 4b shows the quantification of TNF-α, IL-6 and IL-10 secretion levels following LPS and/or EV treatment of macrophages (* p-value <0.05, *** <0.0001, ns non-significant) .

The inventors of the present invention confirmed that the exosomes derived from human epidural adipose tissue-derived stem cells had lower levels of pro-inflammatory cytokines and chemokines compared to exosomes derived from human skin epithelial cells, whereas the expression of anti-inflammatory cytokines was increased. Thus, the present invention was completed.

Accordingly, the present invention provides a pharmaceutical composition for treating or preventing inflammatory diseases, comprising exosomes derived from human epidural adipose tissue-derived stem cells as an active ingredient.

In the present invention, epidural adipose tissue is an anatomically present adipose tissue around the spinal cord, has less connective tissue compared to subcutaneous fat, and is functionally not known except that it facilitates movement during pendulum movement of the spinal cord.

Adipose tissue-derived stem cells (ADSC) are generally easy to collect tissue and can be obtained in sufficient quantity for research, so they are widely used in regeneration studies using stem cells (Paschos and Sennett, 2017; Oberringer et al., 2018). However, it is known that ADSC has different characteristics depending on the location of the adipose tissue and has different characteristics depending on the extraction process and the donor's physical condition (Reina et al., 2006). In particular, the differentiated regeneration and differentiation ability of ADSCs was confirmed according to the location of the harvested adipose tissue, and for example, subcutaneous fat was used in studies related to the prevention of arteriosclerosis, and in the case of visceral fat, insulin resistance and cardiovascular disease were identified. used in related research.

Through this, it can be expected that the characteristics of the stem cells are also different depending on the location of the adipose tissue collected, and the components and functions of the exosomes derived therefrom are also different.

As used herein, the term “exosomes” is a cell-derived vesicle that exists in eukaryotes, and is released from cells when multivesicular bodies (MVBs) fuse with the plasma membrane or are directly released from the plasma membrane. do.

As used herein, the term “stem cells” refers to cells having the ability to differentiate into various tissues, that is, “undifferentiated cells”.

In the present invention, epidural adipose tissue can be obtained incidentally, for example, during a posterior surgical procedure of the spine, has the advantage of easily obtaining and culturing stem cells, and can secure superior safety compared to bone marrow harvesting.

As used herein, the term “inflammatory disease” refers to a disease caused by a chain bioreaction caused by a direct reaction of a humoral mediator constituting the immune system or stimulation of a local or systemic effector system. means

As used herein, the term “prevention” refers to any action that suppresses or delays the onset of an inflammatory disease by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms due to an inflammatory disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention includes an exosome derived from human epidural adipose tissue-derived stem cells as an active ingredient, and may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives, such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection or oral ingestion.

The pharmaceutical composition of the present invention may be administered orally or administered parenterally (eg, intravenously or subcutaneously) according to a desired method, and the dosage may vary depending on the individual's condition and weight, degree of disease, drug form, Although it varies depending on the route and time of administration, it may be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug It can be determined according to factors including sensitivity to, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the individual, and may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like.

In the present invention, the exosome may include anti-inflammatory cytokines.

Specifically, the anti-inflammatory cytokine may be one or more selected from the group consisting of IL-4, IL-10 and IL-13. Since the IL-4, IL-10 and IL-13 correspond to anti-inflammatory cytokines, they exhibit an anti-inflammatory effect when treated in an individual.

In another aspect, the present invention provides a food composition for improving or preventing inflammatory diseases, comprising an exosome derived from human epidural adipose tissue-derived stem cells as an active ingredient.

The food composition is a concept including health functional food.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of symptoms.

In the food composition of the present invention, exosomes derived from human epidural adipose tissue-derived stem cells may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the exosome derived from human epidural adipose tissue-derived stem cells may be suitably determined according to the purpose of its use (for prevention or improvement). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than or equal to the above range.

