WO2016048107A1 - Pharmaceutical composition for preventing or treating immune diseases or inflammatory diseases, comprising interferon-gamma or interleukin-1 beta treated stem cell or culture thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating immune diseases and inflammatory diseases, comprising a stem cell or a culture thereof, the stem cell being cultured by adding, to a stem cell, inflammatory cytokines of interferon-gamma and interleukin-1 beta.

Description

Pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases comprising stem cells treated with interferon-gamma or interleukin-1 beta or cultures thereof

The present invention is directed to the prevention of immune diseases and inflammatory diseases, including stem cells cultured by adding cytokines of interferon-gamma (IFN-γ) and / or interleukin-1 beta (IL-1β) to stem cells or their cultures. Or to a pharmaceutical composition for treatment.

The immune system is building a defense system composed of various cells to protect the human body from internal and external pathogens, which is achieved through the specific function of each cell and signal transmission between each cell. The body's immune system can be broadly divided into immune tolerance, which suppresses and regulates immunity, and immune response, which promotes immunity. The two immune functions are balanced to achieve immunological homeostasis. You lose. However, for some reason, their balance is broken, resulting in immune and inflammatory diseases if the immune response is more advanced than immune tolerance.

Immunosuppression for the prevention or treatment of autoimmune diseases or inflammatory diseases include specific inhibitors and nonspecific inhibitors that inhibit only the response, and theoretically, the action of the specific inhibitor should be excellent, but nonspecific inhibitors are mainly used. The most commonly used immunosuppressive agents are cyclosporine (Neoral, Cipol A), azacioprine (imuran) and prednisolone (a type of steroid). The three were found to have fewer side effects and the highest immunosuppressive effects when used together when used separately. Recently, various immunosuppressive agents such as FK 506, RATG, OKT3, and Cellcept have been developed and used.

These immunosuppressive agents inhibit immunosuppression by inhibiting several processes such as phagocytosis of antigen by macrophages, antigen recognition by lymphocytes, cell division, T cell and B cell division, and antibody production. Cause. Most of them have antitumor activity because they inhibit cell division through the mediation of DNA disorders and the inhibition of DNA synthesis.

However, the typical side effects are hypertension and nephrotoxicity (decrease in kidney function), and the high incidence of these side effects has caused problems such as observing the course when used sufficiently. Other side effects rarely include tremor, seizures, hepatitis, biliary retention, increased blood uric acid, decreased muscle strength, hypertrichosis, and gingival hypertrophy. Among the commonly used inhibitors, azachioprine inhibits bone marrow function, such as decreased white blood cell count, anemia, and platelet reduction. In addition, pancreatitis, hepatitis, and biliary retention may rarely cause hair loss and fever. Prednisolone, one of the steroid agents, was the first to be used among immunosuppressants and has the broadest inhibitory action. It may increase appetite, increase muscles in the shoulders and back, and temporarily bring happiness, but these steroids not only promote atherosclerosis, but also cause high blood pressure, gastric ulcers, diabetes, growth inhibition, osteoporosis, cataracts, and glaucoma. The drug to watch out for.

As a therapeutic agent to overcome the side effects, biopharmaceuticals have been in the spotlight, and the representative ones are stem cell therapeutics. Mesenchymal stem cell research has been reported for the application of stem cell differentiation ability to treat damaged joint tissues for the treatment of regeneration and anti-inflammatory (US Pat. No. 6835377), but the mechanism of treatment is clearly identified. The cure is a rare reality.

On the other hand, endotoxins, such as lipopolysaccharide (LPS) is a substance that induces the production of proinflammatory factors in the cell, and promotes the production of inflammatory cytokines (inflammatory cytokines) causing an inflammatory response. That is, when an external stimulus capable of causing an inflammatory response is applied, expression of inflammatory cytokines such as TNF-α is induced, and the generated inflammatory cytokines stimulate expression of genes encoding iNOS and COX-2, Induces an inflammatory response by producing NO and PGE ₂ substances involved in the inflammatory response. However, to date, pretreatment of these inflammatory cytokines to stem cells has yet to be described for the mechanisms that prevent or treat inflammatory or immune diseases by increasing the expression of inflammatory regulators or cell migration-related factors secreted from stem cells. Not known until.

The inventors of the present invention, when cultured (pretreatment) by adding a cytokine such as interferon-gamma or interleukin-1 beta to stem cells, the expression of inflammatory regulators or cell migration-related factors secreted from stem cells significantly increased, and thus The present invention has been completed by confirming that stem cells can be used as a cell therapy for the prevention or treatment of immune diseases or inflammatory diseases.

One object of the present invention is 10 to 20 ng / ml interferon-gamma (IFN-γ) and 2.5 to 10 ng / ml in stem cells, which is economical and free of side effects that can replace conventional immunosuppressive and inflammatory inhibitors. Stem cells or cultures thereof for 12 to 36 hours by adding interleukin-1 beta (IL-1β); Or to provide a pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases comprising the same.

Another object of the present invention is a method for preparing stem cells or cultures thereof, the method comprising culturing by adding inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). It is to provide a method for producing a stem cell or a culture thereof characterized in that the cultivation of from 20 to 20 ng / ml of IFN-γ and 2.5 to 10 ng / ml of IL-1β for 12 to 36 hours.

Another object of the present invention is 1 × 10 ⁶ to 1 × 10 ⁹ stem cells cultured with the addition of inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β); And a composition for single administration of stem cells containing 1 to 100 ml of a culture medium in which stem cells are cultured by treating inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). The culture medium to provide a stem cell treatment effect of two to three times compared to a single dose of stem cells, to provide a composition for administration of stem cells first.

Still another object of the present invention is to provide a composition for single administration of stem cells containing the stem cells 1 × 10 ⁶ to 1 × 10 ⁹ .

