Method for enhancing the efficacy of stem cells by using ethionamide

Abstract

The present invention relates to a culture medium composition for enhancing stem cell efficacy, comprising ethionamide; a method for enhancing stem cell efficacy and a method for producing stem cells with enhanced efficacy, comprising the step of culturing stem cells in the culture medium composition; stem cells produced by the method and uses thereof. According to the present invention, a simple process of treating mesenchymal stem cells with ethionamide can effectively enhance the anti-inflammatory effect of the mesenchymal stem cells and the expression level of paracrine factors, and the stem cells obtained by the method can be used to prevent or treat inflammatory diseases or degenerative brain diseases.

Description

Technical Field

[0001] The present invention relates to a culture medium composition for enhancing stem cell efficacy, comprising ethionamide; a method for enhancing stem cell efficacy, comprising culturing stem cells in the culture medium composition; a method for preparing stem cells with enhanced efficacy, stem cells prepared by the method and uses thereof. Background Art

[0002] Inflammation is one of the biological responses to harmful stimuli of biological tissues such as pathogens and damaged cells, and a protective response involving immune cells, blood vessels and molecular biological intermediates. However, abnormal inflammation is associated with various human diseases and causes various diseases such as allergies, atopy, arthritis, heart disease, brain disease, circulatory disorders and cancer. The occurrence of various inflammatory diseases is associated with the activation of macrophages, which leads to excessive production of inflammatory factors, and representative inflammatory factors include interleukin-1β (interleukin-1β; IL-1β), tumor necrosis factor-α (tumornecrosis factor-α; TNF-α) and nitric oxide (nitrogen oxide; NO).

[0003] Due to rapid aging, degenerative brain diseases represented by Alzheimer's disease and Parkinson's disease are becoming a serious social problem. According to data from the Alzheimer's Association, Alzheimer's disease, which occurs every 68 seconds in the United States, is expected to occur every 33 seconds in 2050. In the United States, Alzheimer's disease is the third most expensive disease to treat after heart disease and cancer, the sixth leading cause of death in all age groups, and the fifth leading cause of death in the elderly over 65 years old. In South Korea, it is estimated that the number of dementia patients will increase from 470,000 (8.8% of the population aged 65 and above) in 2010 to 750,000 (9.7%) in 2020, and cerebrovascular disease has been the second leading cause of death in South Korea for the past decade.

[0004] Recently, research results have shown that excessive encephalopathy is the main cause of these degenerative brain diseases, which has been reported worldwide. Encephalopathy is one of the pathological symptoms that occurs in most degenerative brain diseases such as Alzheimer's disease and Parkinson's disease, and the death of nerve cells is promoted by inflammatory mediators such as inflammatory cytokines or oxidative substances produced by immune cells due to encephalopathy. Therefore, research on the treatment of degenerative brain diseases by inhibiting encephalopathy is actively being conducted.

[0005] Meanwhile, mesenchymal stem cells (mesenchymal stem cell) are referred to as participating in tissue regeneration, treatment and immune response and multipotent cells, therefore, efforts have been made to separate and cultivate mesenchymal stem cells from umbilical cord blood or bone marrow, to utilize the above-mentioned characteristics to develop therapeutic agents for various diseases. For example, mesenchymal stem cells appear as new substitutes for the treatment of autoimmune diseases, and their immunosuppression, anti-inflammatory effects and T cell activation and proliferation inhibition have been reported. In addition, mesenchymal stem cells have been reported to show neuroprotective effects, and secrete multiple nerve growth factors to promote the survival of nerve cells and the regeneration of nerve fibers in the degenerative nervous system environment, and because mesenchymal stem cells have immunomodulatory ability, multiple immune responses are regulated. In addition, it is known to regulate degenerative nervous system environment and nerve regeneration (Hanyang Med Rev 2012 by differentiation or fusion into nerve cells; 32:145-153).

[0006] However, since only the above-mentioned effects of mesenchymal stem cells are known and the development of mesenchymal stem cells optimized to have more improved therapeutic effects has not yet been fully developed, there is an urgent need to develop stem cell therapeutic agents optimized by enhancing the efficacy of treating inflammatory diseases and degenerative brain diseases.