The health functional food composition of the present invention is not particularly limited in other ingredients other than containing the above ingredients as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in more detail by way of Examples. However, these examples are only provided to help the understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

<Example 1: Method and Materials>

Example 1-1. Cell Isolation and Culture

Human epidural adipose tissue-derived mesenchymal stem cells were obtained from human epidural adipose tissue. Briefly, the skin and adipose tissue were isolated, washed with 70% ethanol (EtOH) and ice-cold PBS, and then treated with 0.45 μm-filtered collagenase type I 2 mg/mL (gibco), followed by an incubator at 37 °C. was digested for 30 min. Then, the digested solution was put into a 70 μm strainer, and the filtered solution was centrifuged at 3000 × g for 5 minutes.

Human dermal fibroblasts and human epidural AD-MSCs (Adipose-Derived Mesenchymal Stem Cells) were obtained from 10% exosome-depleted FBS (gibco), 1% penicillin/streptomycin (gibco), and 10ug/mL recombinant human FGF-basic. (Peprotech), 10 ug/mL of recombinant human PDGF-BB (Peprotech) and 25 ug/mL of plasmocin (InvivoGen) for mycoplasma prophylaxis. All cells were cultured at 37 °C and 5% CO ₂ incubator conditions.

Example 1-2. Isolation of human dermal fibroblasts and human epidural AD-MSC exosomes

The culture medium of AD-MSCs grown in 175 T-flasks was collected after 48 hours of incubation. The exosomes were purified in cell culture medium and cells were removed by centrifugation at 300 x g for 10 min. The supernatant was then centrifuged again at 2500 x g for 25 min to remove cell debris and apoptosis. Then, the supernatant was ultracentrifuged at 100,000 x g for 120 min using a 90Ti rotor (Beckman Coulter). The pellet appeared at the bottom of the ultracentrifuge tube, the supernatant was discarded, and the pellet was resuspended in 200 µL of 0.22 µm filtered PBS. Exosome protein concentration was measured with the Pierce ™ BCA assay kit (Thermo Fisher science 23225).

Examples 1-3. flow cytometry

Flow cytometry was performed using flow cytometry Galios (Beckman Coulter). CD105 (Bio-Rad, MCA1557), CD90 (BioLegend, 555596), CD73 (BioLegend, 344004), CD45 (BioLegend, 555482), CD34 (BioLegend, 343504) and CD14 (Bio-Rad) to determine expressed stem cell markers. , MCA1568) and stained the cells with the same antibody. Antibodies were conjugated with FITC or PE fluorescent dyes.

For exosome analysis, 200 μL of isolated exosomes were incubated with 10 μL of aldehyde/sulfate-latex beads 4% w/v (ThermoFishcer Scientific) at room temperature for 15 minutes. A 1 mL volume of PBS (supplemented with 0.1% BSA) was then added to the exosome/bead mixture. Samples were incubated by rotation overnight. Bead-coupled exosomes were pelleted by centrifugation at 2000 × g for 10 min and washed with 500 μL of PBS.

The pellet was resuspended in 50 µL of PBS containing antibodies CD9 (NOVUS, NBP1-28364), CD81 (NOVUS, NBP1-44859) for 1 h at 4 °C and all antibodies were conjugated with FITC fluorescent dye. Samples were washed with 500 µL of PBS and centrifuged at 2000 x g for 10 min. The pellet was then resuspended in 150 μL of PBS. Exosomes treated with 4 μm diameter beads were gated and analyzed.

Examples 1-4. Transmission electron microscopy (TEM)

Freshly isolated exosomes from human dermal fibroblasts and human epidural AD-MSC were resuspended in cold distilled water. The exosome suspension was loaded onto a Pomba carbon coating grid (Ted Pella Inc.) and fixed in 2% paraformaldehyde for 10 minutes, then the solution was removed and the sample was dried. The grid was observed with bioTEM (Hitachi HT7700).

Examples 1-5. Nanoparticle Tracking Analysis (NTA).

NTA analysis was performed with a PMX120 (Particle Metrix) instrument according to the manufacturer's recommendations.

Example 1-6. Cytokine analysis

Cytokines were quantified in human dermal fibroblasts and human epidural AD-MSC exosome solutions using the Quantibody® Human Cytokine Array Kit (RayBiotech, QAH-CYT-1). Exosomal proteins were diluted to 300 µg/mL for each array and 100 µL of the total sample volume was loaded and performed according to the manufacturer's protocol. Signals were measured with a laser scanner equipped with a Cy3 wavelength (532 nm) from Innopsys Innoscan. Data analysis was quantified with Mapix version 7.2.0.