Still another object of the present invention is a composition for inhibiting proliferation of mononuclear cells, including the stem cells or cultures thereof; Or it provides a method for inhibiting the proliferation ability of monocytes comprising the step of treating the stem cells or cultures thereof to the tissue to be transplanted.

Another object of the present invention is a composition for increasing the expression or activity of IDO (Indoleamine 2,3-dioxygenase), including the stem cells or cultures thereof; Or treating stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β) to increase the expression or activity of IDO. It is.

Still another object of the present invention is a composition for inhibiting differentiation of CD4 + T cells into Th1 cells or Th17 cells, including the stem cells or cultures thereof; Or Th4 of CD4 + T cells comprising treating CD4 + T cells with stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). It is to provide a method for inhibiting differentiation into 1 cell or Th17 cell.

Another object of the present invention is a composition for increasing the migration or recruitment of CD4 + T cells comprising the stem cells or cultures thereof; Or migrating CD4 + T cells, comprising treating CD4 + T cells with stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). It provides a way to increase migration or recruitment.

Still another object of the present invention is a method of targeting stem cells to secondary lymphoid organs (SLO), wherein the stem cells contain inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). Culturing by addition; And administering the stem cells, wherein the stem cells targeted to the secondary lymphoid organs are CCR7 compared to stem cells not treated with interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). (CC-chemokine receptor type 7) A method characterized by increased gene expression.

Another object of the present invention is an inflammatory cytokine of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β), which is administered immediately after the onset of the acute graft-versus-host disease or within 5 days of onset. Pharmaceutical composition for treating acute graft-versus-host disease containing stem cells or cultures thereof added thereto; Or stem cells or cultures thereof cultured with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) immediately after onset or within 5 days of onset of acute graft-versus-host disease. It provides a method for treating acute graft-versus-host disease, comprising the step of administering.

Still another object of the present invention is to prevent or treat Crohn's disease, which contains stem cells cultured with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) or cultures thereof. It is to provide a pharmaceutical composition.

Still another object of the present invention is to provide an IDO-mediated immune disease, comprising stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β). It is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

Still another object of the present invention is to provide a method for preparing an immunosuppressant or anti-inflammatory agent, comprising culturing by adding interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β) to stem cells.

Another object of the present invention is to incubate stem cells in medium containing inflammatory cytokines COX-2 (Cyclooxygenase-2), IDO (Indoleamine 2,3-dioxygenase), PGE ₂ (Prostaglandin E2) and CCR7 It provides a method for mass production of one or more proteins selected from the group consisting of (CC-chemokine receptor type 7).

Stem cells or cultures thereof according to the present invention can be used for the prevention or treatment of immune diseases and inflammatory diseases. In addition, stem cells according to the present invention can be produced in a simple, economical and improved yield without chemical processing factors or factors related to cell migration. Stem cells or cultures thereof according to the present invention is a cell therapy which can be used economically without side effects and replaces existing immunosuppressive agents and inflammatory inhibitors with known side effects such as autoimmune diseases such as Clones disease, rheumatoid arthritis, atopy, etc. It can be used for the prevention or treatment of diseases and inflammatory diseases.

Figure 1a is a diagram showing the expression level of the immunoregulatory factor COX-2 mRNA when complex treatment of cord blood-derived mesenchymal stem cells IFN-γ, IL-1β, TNF-α or IL-2, respectively.

Figure 1b is a diagram showing the expression level of the immunoregulatory factor PGE ₂ when the combination of umbilical cord blood-derived mesenchymal stem cells IFN-γ, IL-1β, TNF-α or IL-2, respectively.

Figure 1c is a diagram showing the immunomodulator IDO activity level when the combination of cord blood-derived mesenchymal stem cells IFN-γ, IL-1β, TNF-α or IL-2, respectively.

1D is a diagram showing the proliferation level of human mononuclear cells when mixed lymphocyte reaction was performed after complex treatment of umbilical cord blood-derived mesenchymal stem cells with IFN-γ, IL-1β, TNF-α or IL-2, respectively. .

Figure 2a is a diagram showing the expression level of mRNA of IDO over each time after treatment with 20ng / ml of IFN-γ and 10ng / ml of IL-1β for 3, 6, 12, 24 hours simultaneously.

Figure 2b is a diagram showing the expression level of PGE ₂ according to each time after treatment with IFN-γ 20ng / ml and IL-1β 10ng / ml for 3, 6, 12, 24 hours at the same time.

Figure 3a is a diagram showing the expression level of mRNA of COX-2 according to each concentration after treatment with IFN- γ 5, 10, 20ng / ml and IL-1β 2.5, 5, 10ng / ml for 24 hours at the same time to be.

Figure 3b is a diagram showing the expression level of mRNA of IDO according to each concentration after treatment with 5 times, IFN- γ 5, 10, 20ng / ml and IL-1β 2.5, 5, 10ng / ml to the stem cells for 24 hours at the same time.

Figure 4 is a diagram showing the results of measuring the expression level of the immunoregulatory factors PGE ₂ and NOD2 of mesenchymal stem cells isolated from different tissues (bone marrow, fat, umbilical cord blood) of human.

Figure 5a is a diagram showing the expression level of PGE ₂ after inhibiting each factor using 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE ₂ inhibitor.

Figure 5b is a diagram showing the expression level of IDO after inhibiting each factor using 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE ₂ inhibitor.

Figure 5c is a diagram showing the level of monocyte proliferation after inhibition of each factor using 1-Methyltryptophan (1-MT) as the IDO inhibitor and Indomethacin as the PGE ₂ inhibitor.

Figure 5d is a diagram showing the expression level of IL-17 after inhibiting each factor using 1-Methyltryptophan (1-MT) as the IDO inhibitor and Indomethacin as the PGE ₂ inhibitor.