[0007] public

[0008] Technical issues

[0009] Therefore, as a result of research on methods for further improving the anti-inflammatory effect of stem cells and the efficacy of expressing paracrine factors, the inventors of the present invention confirmed that when ethionamide, generally known as an antibiotic, is used, the efficacy of stem cells is improved and that stem cells substantially reduce the pathological symptoms of encephalopathy and dementia in vivo, thereby completing the present invention.

[0010] Therefore, the present invention is directed to providing a culture medium composition for enhancing stem cell efficacy, which comprises ethionamide.

[0011] Furthermore, the present invention provides a method for enhancing the efficacy of stem cells, which comprises culturing the stem cells in a culture medium composition.

[0012] In addition, the present invention relates to a method for preparing stem cells with enhanced efficacy, which comprises culturing stem cells in a culture medium composition; stem cells with enhanced efficacy prepared by the method, and uses of the stem cells.

[0013] However, the technical problems to be solved by the present invention are not limited to the above problems, and a person skilled in the art will fully understand other problems not described herein from the following description.

[0014] Technical Solution

[0015] The present invention provides a culture medium composition for enhancing the efficacy of stem cells, comprising ethionamide.

[0016] In one embodiment of the present invention, ethionamide may be included in the culture medium at a concentration of 1 to 200 μM.

[0017] In another embodiment of the present invention, the stem cells may be embryonic stem cells or adult stem cells.

[0018] In another embodiment of the present invention, the adult stem cells may be mesenchymal stem cells derived from one or more tissue types selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amnion and placenta.

[0019] In another embodiment of the present invention, the enhancement of efficacy may be the increased expression of paracrine factors in stem cells.

[0020] In another embodiment of the present invention, the paracrine factor may be one or more selected from the following: brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO1), glutamate-cysteine ​​ligase catalytic subunit (GCLC) and glutamate-cysteine ​​ligase modifier subunit (GCLM).

[0021] Furthermore, the present invention provides a method for enhancing the efficacy of stem cells, comprising culturing the stem cells in a culture medium composition.

[0022] Furthermore, the present invention provides a method for preparing stem cells having enhanced efficacy, which comprises culturing the stem cells in a culture medium composition.

[0023] Furthermore, the present invention provides stem cells with enhanced efficacy prepared by the above method.

[0024] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, which comprises stem cells as an active ingredient.

[0025] In one embodiment of the present invention, the inflammatory disease can be selected from: dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, peritonitis, osteomyelitis, cellulitis, meningitis, encephalitis, pancreatitis, stroke, acute bronchitis, chronic bronchitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, infectious arthritis, periarthritis, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases.

[0026] Furthermore, the present invention provides a method for preventing or treating an inflammatory disease, which comprises treating a subject with a pharmaceutical composition comprising stem cells as an active ingredient.

[0027] In addition, the present invention provides use of the pharmaceutical composition for preventing or treating inflammatory diseases.

[0028] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising stem cells as an active ingredient.

[0029] In one embodiment of the present invention, the degenerative brain disease can be selected from: Parkinson's disease, dementia, Alzheimer's disease, frontotemporal dementia, Huntington's disease, stroke, cerebral infarction, Pick's disease, head injury, spinal cord injury, cerebral arteriosclerosis, Lou Gehrig's disease, multiple sclerosis, senile depression and Creutzfeldt-Jakob disease.

[0030] Furthermore, the present invention provides a method for preventing or treating a degenerative brain disease, which comprises treating a subject with a pharmaceutical composition comprising stem cells as an active ingredient.

[0031] In addition, the present invention provides use of the pharmaceutical composition for preventing or treating degenerative brain diseases.