Example 1-7. LPS and EV Treatment for THP-1 Cells

Human THP-1 cells were differentiated into macrophages in 100 nM PMA (Sigma-Aldrich, Saint Louis, MO, USA) for 24 h. PMA of THP-1 cells was washed before LPS treatment. EVs derived from human dermal fibroblasts and EVs derived from human epidural fat MSCs (50 μg/mL) were simultaneously added with 1 μg/mL LPS (Sigma-Aldrich). As a control, THP-1-derived macrophages were cultured without LPS and EV or with 50 μg/mL of two cell-derived EVs.

Examples 1-8. statistical analysis

Statistical analysis was processed using GraphPad Prism software. All measured data were expressed as mean ± standard deviation, and unpaired t-test was used. A p value <0.05 was considered significant and is described in the description of the figures.

<Example 2: Characterization of human epidural AD-MSC>

Two cell types were cultured to compare stem cells and fibroblasts. Human epidural AD-MSCs were isolated from human epidural adipose tissue, and the isolated human epidural AD-MSCs had typical mesenchymal stem cell characteristics. The cells were attached to a plastic culture plate, and the cell shape was long and thin spindle-shaped like dermal fibroblasts (see Fig. 1a). In addition, flow cytometry analysis confirmed that human epidural AD-MSCs showed positive cell surface marker expression for CD73, CD90 and CD105, and negative cell surface marker expression for CD14, CD34 and CD45 (see Fig. 1b). . For proliferation of stem cells and isolation of exosomes, the cells were subcultured 12 times. These results suggest that isolated human epidural AD-MSCs have typical characteristics of mesenchymal stem cells.

<Example 3: Characterization of human dermal fibroblasts and human epidural AD-MSC exosomes>

We tried to use a serum-free or xeno-free medium for cell culture, but the medium contains a lot of albumin and other proteins. Since the serum albumin and exosomes of normal FBS affect the purity of the isolated exosomes, a cell culture medium containing the exosomes depleted and the isolated exosomes was used. In order to confirm the exosomes purified by TEM, FACS and NTA (Nanoparticles tracking analysis) analysis of the size distribution and number of particles for a specific protein was performed. According to the TEM imaging results, the isolated exosomes exhibited a classical form of exosomes, and it was confirmed that the size of the exosomes was 100-200 nm in diameter and spherical.

In addition, the exosome membrane was composed of a lipid bilayer, and dark and thick exosome membranes were observed by TEM (see Fig. 2a). Exosomes made from multivesicular bodies (MVBs) have several biomarkers such as tetraspanins, fusion proteins, and MVB biogenesis markers. In particular, tetraspanins such as CD9, CD63 and CD81 were expressed in the exosome membrane. Thus, tetraspanins CD63 and CD81 were detected using flow cytometry.

Although flow cytometry is usually performed on cells, exosomes are much smaller than normal cells, so flow cytometry was performed using aldehyde/latex beads with a diameter of about 4 μm. The bead-bound exosomes were analyzed by flow cytometry, and dermal fibroblasts and human epidural AD-MSC-derived exosomes exhibited about 60% and 90% of CD63 and CD81 expression, respectively (see FIG. 2b ).

NTA data represent exosome size distribution and concentration. 99.7% of isolated human dermal fibroblast-derived exosomes had an average size of 144.4 nm and a concentration of 1.06 x 10 ¹⁰ particles/mL. 99.1% of isolated human epidural AD-MSC-derived exosomes had a diameter of 142.8 nm and a concentration of 1.27 x 10 ¹⁰ particles/mL (see Fig. 2c).