Figure 6 shows the differentiation of IFN-γ and Th17 cells secreted during the differentiation process to Th1 cells in the experiment to confirm the effect of differentiation of Th4 cells or Th17 cells of CD4 + T cells co-cultured with mesenchymal stem cells Figure showing the secretion amount of interleukin-17 (IL-17) secreted in the process.

Figure 7 is a diagram showing the results of co-culture by fluorescent staining of mesenchymal stem cells and CD4 + T cells cultured by pretreatment with IFN-γ or / and IL-1β.

8 is a diagram showing the mobility of CD4 + T cells with respect to the culture medium secreted from the cells through the culture for 3 days after replacing the mesenchymal stem cells pre-treated with IFN-γ or / and IL-1β with fresh medium.

9 is a diagram showing the expression level of CCR7 with each time after treatment with IFN-γ and / or IL-1β for 6, 12, 24 hours.

FIG. 10 is a diagram illustrating a manufacturing process of an animal model of acute graft-versus-host disease, that is, a process of inducing acute graft-versus-host disease and administering stem cells pretreated with IFN-γ and / or IL-1β.

FIG. 11 is a diagram showing the change in survival rate following administration of mesenchymal stem cells pretreated with IFN-γ and / or IL-1β in an acute graft-versus-host disease animal model.

12 is a diagram showing the change in GVHD score according to the administration of mesenchymal stem cells pretreated with IFN-γ and / or IL-1β in acute graft-versus-host disease animal model.

13A-13D show IFN-γ and / or IL-1β in skin tissue (a), liver tissue (b), small intestine tissue (c) and their total tissue (d), respectively, of the acute graft-versus-host disease animal model. The therapeutic effect of pretreatment with mesenchymal stem cells is shown through histopathological evaluation.

14 is a diagram showing changes in survival rate according to administration of mesenchymal stem cells pretreated with IFN-γ and / or IL-1β in Crohn's disease animal model.

FIG. 15 is a diagram showing the effect of weight loss upon administration of mesenchymal stem cells pretreated with IFN-γ and / or IL-1β to Crohn's disease animal model.

As one aspect for achieving the above object, the present invention is a stem cell cultured by treating the inflammatory cytokine in the stem cell or its culture; Or it provides a pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases comprising the same.

In the present invention, when inflammatory cytokines, specifically interferon-gamma (IFN-γ), interleukin-1 beta (IL-1β), or both of these factors are treated to stem cells, inflammatory cell inhibitors secreted from the stem cells Expression of (COX-2, PGE ₂ , IDO) and factors related to cell migration (migration) (CCR7) was increased, thereby regulating immunity and inflammation. In addition, it was confirmed that the stem cells pre-treated with the two cytokines have a therapeutic effect when administered to graft-versus-host disease or Crohn's disease animal model. That is, the present invention confirms that the stem cells treated with inflammatory cytokines and their cultures can be used as cell therapy for immunoregulation and inflammation control, stem cells cultured by adding inflammatory cytokines to stem cells or It can be used as a pharmaceutical composition for the prevention or treatment of immune diseases or inflammatory diseases comprising the culture.

In the present invention, "proinflammatory cytokine" is a generic name for cytokines that cause inflammatory reactions occurring in the body, typically tumor necrosis factor-alpha (TNF-α), interferons (IFNs), Interleukins (ILs), monocyte chemoattractant protein, and the like. In the present invention, the inflammatory cytokine may be IFN-γ and / or IL-1β, but is not limited thereto.

In the present invention, "adding and culturing" an inflammatory cytokine may be, for example, by culturing by adding the inflammatory cytokine to a medium of stem cells. The cytokine is specifically 0.1 to 100 ng / ml , 1 to 50 ng / ml, 2 to 25 ng / ml, 2.5 to 20 ng / ml, or 5 to 10 ng / ml. For example, IFN-γ may be treated at 10 to 20 ng / ml, and IL-1β at 2.5 to 10 ng / ml. When the concentration of the treated cytokine is lower than the concentration, the secretion of immunomodulators secreted from stem cells such as IDO and PGE ₂ may not be sufficient, resulting in a slight or no increase in immunomodulatory capacity. On the other hand, when the concentration of the treated cytokine is higher than the concentration, there is a disadvantage that it may affect the characteristics of the cells, thereby promoting aging of stem cells, suppression of proliferation, reduction of differentiation capacity, and the like.

In addition, the treatment (culture) time of these cytokines can be incubated for 0.5 to 96 hours, 2 to 72 hours, 3 to 48 hours, 6 to 36 hours, 12 to 36 hours, or 12 to 24 hours. If the cytokine treatment time is shorter than the above, the secretion of immunomodulators secreted from stem cells such as IDO and PGE ₂ may not be sufficient, resulting in a slight or no increase in immunomodulatory capacity. On the other hand, if the treatment time of the cytokine is longer than the above, there is a disadvantage in that it may affect the characteristics of the cells, thereby promoting aging of stem cells, suppressing proliferation, and decreasing the differentiation ability.

Preferably, the stem cells of the present invention may be cultured 10 to 20 ng / ml of IFN-γ and 2.5 to 10 ng / ml of IL-1β for 12 to 36 hours.

In addition, after culturing by adding inflammatory cytokines, it may be to culture by replacing the medium.

In the present invention, "stem cells" may be cells having the ability to differentiate into various tissues, that is, 'undifferentiated cells'.

In the present invention, the stem cells are human adult stem cells, human pluripotent stem cells, human pluripotent stem cells, induced pluripotent stem cells (induced pluripotent stem cells), animal embryonic stem cells or animal adult stem cells Can be.

Multipotent stem cells are multipotent stem cells derived from various adult cells such as bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells), and the like. For example, various studies have been conducted for the development of mesenchymal stem cells derived from bone marrow as cell therapeutics due to the differentiation that can be differentiated into adipose tissue, bone / cartilage tissue, and muscle tissue.