[0032] Beneficial Effects

[0033] The inventors of the present invention have experimentally confirmed that when inflammation-induced microglia are co-cultured with mesenchymal stem cells sensitized by ethionamide, the anti-inflammatory effect caused by mesenchymal stem cells is improved, and the paracrine activity of various growth factors and antioxidant factors is improved in mesenchymal stem cells sensitized by ethionamide, and the effect of significantly reducing amyloid beta, encephalopathy and phosphorylated tau is also confirmed by administering mesenchymal stem cells in a dementia animal model. Therefore, the present invention can effectively improve the anti-inflammatory effect of mesenchymal stem cells and the expression level of paracrine factors through a simple process of treating mesenchymal stem cells with ethionamide, and the stem cells prepared by the above method can be effectively used to prevent or treat inflammatory diseases or degenerative brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1a Shown are the expression levels of iNOS after co-culture of inflammation-induced microglia (BV2) and mesenchymal stem cells sensitized or not with ethionamide.

[0035] Figure 1b Shown are the expression levels of NO after co-culture of inflammation-induced microglia (BV2) and mesenchymal stem cells sensitized or not with ethionamide.

[0036] Figure 1c Shown are the expression levels of ROS after co-culture of inflammation-induced microglia (BV2) and mesenchymal stem cells sensitized or not with ethionamide.

[0037] Figure 2a Shown are the mRNA expression levels of the inflammatory cytokine IL-6 after co-culture of inflammation-induced microglia (BV2) and mesenchymal stem cells sensitized or not with ethionamide.

[0038] Figure 2b Shown are the mRNA expression levels of the inflammatory cytokine TNF-α after co-culture of inflammation-induced microglia (BV2) and mesenchymal stem cells sensitized or not with ethionamide.

[0039] Figure 3 Shown are NF-kB activity and its quantitative expression levels measured by Western blotting after co-culture of inflammation-induced microglia (BV2) and mesenchymal stem cells sensitized or not with ethionamide.

[0040] Figure 4a Shown are the expression levels of growth factors BDNF, VEGF, IGF-1, and HGF as paracrine factors after treatment of mesenchymal stem cells with ethionamide at concentrations (50 and 100 μM).

[0041] Figure 4b Shown are the expression levels of antioxidant-related factors HO-1, NQO1, GCLC, and GCLM as paracrine factors after treatment of mesenchymal stem cells with ethionamide at concentrations (50 and 100 μM).

[0042] Figure 5a Shown are the results of immunostaining on brain tissue sections obtained one week after administration of ethionamide-sensitized or non-sensitized mesenchymal stem cells to the ventricles of dementia mouse models using an amyloid β antibody (anti-6E10), and the quantitative results thereof.

[0043] Figure 5b Shown by Figure 5a The levels of amyloid β were measured by ELISA using the soluble and insoluble fractions obtained from homogenized brain tissues after administration of mesenchymal stem cells to a dementia mouse model using the same method used in .

[0044] Figure 6 showed that the use of GFAP antibody in Figure 5a Results of immunostaining performed on brain tissue sections obtained by the same method as used in , and their quantitative results.

[0045] Figure 7 The levels of tau protein are shown, in which the threonine residues 181 and 231 are phosphorylated by Figure 5b The same method used in was performed by ELISA on brain tissue samples obtained 2 weeks after administration of ethionamide-sensitized or non-sensitized mesenchymal stem cells to the ventricle of the dementia mouse model.

[0046] Mode for the Invention

[0047] The present invention provides a culture medium composition for enhancing the efficacy of stem cells, comprising ethionamide.

[0048] Generally, 2-ethylpyridine-4-carbothioamide having the following chemical structure is a thioamide-based antibiotic and is known to be used for treating bacterial infection diseases, but in the present invention, the effect of ethionamide for enhancing the efficacy of stem cells was first discovered.

[0049] [Ethionamide]

[0050]

[0051] The inventors of the present invention have demonstrated the effect of enhancing the efficacy of stem cells sensitized by ethionamide through specific examples.

[0052] In one embodiment of the present invention, as a result of measuring the levels of iNOS, NO and ROS after co-culture of inflammation-induced microglia and mesenchymal stem cells sensitized or not sensitized with ethionamide, the expression of factors can be further reduced in the group co-cultured with mesenchymal stem cells sensitized with ethionamide compared with the control or ethionamide-unsensitized group, which shows that the anti-inflammatory effect of mesenchymal stem cells is improved by ethionamide treatment (see Example 3).