<Example 4: Confirmation of immunosuppressive effect of exosomes derived from human epidural AD-MSC>

Cytokine and chemokine levels were compared between human dermal fibroblast-derived exosomes and human epidural AD-MSC-derived exosomes. To compare accurate measurements of inflammatory factor levels, the protein concentration was adjusted to 300 μg/mL according to the cytokine array manual. Pro-inflammatory factors such as GM-CSF, MIP-1 alpha, MMP-9, IL-5, GRO and VEGF were confirmed to have low expression levels in human epidural AD-MSC-derived exosomes. On the other hand, it was confirmed that the anti-inflammatory factors IL-4, IL-10 and IL-13 were increased in human epidural AD-MSC-derived exosomes, and in particular, IL-13 expression was significantly increased (FIG. 3). These results suggest that MSC-derived exosomes play an important role in immune defense compared to fibroblast-derived exosomes.

<Example 5: Confirmation of anti-inflammatory effect of EV derived from human epidural fat MSC>

The THP-1 human monocytic leukemia cell line was used to determine if epidural adipose MSC-derived EVs induce anti-inflammatory effects. THP-1 cells cultured without PMA showed sufficient proliferation. PMA was added to THP-1 cells grown in culture plates. THP-1 cells treated with PMA stopped proliferating, adhered to the bottom of the culture dish, and then differentiated into macrophage-like cells (Fig. 4a).

Lipopolysaccharides (LPS) constitute the outer membrane of Gram-negative bacteria and are considered one of the most characterized pathogen-associated molecular patterns to interact with CD14/TLR4 and activate intracellular signaling. Treatment of THP-1-derived macrophages with LPS released inflammatory factors and arachidonic acid products such as PGE2 and PGF2α.

LPS stimulation was attempted to detect LPS-induced inflammation and changes in cytokine expression. THP-1-derived macrophages stimulated with LPS in EV- were also treated to confirm the anti-inflammatory effect of EVs on macrophages (Fig. 4a). After LPS stimulation, TNF-α, IL-6 and IL-10 in the conditioned medium were quantified by cytokine array and ELISA (Fig. 4b). LPS significantly induced TNF-α (973.6 ± 0.69 pg/mL) in THP-1-derived macrophages. Conversely, there was no difference in TNF-α production between the group not treated with LPS and the group treated with EV. Dermal fibroblasts and epidural adipose MSC-derived EVs treated with LPS had values of 68.55 ± 49.92 pg / mL and 53.64 ± 33.20 pg / mL, respectively, indicating a significant decrease in TNF-α production.

In the group not treated with LPS, the cytokine IL-6 was not produced. However, IL-6 production was increased in the LPS-treated group (1489.39 ± 121.92 pg/mL). Moreover, LPS-treated dermal fibroblast-derived EVs increased IL-6 production levels (1321.79 ± 203.60 pg/mL). However, simultaneous treatment of LPS and epidural MSC-derived EVs significantly blocked IL-6 production (167.78 ± 7.69 pg/mL).

When LPS and epidural MSC-derived EV were treated simultaneously, IL-10 production was confirmed as 406.96 ± 67.94 pg/mL. This is an increased value compared to the case where only LPS (77.49 ± 15.80 pg / mL) and LPS and dermal fibroblast EV (133.23 ± 15.80 pg / mL) were treated.

These results demonstrate that EVs reduced TNF-α production in THP-1 derived macrophages. In addition, unlike dermal fibroblast-derived EVs, epidural MSC-derived EVs showed reduced production of the pro-inflammatory factor IL-6. In addition, the production of IL-10, a known anti-inflammatory factor, was increased in epidural MSC-derived EVs. That is, the treatment of epidural MSC-derived EVs suppressed LPS-induced inflammation in THP-1 macrophages compared to dermal fibroblast-derived EVs.

Claims (6)

A pharmaceutical composition for treating or preventing inflammatory diseases, comprising, as an active ingredient, an exosome derived from human epidural adipose tissue-derived stem cells.
The pharmaceutical composition according to claim 1, wherein the exosome comprises an anti-inflammatory cytokine.
The pharmaceutical composition of claim 2, wherein the anti-inflammatory cytokine is at least one selected from the group consisting of IL-4, IL-10 and IL-13.
A food composition for improving or preventing inflammatory diseases, comprising an exosome derived from human epidural adipose tissue-derived stem cells as an active ingredient.
The food composition of claim 4, wherein the exosomes contain anti-inflammatory cytokines.
The food composition according to claim 5, wherein the anti-inflammatory cytokine is at least one selected from the group consisting of IL-4, IL-10 and IL-13.

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