Meanwhile, adult stem cells may be mesenchymal stem cells, mesenchymal stromal cells derived from human tissues, mesenchymal stem cells derived from human tissues, multipotent stem cells, or amniotic epithelial cells, and mesenchymal stem cells may be umbilical cord cells. , Umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membrane and placenta can be derived from mesenchymal stem cells (mesenchymal stem cells), in particular, may be derived from human, especially human umbilical cord blood It may be derived from Human Umbilical Cord Blood Mesenchymal Stem Cell (hUCB-MSC). In the present invention, it was confirmed that the expression level of immune and inflammatory regulators (COX-2, PGE ₂ ) is significantly higher in cord blood-derived mesenchymal stem cells derived from humans than in other tissues (FIG. 4). The method of obtaining stem cells from each of the derivatives may be by a method known in the art, and is not limited to the method of the embodiment of the present invention.

In the present invention, "culture" may include both cell culture medium containing stem cells, culture supernatants from which stem cells are removed from the cell culture solution, and dilutions thereof. The composition of the culture may further include not only components necessary for conventional stem cell culture, but also components that synergistically act on stem cell proliferation, and thus the composition may be easily carried out by those skilled in the art. Can be selected.

In addition, when preparing a stem cell or a culture thereof according to the present invention, IFN-γ and IL-1β may be added and cultured at the same time, or after the addition of IFN-γ, IL-1β may be added, or after IL-1β addition. It is also possible to culture by adding -γ, and the order of addition is not limited.

As a medium used for culturing the stem cells, a conventional medium known in the art to be suitable for culturing stem cells may be used. For example, DMEM (Dulbecco's modified Eagle medium) or Keratinocyte-SFM (Keratinocyte serum free medium) may be used. Can be used.

The stem cell medium may be supplemented with an additive. Generally, it may contain neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt) Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as β-mercaptoethanol.

According to an embodiment of the present invention, adult stem cells treated with IFN-γ and / or IL-1β significantly increase the expression of COX-2, IDO, PGE ₂ and CCR7, which are known as inflammatory regulators or cell migration factors. Confirmed. In particular, the immune suppression effect of IFN-γ and / or IL-1β treatment in the present invention was confirmed that the main effect of the mixed lymphocyte inhibitory effect by increasing the expression of IDO (Fig. 5c).

Thus, the stem cells of the present invention or the culture thereof has a mononuclear cell proliferation inhibitory effect according to the mononuclear cell proliferation inhibitory composition comprising the same, or the step of treating the stem cells or the culture of the present invention to the tissue to be transplanted It is possible to provide a method of inhibiting the proliferative ability of a mononuclear cell containing (Example 6).

In addition, since the stem cells of the present invention or the culture thereof have an activity of inhibiting the differentiation of CD4 + T cells into Th 1 cells or Th17 cells, interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) Stem cells cultured with the addition of inflammatory cytokines or their cultures are in the form of a composition for inhibiting differentiation of CD4 + T cells into Th 1 cells or Th17 cells, or the stem cells or their cultures as CD4 + T cells It can be provided in the form of a method for inhibiting the differentiation of CD4 + T cells into Th 1 cells or Th 17 cells comprising the step of (Example 7).

In addition, since the stem cells of the present invention or cultures thereof have an activity of increasing migration or recruitment of CD4 + T cells, interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β). Stem cells or culture thereof added with an inflammatory cytokine of) is in the form of a composition for increasing migration or recruitment of CD4 + T cells, or treating the stem cells or cultures thereof with CD4 + T cells. It may be provided in the form of a method for increasing the migration (migration) or recruitment (recruitment) of CD4 + T cells comprising a,

On the other hand, the present invention is a method for targeting stem cells to secondary lymphoid organs (SLO), the stem cells by adding inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) Culturing; And administering the stem cells, wherein the stem cells targeted to the secondary lymphoid organs are CCR7 compared to stem cells not treated with interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). (CC-chemokine receptor type 7) A method characterized by increased gene expression can be provided (Example 8). Stem cells that are targeted may include artificially genetically engineered.

On the other hand, stem cells cultured by the addition of the inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) of the present invention or cultures thereof, for increasing the expression or activity of IDO It may be provided in the form of a composition or in the form of a method of increasing the expression or activity of IDO by treating the stem cells or cultures thereof (Example 5).

Therefore, the stem cells and the cultures thereof according to the present invention are useful for the prevention and treatment of immune diseases or inflammatory diseases, particularly IDO-mediated immune diseases or inflammatory diseases. Here, the IDO-mediated disease refers to an immune disease or an inflammatory disease that may occur due to the inability to normally express an IDO gene or to secrete a protein thereof in an immune or inflammation-related mechanism.

In the present invention, "inflammatory disease" is a generic term for inflammation as the main lesion, but is not limited thereto, specifically, swelling, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, Gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, spondylitis spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome or multiple sclerosis.

In the present invention, "immune disease" is a disease that is a problem when a specific immune response occurs, but is not limited thereto, and specifically, may be autoimmune disease, transplant rejection, and graft-versus-host disease.

The immune disease or inflammatory disease may be autoimmune disease, transplant rejection, graft-versus-host disease, arthritis, bacterial infection, sepsis or inflammation, and the autoimmune disease is Crohn's disease, erythema disease, atopic dermatitis, rheumatoid arthritis, Hashimoto's thyroiditis , Pernicious anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hypertension, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome , Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, epithelial scleroderma, asthma or ulcerative colitis.

In the present invention, "prevention" is any action that inhibits or delays the onset of an immune disease or inflammatory disease by administration of the composition, and "treatment" is any improvement or benefit of the symptoms of an immune disease or inflammatory disease by administration of the composition. It is an act.

In addition, the stem cells or cultures thereof according to the present invention is 1.0 × 10 ⁵ to 1.0 × 10 ⁹ , specifically 1.0 × 10 ⁶ to 1.0 × 10 ⁸ , more specifically 1.0 × 10 ⁷ per ml. Cells may be included.