[0053] In another embodiment of the present invention, as a result of measuring the expression levels of inflammatory cytokines IL-6 and TNF-α after co-culture of inflammation-induced microglia and mesenchymal stem cells sensitized or not sensitized with ethionamide, it was confirmed that the inhibitory effect on the expression of inflammatory cytokines was improved in the group co-cultured with mesenchymal stem cells sensitized with ethionamide compared with the control or ethionamide-unsensitized group (see Example 4).

[0054] In another embodiment of the present invention, as a result of measuring NF-κB activity after co-culturing inflammation-induced microglia and mesenchymal stem cells sensitized or not sensitized with ethionamide, the inhibitory effect on NF-κB activity was improved in the group co-cultured with mesenchymal stem cells sensitized with ethionamide compared to the control or ethionamide-unsensitized group (see Example 5).

[0055] In another embodiment of the present invention, as a result of treating mesenchymal stem cells with different concentrations of ethionamide and measuring the secretion amounts of various growth factors and antioxidant factors, it was confirmed that the expression levels of paracrine factors increased in proportion to the treatment concentration of ethionamide (see Example 6).

[0056] In another embodiment of the present invention, the in vivo effect of stem cells sensitized by ethionamide was confirmed. In particular, as a result of immunostaining or ELISA performed after administering mesenchymal stem cells sensitized or not sensitized by ethionamide to the ventricle of a dementia mouse model and then extracting brain tissue, it was confirmed that mesenchymal stem cells sensitized by ethionamide had a significant effect of reducing amyloid β, encephalopathy, and phosphorylated tau protein (see Examples 7 to 9).

[0057] Therefore, the above results indicate that the efficacy of human mesenchymal stem cells can be further enhanced by ethionamide treatment.

[0058] The concentration of ethionamide included in the culture medium of the present invention may be, but is not limited to, 1 to 200 μM, preferably 50 to 200 μM, and more preferably 50 to 150 μM.

[0059] The term "stem cell" as used herein is an undifferentiated cell that has the ability to self-replicate and differentiate into two or more different types of cells. The stem cells of the present invention may be autologous or allogeneic stem cells, may be derived from any type of animal (including humans and non-human mammals), and may be derived from adult animals or embryos, but the present invention is not limited thereto.

[0060] In the present invention, the adult stem cells derived from adult animals can be mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, pluripotent stem cells or amniotic epithelial cells, preferably mesenchymal stem cells, but the present invention is not limited thereto. Mesenchymal stem cells can be mesenchymal stem cells derived from the following: umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion and placenta, but the present invention is not limited thereto.

[0061] In the present invention, the separation and culture of mesenchymal stem cells can be performed by methods well known to those of ordinary skill in the art, and there is no limitation to the method as long as it can proliferate mesenchymal stem cells without changing their properties while maintaining stem cell properties.

[0062] The enhanced efficacy of stem cells in the present invention refers to the improvement of the therapeutic properties and effects of stem cells against inflammatory diseases or degenerative brain diseases, and more specifically, the inhibition of the production of nitric oxide, its related factors or reactive oxygen species, the inhibition of the expression of inflammatory cytokines, the antioxidant and anti-inflammatory effects, and the improvement of the efficacy of expressing paracrine factors such as growth factors and antioxidant factors according to the reduction of NF-κB activity.

[0063] The paracrine factors are more specifically selected from one or more of the following: brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO1), glutamate-cysteine ​​ligase catalytic subunit (GCLC) and glutamate-cysteine ​​ligase modifier subunit (GCLM), but the present invention is not limited thereto.

[0064] As another aspect of the present invention, the present invention provides a method for enhancing the efficacy of stem cells, which comprises culturing the stem cells in a culture medium composition.

[0065] As another aspect of the present invention, the present invention provides a method for preparing stem cells with enhanced efficacy, which comprises culturing the stem cells in a culture medium composition.

[0066] Furthermore, the present invention provides stem cells with enhanced efficacy prepared by the above method.

[0067] As another aspect of the present invention, the present invention provides an anti-inflammatory composition comprising stem cells as an active ingredient.

[0068] As another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or degenerative brain diseases, which comprises stem cells as an active ingredient.

[0069] The term "prevention" as used herein refers to all effects of inhibiting or delaying the onset of an inflammatory disease or a degenerative brain disease by administering the pharmaceutical composition according to the present invention.

[0070] The term "treatment" as used herein refers to all actions involved in alleviating or beneficially altering the symptoms of inflammatory diseases or degenerative brain diseases by administering a pharmaceutical composition according to the present invention.

[0071] The term "inflammatory disease" as used herein is a general term for diseases with inflammation as the main lesion. More preferably, in the present invention, the inflammatory disease can be selected from: dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, peritonitis, osteomyelitis, cellulitis, meningitis, encephalitis, pancreatitis, stroke, acute bronchitis, chronic bronchitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, infectious arthritis, periarthritis, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases, but the present invention is not limited thereto.

[0072] The term "degenerative brain disease" used herein is a disease occurring in the brain among the degenerative diseases occurring with age. More preferably, the degenerative brain disease in the present invention can be selected from: Parkinson's disease, dementia, Alzheimer's disease, frontotemporal dementia, Huntington's disease, stroke, cerebral infarction, Pick's disease, head injury, spinal cord injury, cerebral arteriosclerosis, Lou Gehrig's disease, multiple sclerosis, senile depression and Creutzfeldt-Jakob disease, but the present invention is not limited thereto.

[0073] The pharmaceutical composition according to the present invention may include stem cells with enhanced efficacy treated by ethionamide, and also include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are commonly used in preparations, including saline, distilled water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., but the present invention is not limited thereto. If necessary, the pharmaceutical composition may also include other conventional additives, including antioxidants, buffers, etc. In addition, by adding a diluent, a dispersant, a surfactant, an adhesive or a lubricant, the pharmaceutical composition may be formulated into an injectable form, such as an aqueous solution, an emulsion or suspension, a pill, a capsule, a granule or a tablet. Suitable pharmaceutically acceptable carriers and their preparations can be formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not limited in dosage form, and therefore can be formulated as an injection, an inhalant or an external skin preparation.

[0074] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and is preferably administered intracerebrally, and the dosage of the pharmaceutical composition of the present invention can be appropriately selected by a person of ordinary skill in the art according to the patient's condition and weight, the severity of the disease, the dosage form, the administration route and the duration.

[0075] The pharmaceutical composition of the present invention is used in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" used herein refers to the amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and effective doses can be determined by parameters, including the type of the patient's disease, severity, drug activity, sensitivity to the drug, time of application, route of administration and excretion rate, duration of treatment and concurrently used drugs and other parameters well known in the medical field. The pharmaceutical composition of the present invention can be used alone or in combination with other therapeutic agents, and can be used sequentially or simultaneously with conventional therapeutic agents, or with a single dose or multiple doses. Considering all of the above parameters, it is important to achieve maximum effect with a minimum dose in the absence of side effects, and such dosage can be easily determined by a person of ordinary skill in the art.

[0076] Specifically, the effective amount of the pharmaceutical composition of the present invention can be varied according to the patient's age, sex, condition, body weight, absorption, inactivation rate and excretion rate of the active ingredient in the body, disease type and drugs used in combination, and usually, 5×10 2 cells to 5×10 8 Cells / kg body weight. For multiple administrations, administration can be performed weekly or monthly. However, the effective amount can be increased or decreased according to the administration route, severity of obesity, sex, body weight or age, and therefore does not limit the scope of the present invention in any way.

[0077] As another aspect of the present invention, the present invention provides a method for preventing or treating an inflammatory disease or a degenerative brain disease, which comprises administering a pharmaceutical composition to a subject.

[0078] The term "subject" as used herein refers to a target in need of treatment, and more specifically, refers to a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse or cow.

[0079] In addition, the present invention provides use of the pharmaceutical composition for preventing or treating inflammatory diseases or degenerative brain diseases.