Stem cells or cultures thereof according to the invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife cell freezing medium (Cascade Biologics)) can be added to the cell population before freezing.

In addition, the stem cells or cultures thereof according to the present invention may be administered in a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, the formulation may be administered once or several times. Dosages include effective dosages. Suitable formulations for this purpose are parenteral formulations such as injections such as ampoules for injection, injections such as infusion bags, and sprays such as aerosol formulations. The injection ampoule may be mixed with the injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution. The infusion bag may also be made of polyvinyl chloride or polyethylene and may be Baxter, Becton Dickinson, Medcep, National Hospital Products or Terumo. The injection bag of the yarn can be illustrated.

In the pharmaceutical preparations, in addition to the active ingredient, one or more pharmaceutically acceptable inert carriers such as preservatives, analgesic agents, solubilizers or stabilizers in the case of injections, etc. It may further include a base, excipients, lubricants or preservatives.

The stem cells, cultures or pharmaceutical preparations thereof according to the present invention thus prepared are prepared with or in combination with other stem cells used for transplantation and other uses using administration methods commonly used in the art. It may be administered in the form, specifically, it is possible to engraft or implant directly to the disease site of the patient in need of treatment, or to implant or inject directly into the abdominal cavity, but is not limited thereto. In addition, the administration can be both non-surgical administration using a catheter and surgical administration such as injection or transplantation after dissection of the disease site, but non-surgical administration using a catheter is more appropriate. In addition to parenteral administration, for example, in addition to direct lesions, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible according to a conventional method.

The single dose of the stem cells is 1.0 × 10 ⁴ to 1.0 × 10 ¹⁰ cells / kg body weight, specifically 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cells / kg body weight, more specifically 1.0 × 10 ⁶ to 1.0 × 10 ⁸ cells / kg body weight may be administered once or in several doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore the dosage may It does not limit the scope of the present invention in terms of aspects.

In particular, one containing 1.0 × 10 ⁶ to 1.0 × 10 ⁹ stem cells cultured with the addition of inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) of the present invention It may be provided in a dosage.

In addition, when containing not only the stem cell but also its culture solution, as a single dose, 1.0 × incubated with the addition of inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) 10 ⁶ to 1.0 × 10 ⁹ stem cells; And 1 to 100 ml of a culture medium in which stem cells are cultured by treating inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β).

That is, when the stem cell culture is included in the means of administration, such as a cell therapy agent, the number or amount of stem cells required to have an equivalent effect is remarkably at the level of 1/2 to 1/10, 1/2 to 1/3. Reduced, which may have a much more economical effect on formulation (Example 9).

In addition, with respect to the time of administration, the stem cells or cultures thereof may be treated immediately after the onset of the disease to be treated, such as an immune disease or an inflammatory disease, in particular, an inflammatory bowel disease such as acute graft-versus-host disease or Crohn's disease. It can be administered within 5 days, 3 days, 2 days or 1 day. In an embodiment of the present invention, interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β) were treated within 3 days and 1 day from the induction of each disease in an animal model of acute graft-versus-host and Crohn's disease. As a result of administration of stem cells or cultures thereof, it was confirmed that the symptoms of these lesions showed improved therapeutic activity (Examples 9-1 and 10-1). This is expected to be related to the significantly increased expression of inflammatory regulators 12 to 24 hours after treatment with interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β).

In another embodiment, the inflammatory cytokine-treated stem cells according to the present invention can be inhibited by administering a stem cell and its culture, and the inflammation can be controlled. The present invention provides a culture by adding inflammatory cytokines to stem cells. Provided are a method for inhibiting an immune response or an inflammatory response of a subject or a method for preventing or treating an immune disease or an inflammatory disease, comprising administering one stem cell or a culture thereof to the subject.

As used herein, "subject" or "individual" includes, but is not limited to, mammals including cattle, dogs, pigs, chickens, sheep, horses, and humans. In addition, specifically, administration of stem cells or cultures thereof added with inflammatory cytokines may be intraperitoneal or vascular administration, direct administration to a lesion, or administration in a synovial cavity.

Inhibition of the immune response or inflammatory response may be characterized in that the prevention or treatment of immune diseases or inflammatory diseases.

As another aspect, the present invention provides a method for preparing an immunosuppressant or anti-inflammatory agent, comprising culturing by adding inflammatory cytokines to stem cells. The immunosuppressant or anti-inflammatory agent may be in the form of a cell therapy.

In the present invention, as described above, an "immunosuppressant" is an agent capable of treating an immune disease by inhibiting an immune response with an agent comprising a stem cell or a culture obtained by adding an inflammatory cytokine to the stem cell and culturing the stem cell. You can be the best.

In the present invention, as described above, "an anti-inflammatory agent" may be an agent capable of treating inflammatory diseases by inhibiting inflammation with a stem cell obtained by culturing by adding inflammatory cytokines to stem cells or an agent comprising the culture thereof. have.

As another aspect, the present invention includes the step of culturing stem cells in medium containing inflammatory cytokines, COX-2 (Cyclooxygenase-) characterized in that the expression of inflammatory regulators in the stem cells is increased during the culture 2), a mass production method of one or more proteins selected from the group consisting of IDO (Indoleamine 2,3-dioxygenase), PGE ₂ (Prostaglandin E2) and CCR7 (CC-chemokine receptor type 7).

In the present invention, the recovery of the protein can be obtained by collecting the culture medium of the stem cells, centrifugation and filtration to remove the cells and suspended solids, and recovering the remaining supernatant.

In another aspect, the present invention provides an implant comprising a stem cell and an inflammatory cytokine.