[0080] Hereinafter, in order to help understand the present invention, exemplary embodiments will be presented. However, the following embodiments are provided only to make it easier to understand the present invention, rather than to limit the present invention. Example

[0081] Example 1. Preparation of human umbilical cord mesenchymal stem cells

[0082] After obtaining the umbilical cord according to the standards approved by the IRB of Samsung Medical Center (IRB #2015-09-023-003), human umbilical cord mesenchymal stem cells were isolated by the following method.

[0083] First, a piece of umbilical cord tissue of 3 to 4 cm was fragmented and treated with collagenase solution (Gibco, USA) for 60-90 minutes to digest the extracellular matrix, and then digested with 0.25% trypsin (Gibco, USA) at 37° C. for 30 minutes. Subsequently, cells were obtained by adding fetal bovine serum (FBS) (BioWest, USA) to the resulting product, then centrifuged at 1000×g for 10 minutes, and then cultured in MEM medium (Minimum Essential Media, Minimum Essential Media) (Gibco, USA) supplemented with 10% FBS and 50 μg / ml gentamicin (Gibco, USA) at 37° C., 5% CO2 environment, so that mesenchymal stem cells that were passaged 5 or 6 times were used for experiments.

[0084] Example 2. Preparation of human umbilical cord mesenchymal stem cells sensitized with ethionamide

[0085] 6×10 3 cells / cm 2 The human umbilical cord mesenchymal stem cells prepared according to the method of Example 1 were seeded in a cell culture container and treated with ethionamide at a concentration of 50 μM, 100 μM or 150 μM, and then cultured for 72 hours.

[0086] Example 3. Verification of the anti-inflammatory effect of stem cells sensitized by ethionamide

[0087] In order to confirm whether the anti-inflammatory effect of mesenchymal stem cells is improved when treated with ethionamide, the following experiment was performed. Specifically, the inflammation model was induced by treating microglial BV2 cells with LPS (lipopolysaccharide), and co-cultured with mesenchymal stem cells (hMSCs) sensitized (primed) or not sensitized with ethionamide, and then the expression levels of nitric oxide (NO), NO-related factors and reactive oxygen species were measured.

[0088] Results, such as Figure 1aAs shown in , in the case of iNOS (inducible nitric oxide synthase), a representative NO-related inflammatory factor, mRNA expression was significantly reduced in the group co-cultured with mesenchymal stem cells sensitized with ethionamide compared to the control or the group co-cultured with mesenchymal stem cells not sensitized with ethionamide. Figure 1b and 1c As shown, according to the results of measuring the expression levels of NO and reactive oxygen species (ROS), it was confirmed that the expression levels were significantly reduced in the co-culture group with mesenchymal stem cells sensitized with ethionamide compared with the control group or the co-culture group with mesenchymal stem cells not sensitized with ethionamide. These results indicate that the anti-inflammatory effect of stem cells is improved by treating mesenchymal stem cells with ethionamide.

[0089] Example 4. Verification of the effect of ethionamide sensitizing stem cells to inhibit cytokine expression

[0090] Based on the results of Example 1, the inventors of the present invention induced the same inflammatory model as in Example 1 and co-cultured it with mesenchymal stem cells primed or not primed with ethionamide, and then measured the expression levels of representative inflammatory cytokines IL-6 and TNF-α.

[0091] Results, such as Figure 2a and 2b As shown in , compared with the control group or the group co-cultured with mesenchymal stem cells not sensitized with ethionamide, the expression of IL-6 and TNF-α mRNA in the group co-cultured with ethionamide-sensitized stem cells was slightly reduced, and the expression level of IL-6 decreased in proportion to the treatment concentration of ethionamide (100, 150 μM). In addition, as Figure 2b As shown in, the results of measuring TNF-α protein levels by ELISA confirmed that the group co-cultured with mesenchymal stem cells sensitized with ethionamide showed a significant decrease in protein levels. These results indicate that by treating mesenchymal stem cells with ethionamide, the effect of inhibiting the expression of inflammatory cytokines in stem cells is improved.