In the present invention, a "graft" is a substance that can be transplanted into a human body or an animal, which isolates the damaged site from the outside or contains transplanted cells or secreted therapeutic material. Such implants may be provided with various materials used in the art such as synthetic polymers and natural materials having biodegradability as a support for tissue engineering. Since the implant of the present invention contains stem cells and inflammatory cytokines, there is an advantage of not causing an immune rejection reaction or an inflammatory response due to the implant. Therefore, even without the administration of a separate immune rejection inhibitor or anti-inflammatory agent to suppress the immune rejection or inflammatory response that may occur when various implants are implanted in the body, the transplanted implant is stable without the immune rejection reaction or inflammation in vivo It can engulf as.

In another aspect, the present invention provides a transplant in which the stem cells are removed after culturing stem cells by adding inflammatory cytokines on a support.

As another aspect, the present invention provides a method for producing an implant comprising culturing by adding inflammatory cytokines to stem cells on a support.

Specifically, the method may further include removing stem cells after the culturing step.

As another aspect, the present invention is a stem cell; And a cytokine of 10-20 ng / ml interferon-gamma (IFN-γ) and 2.5-10 ng / ml interleukin-1 beta (IL-1β). The complex can be used as a cell therapy.

As another embodiment, the present invention is added to the stem cells by adding a cytokine of 10 to 20 ng / ml of interferon-gamma (IFN-γ) and 2.5 to 10 ng / ml of interleukin-1 beta (IL-1β) It provides a cultured stem cell or a culture thereof.

The culture may exert an effect capable of preventing or treating an immune disease or an inflammatory disease, and an inflammation regulator or cell migration-related factors such as COX-2, IDO, PGE ₂ and CCR7 may be present.

In another aspect, the present invention provides a 10 to 20 ng / ml interferon-gamma (IFN-γ) and 2.5 to 10 ng in stem cells for the preparation of a pharmaceutical composition for the prevention or treatment of immune or inflammatory diseases Provided is the use of stem cells or cultures thereof cultured with the addition of / ml cytokine of interleukin-1 beta (IL-1β).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Preparation Example: Isolation of Mesenchymal Stem Cells from Umbilical Cord Blood

Umbilical cord blood was mixed with Hetasep ^™ solution at a ratio of 5: 1 and left at room temperature for 40 minutes. Then, the supernatant was carefully placed on Ficoll and centrifuged at 2500 rpm for 20 minutes. After centrifugation, the monocyte layer was collected, washed twice with PBS, and coated with fibronectin at a concentration of 2 × 10 ⁵ to 2 × 10 ⁶ cells / cm ² using EGM-2 medium containing 20% fetal bovine serum. Seeded on. After 3 days, the non-adherent cells were removed and colony formation was observed by replacing the medium every 2-3 days. The colonies thus formed were passaged to obtain mesenchymal stem cells. As a result of observing expression markers using FACS on the obtained mesenchymal stem cells, the mesenchymal stem cells were characterized by expressing ZNF281 (zinc finger protein 281) in addition to the markers of normal mesenchymal stem cells.

Example 1 Analysis of Expression Level of Immunomodulators According to Inflammatory Cytokine Treatment

In order to confirm whether the immunosuppressive effect or anti-inflammatory effect of stem cells by the pretreatment of four inflammatory cytokines such as IFN-γ, IL-1β, TNF-α and IL-2 to stem cells, The expression level of the immunoregulatory factors COX-2 and IDO mRNA in the combination of IFN-γ, IL-1β, IL-2 or TNF-α in cord blood-derived mesenchymal stem cells, respectively, was measured by real-time RT-PCR. Comparative analysis using Specifically, 20 ng / ml of IFN-γ, 5 ng / ml of IL-1β, 20 pg / ml of IL-2 or 20 ng / ml of TNF- in the culture medium of cord blood-derived mesenchymal stem cells of the above preparation After the addition of α, the mixture was incubated for 24 hours.

Then, expression levels of mRNA of COX-2 and IDO in cultured mesenchymal stem cells were analyzed using real-time RT-PCR. Primer sequences for each gene used for this are shown in Table 1 below.

gene	direction	Primer sequence	SEQ ID NO:
COX-2	Forward direction	5'-TGAGCATCTACGGTTTGCTG-3 '	SEQ ID NO: 1
	Reverse	5'-TGCTTGTCTGGAACAACTGC-3 '	SEQ ID NO: 2
IDO	Forward direction	5'-ACTGTGTCCTGGCAAACTGGAAG-3 '	SEQ ID NO: 3
	Reverse	5'-AAGCTGCGATTTCCACCAATAGAG-3 '	SEQ ID NO: 4

In addition, comparative analysis was performed using ELISA to determine the expression level of PGE ₂ . IDO converts L-tryptophan to N-formylkynurenine, which is produced by hydrolysis by reaction with 30% (wt / vol) trichloroacetic acid. The amount of secretion of IDO, ie the activity of IDO, was determined by measuring the amount of kynurenine.

Furthermore, mixed lymphocyte reaction was performed to confirm the proliferation level of human mononuclear cells (hMNC) obtained through primary culture. The mixed lymphocyte reaction is described in Example 5 below.

As a result, COX-2, a representative immunomodulator, showed higher expression level when combined with IL-1β compared to other cytokines (FIG. 1A), and PGE ₂ , an immunomodulator regulated by COX- ₂ When confirming the expression level of was also confirmed that the secreted to the highest level when combined with IL-1β (Fig. 1b). In addition, the activity of another immunomodulator, IDO, was also highest when combined with IL-1β (FIG. 1C), which was combined with IFN-γ and IL-1β to increase the immunomodulatory capacity of mesenchymal stem cells. By pretreatment, it was confirmed that these can be further maximized at the RNA and protein levels. In addition, as a result of confirming the inhibition of the proliferation capacity of mononuclear cells through co-culture with mixed lymphocytes, it was confirmed that the effect of further inhibiting the proliferative capacity under complex treatment conditions by the combination of IFN-γ and IL-1β (Fig. 1D).

By maximizing the expression of immune regulators COX-2 and IDO from stem cells through pretreatment of stem cells with IFN-γ and IL-1β, they can be used as cell therapeutics with enhanced immunosuppressive and anti-inflammatory effects. It suggests that there is.

Example  2: IFN -γ and IL- 1β  According to processing time Immunomodulatory factors  Expression level analysis

In order to confirm whether the immunosuppressive effect or anti-inflammatory effect of stem cells according to the treatment time of inflammatory cytokines of IFN-γ and IL-1β to cord blood-derived mesenchymal stem cells obtained in the above preparation, IFN- γ 20ng / ml and IL-1β 10ng / ml were treated simultaneously with stem cells for 3, 6, 12, and 24 hours, and then the expression levels of mRNA of IDO were compared using real-time RT-PCR. In addition, comparative analysis was performed using ELISA to determine the expression level of PGE ₂ .

As a result, it was confirmed that the expression level of IDO showed the highest expression level 24 hours after treatment (FIG. 2A). On the other hand, the expression level of PGE ₂ was confirmed to show the highest expression level 12 hours after treatment (Fig. 2b). This resulted in the highest amount of PGE ₂ secreted in IFN-γ and IL-1β pretreated stem cells at 12 hours, and when treated for more than 12 hours, the secretion could be lowered. The longer the hour, the more the secretion increases in time within 24 hours.

Example  3: IFN -γ and IL- 1β  Treatment concentration Immunomodulatory factors  Expression level analysis

In order to confirm whether the immunosuppressive effect or anti-inflammatory effect of stem cells according to the concentration of inflammatory cytokines of IFN-γ and IL-1β in the cord blood-derived mesenchymal stem cells obtained in the preparation example, IFN- Simultaneous treatment of γ 5, 10, 20ng / ml and IL-1β 2.5, 5, 10ng / ml with stem cells for 24 hours was followed by real-time RT-PCR expression of mRNA levels of COX-2 and IDO mRNA. Comparative analysis.

As a result, it was confirmed that the expression level of COX-2 and IDO showed the highest expression level in the IFN-γ of 20ng / ml and the complex treatment group of 2.5 or 5ng / ml (FIGS. 3A and 3B).

Example 4 Analysis of Expression Levels of Immune Regulators According to Stem Cell-Derived Tissues

In order to analyze the expression level of immunomodulators according to stem cell-derived tissues, mesenchymal stem cells were isolated from different tissues (bone marrow, fat, umbilical cord blood) of human.

Cord blood-derived mesenchymal stem cells were obtained by the method described in Preparation Example, and bone marrow-derived mesenchymal stem cells were distributed from Yonsei University College of Medicine Severance Hospital Cell Therapy Center and used. In addition, fat-derived mesenchymal stem cells are described in Ji-Won Jung et al , Cell Mol Life Sci. 2010 Apr; 67 (7): 1165-1176. Obtained by a method known in the art.

Expression levels of immunomodulators such as "PGE ₂ " and "NOD2" in these mesenchymal stem cells were measured by ELISA and western blot, respectively. Expression of these factors in cord blood derived stem cells was confirmed to be significantly high (Fig. 4). Therefore, it is suggested that stem cells derived from other tissues secrete immunomodulators or inhibit immune cells to a higher level when treated with umbilical cord blood-derived stem cells than when treated with inflammatory cytokines.

Example  5: IFN -γ and IL- 1β  Stem Cells by Concurrent Treatment Immunosuppressive ability  Enhancement analysis

Treatment of these inhibitors with mesenchymal stem cells to determine which factors of IDO and PGE ₂ are involved in stem cell immune regulation through the IFN-γ and IL-1β treatment. After the expression was reduced, 20 ng / ml of IFN-γ and / or 10 ng / ml of IL-1β were added to the culture and analyzed for mixed lymphocyte reaction after 24 hours. In addition, 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE ₂ inhibitor were used to inhibit respective cytokines and mixed lymphocyte reactions were confirmed (FIGS. 5A and 5B). Specifically, 100 μM 1-MT or 20 μM Indometacin was treated with stem cells for 24 hours to inhibit the expression or activity of each gene, and mixed lymphocyte reaction proceeded as follows.

Mesenchymal stem cells (hMSC) obtained in the above preparations were seeded in 6-well plates at a concentration of 3 × 10 ⁵ cells / well, and after 24 hours, 2 μM of 5-aza was treated for 24 hours. For direct coculture, the mesenchymal stem cells were treated with 25 mg / ml mitomycin C, and the cells were plated in 96-well plates at a concentration of 1 × 10 ⁴ cells / well. Monocytes (MNC) were activated using Concanavalin A (ConA) and seeded onto plates at a concentration of 5 × 10 ⁴ cells / well.

For indirect coculture, transwells (24-well plates, Corning, Corning, NY) with pores of 0.4 μm size were used. Monocytes (MNC) were activated using ConA and plated at a concentration of 1 × 10 ⁶ cells / well in the lower chamber. Mesenchymal stem cells treated with 5-aza were seeded at a concentration of 1 × 10 ⁵ cells / well in the upper chamber. After 3 days, the proliferation rate of monocytes was analyzed using BrdU assay (Bromodeoxyuridine kit, Roche, Upper Bavaria, Germany).

As a result, IFN-γ-treated mesenchymal stem cells showed an effect of further inhibiting the mixed lymphocyte response regardless of the simultaneous treatment of IL-1β (Fig. 5c). In addition, the inhibitory effect was reduced to the control level when treated with IDO inhibitor 1-MT, while the inhibitory effect was partially reduced when treated with Indomethacin, an inhibitor of PGE ₂ (FIG. 5C).

This suggests that the mixed lymphocyte inhibitory effect through IFN-γ and / or IL-1β treatment is related to both factors of IDO and PGE ₂ , but that the mixed lymphocyte inhibitory effect by IDO increase is the main mechanism (FIG. 5C). This trend was confirmed to be the same in the inhibitory effect on Th17 cells (FIG. 5D).