[0092] Example 5. Verification of the effect of sensitizing stem cells with ethionamide on inhibiting NF-κB activity

[0093] NF-κB (nuclear factor-κB) is considered to be the most important transcription factor involved in the inflammatory mechanism in immune cells, and abnormal NF-κB activation caused by various reasons is reported as a mechanism for the development of various inflammatory diseases (including degenerative brain diseases). Therefore, based on the results of Examples 1 and 2, it is intended to verify whether ethionamide treatment can improve the effect of inhibiting NF-κB activity in mesenchymal stem cells. To this end, similar to Examples 1 and 2, after inducing inflammation, microglia BV2 were co-cultured with mesenchymal stem cells sensitized or not sensitized by ethionamide, and the activity of NF-κB was measured.

[0094] Results, such as Figure 3 As shown in, in the group co-cultured with mesenchymal stem cells sensitized with ethionamide, the expression of NF-κB protein decreased in proportion to the treatment concentration (100, 150 μM) of ethionamide, compared with the control or the group co-cultured with mesenchymal stem cells not sensitized with ethionamide, and the quantitative results obtained by Western blotting confirmed that the expression was significantly reduced compared with the control. These results indicate that the effect of stem cells inhibiting NF-κB activity is improved by treating mesenchymal stem cells with ethionamide.

[0095] Example 6. Verification of the effect of sensitizing stem cells with ethionamide on increasing the expression of paracrine factors

[0096] To verify whether the expression levels of paracrine factors of mesenchymal stem cells were changed by ethionamide treatment, mesenchymal stem cells were treated with 50 or 100 μM ethionamide, and then the expression levels of brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF), which are known paracrine factors, were measured. Figure 4a As shown in , it was confirmed that the expression of paracrine factors increased in proportion to the treatment concentration of ethionamide.

[0097] Subsequently, mesenchymal stem cells were treated with ethionamide using the same method as above, and the mRNA expression levels of antioxidant-related factors such as HO-1 (heme oxygenase-1), NQO1 (NAD(P)H:quinone oxidoreductase), GCLC (glutamate-cysteine ​​ligase catalytic subunit), and GCLM (glutamate-cysteine ​​ligase modifier subunit) were measured. Figure 4b As shown in , it was confirmed that the expression of HO-1, NQO1, GCLC, and GCLM genes in mesenchymal stem cells was significantly increased in proportion to the treated ethionamide concentration.

[0098] These results suggest that treatment of MSCs with ethionamide increases the expression of stem cell paracrine factors.

[0099] Example 7. Confirmation of the effect of ethionamide-sensitized stem cells on reducing amyloid β in a dementia mouse model recognize

[0100] In order to examine whether mesenchymal stem cells sensitized with ethionamide actually have a therapeutic effect on degenerative brain diseases, the present inventors measured the expression level of amyloid β according to the administration of mesenchymal stem cells using a dementia mouse model.

[0101] Specifically, in a dementia mouse model, mesenchymal stem cells (hMSC) primed or not primed with ethionamide were administered to the mouse ventricle, and then the brain was extracted one week after administration. The extracted brain was fixed with 4% paraformaldehyde and then processed to obtain tissue sections, and the tissue sections were treated with anti-6E10, a representative antibody against amyloid β, for immunostaining, and then the tissue was observed with a fluorescence microscope. In addition, the fluorescence intensity was quantified and plotted on the graph.

[0102] Results, such as Figure 5a As shown in , the expression of amyloid β was significantly decreased in the group co-cultured with mesenchymal stem cells sensitized with ethionamide, compared with the control group or the group co-cultured with mesenchymal stem cells not sensitized with ethionamide.

[0103] In addition, using the same dementia mice as above, mesenchymal stem cells (hMSC) sensitized or not sensitized by ethionamide were administered to the ventricles of the mice, and the brain was extracted one week after administration. Subsequently, ELISA was performed using samples obtained from the brain extracted by homogenization to measure the level of amyloid β. Here, tissue samples were separated into soluble and insoluble parts to confirm the level of amyloid β.

[0104] Results, such as Figure 5b As shown in , it was confirmed that the expression of amyloid β was significantly decreased in the group co-cultured with mesenchymal stem cells sensitized with ethionamide, compared with the control group or the group co-cultured with mesenchymal stem cells not sensitized with ethionamide.

[0105] Through the above experiments, it can be seen that stem cells sensitized with ethionamide have the effect of reducing amyloid β, which indicates that stem cells have a therapeutic effect by reducing the symptoms of degenerative brain diseases.

[0106] Example 8. Confirmation of the effect of sensitizing stem cells with ethionamide on alleviating brain disease in a dementia mouse model

[0107] Based on the results obtained in Example 7, it was intended to examine whether stem cells sensitized with ethionamide have the effect of alleviating encephalopathy in a dementia mouse model. Figure 7 In the same method as in the dementia mouse model, mesenchymal stem cells (hMSC) sensitized or not sensitized by ethionamide were administered to the mouse ventricle, and the brain was extracted one week after administration and then fixed with 4% paraformaldehyde. Subsequently, the tissue sections obtained by processing the brain tissue were treated with GFAP (glial fibrillary acidic protein) encephalopathy-related antibodies for immunostaining and then observed using a fluorescence microscope. In addition, the fluorescence intensity was quantified and compared.

[0108] Results, such as Figure 6 As shown in, in the complete dementia mice, GFAP expression was confirmed to be significantly increased compared with wild-type mice (WT). In contrast, when mesenchymal stem cells sensitized or not sensitized with ethionamide were administered, GFAP expression was decreased, and in particular, when mesenchymal stem cells sensitized with ethionamide were administered, GFAP expression was decreased at a higher level.

[0109] From these results, it can be seen that stem cells sensitized by ethionamide have the effect of alleviating encephalopathy.

[0110] Example 9. Confirmation of the tau-lowering effect of ethionamide-sensitized stem cells in a dementia mouse model

[0111] In addition, based on the results of Examples 7 and 8, the inventors of the present invention aim to examine whether the stem cells sensitized by ethionamide according to the present invention have the effect of reducing tau protein, which is another pathological symptom, in a dementia mouse model. Specifically, in a dementia mouse model, mesenchymal stem cells (hMSCs) sensitized or not sensitized by ethionamide were administered to the ventricles of mice, and the brain was extracted two weeks after administration and then homogenized. Then, ELISA was performed using the obtained brain tissue samples to measure the expression level of tau protein. Here, the amount of tau with the 181st and 231st threonine residues of phosphorylated tau protein was confirmed.

[0112] As a result of the analysis, Figure 7 As shown in , the expression of phosphorylated tau was confirmed to be significantly reduced in the group co-cultured with mesenchymal stem cells sensitized with ethionamide, compared with the control group or the group co-cultured with mesenchymal stem cells not sensitized with ethionamide.

[0113] It should be understood by those skilled in the art that the above description of the present invention is exemplary, and that the exemplary embodiments disclosed herein can be easily modified into other specific forms without departing from the technical spirit or basic features of the present invention. Therefore, the above exemplary embodiments should be interpreted as illustrative, rather than limiting in any aspect.

[0114] Industrial Applicability

[0115] It has been confirmed that ethionamide according to the present invention enhances the efficacy of stem cells (such as anti-inflammatory effect and paracrine effect of stem cells), and that stem cells sensitized with ethionamide exhibit therapeutic effects through substantial lesion improvement in a dementia model, and therefore ethionamide and ethionamide-sensitized stem cells enhanced in various efficacy are expected to be used in various fields, including the development of therapeutic agents for inflammatory diseases and degenerative diseases.

Claims

1. Use of stem cells in the preparation of a medicament for preventing or treating dementia, wherein the stem cells are obtained by culturing in a culture medium composition comprising ethionamide, and the expression of paracrine factors contained in the stem cells is increased, and wherein the stem cells are mesenchymal stem cells derived from umbilical cord.

2. The use according to claim 1, wherein the ethionamide is contained in the culture medium at 1 to 200 μM.

3. The use according to claim 1, wherein the paracrine factor is one or more selected from the following: brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), heme oxygenase-1 (HO-1), NAD(P)H:quinoneoxidoreductase (NQO1), glutamate-cysteine ​​ligase catalytic subunit (GCLC) and glutamate-cysteine ​​ligase modifier subunit (GCLM).

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