Example  6: IFN -γ and IL- 1β  Stem Cells by Concurrent Treatment Th1 Of Th17  Immunosuppressive assay

In order to confirm the immunosuppressive ability of the IFN-γ and / or IL-1β-treated stem cells to the differentiation of Th4 or Th17 cells of CD4 + T cells, 20 ng / ml of IFN-γ and 5 ng / Co-culture of CD4 + T cells isolated from umbilical cord blood-derived mesenchymal stem cells and mononuclear cells (MNCs) treated with 24 ml of IL-1β at 37 ° C and 5% CO ₂ incubation by MACs sorting It was.

In the lower chamber of the co-culture plate, which is a 24-well plate, 1 × 10 ⁶ sorted CD4 + T cells were placed in the upper chamber, and the mesenchymal stem cells of each culture condition were placed in the upper chamber. At this time, the lower chamber is equipped with a composition of a suitable medium so that CD4 + T cells are differentiated into Th1 cells or Th17 cells, and then cultured for 4 days for Th1 cells and 7 days for Th17 cells, and then recovered the culture medium to measure the secreted factors. The differentiation of T cells was confirmed. The composition of each culture medium is shown in Table 2 below.

Differentiated cells	Badge composition
Th1	5 × 10 5 CD3 / 28 beads, rIL-2 (2.78 pg / ml), rIL-12 (2.5 ng / ml), anti-IL-4 (5 μg / ml)
Th17	5 × 10 5 CD3 / 28 beads, IL-1β (20 ng / ml), IL-6 (30 ng / ml), IL-23 (30 ng / ml), TGF-β1 (2.25 ng / ml), anti -IFN-γ (1 μg / ml), anti-IL-4 (2.5 μg / ml)

Then, in order to confirm the inhibitory effect of differentiation of CD4 + T cells into Th1 cells (after 6 days of co-culture) or Th17 cells, IFN-γ secreted in the differentiation process to Th1 cells and in the differentiation process to Th17 cells The amount of secreted interleukin-17 (IL-17) was measured.

As a result, IFN-γ and / or IL-1β-treated mesenchymal stem cells and CD4 + T cells were pre-treated with mesenchymal stem cells and CD4 + T cells pretreated with IFN-γ and / or IL-1β. When co-cultured, it was confirmed that the secretion amount of IFN-γ and IL-17 was further reduced, so that the effect of inhibiting the differentiation of CD4 + T cells into Th1 cells and Th17 cells was better (FIG. 6).

Example 7: Change in T Cell Mobility by Stem Cells Treated with IFN-γ and IL-1β

In order to evaluate the effect of the IFN-γ and / or IL-1β-treated stem cells on the migration of CD4 + T cells, mesenchymal stem cells and monocytes cultured in culture medium containing the two cytokines ( CD4 + T cells isolated from MNC) were co-cultured for 4 hours by fluorescent staining.

As a result, the mesenchymal stem cells pre-treated with the IFN-γ and / or IL-1β were confirmed to recruit more CD4 + T cells than the mesenchymal stem cells cultured in the non-treated medium. (FIG. 7).

In addition, after replacing the two cytokine pretreated mesenchymal stem cells with fresh medium containing no cytokines, the mobility of CD4 + T cells with respect to the culture medium secreted from the cells was evaluated by the same method as above. Mobility was obtained by obtaining three or more images taken at random and counting the number of fluorescent cells by calculating the average value.

As a result, it was confirmed that the number of CD4 + T cells migrated into the culture medium of the two cytokines pretreated (Fig. 8).

This suggests that factors affecting T cell mobility are secreted from mesenchymal stem cells treated with IFN-γ and IL-1β.

Example  8: In Stem Cells by Treatment of Inflammatory Cytokines CCR7 Expression amount  increase

Recently, expression of CC-chemokine receptor 7 (CCR7) factor has been suggested as one of the development of Graft-versus-host disease (GVHD) therapeutics (Coghill JM et al ., Blood. 2010 Jun 10; 115). (23): 4914-22). CCR7 is a G protein-coupled receptor, secondary lymphoid organs (SLOs) that are expressed in various immune cells, such as dendritic cells (DCs) and naive and central memory T cells, and accumulate lymphocytes. This is an important factor in moving to. Specifically, it reacts with CCL21 secreted from the secondary lymphoid organs to induce migration of undifferentiated T cells to the secondary lymphoid organs, thereby allowing differentiation into CD4 or CD8 T cells. Thus, CCR7 can enhance the immune regulatory function by moving the mesenchymal stem cells to secondary lymphoid organs.

Therefore, in order to confirm whether the expression of CC-chemokine receptor 7 (CCR7) is increased in stem cells by pretreatment of IFN-γ and IL-1β, treatment with IFN-γ and IL-1β alone or a combination thereof After the test, the expression level of CCR7 was confirmed by RT-PCR at each time. Primer sequences used therein are shown in Table 3 below.

gene	direction	Primer sequence	SEQ ID NO:
CCR7	Forward direction	5'-GTGGTTTTACCGCCCAGAGA-3 '	SEQ ID NO: 5
	Reverse	5'-CACTGTGGTGTTGTCTCCGA-3 '	SEQ ID NO: 6

As a result, when treated with interleukin-1 beta (IL-1β), it was confirmed that the expression of the CCR7 gene at the RNA level is increased (Fig. 9).

Example  9: acute Graft-versus-host disease (Acute GVHD)  In the induced animal model IFN to determine the therapeutic effect of stem cells treated with -γ and IL-1β

9-1: Induction of Acute Graft-versus-host Disease and Infusion of Stem Cells

The animals used in the Acute GVHD-induced model experiment were mice (C57 / BL6 mouse (Donor), BDF1 (Recipient)) that purchased 8-week-old mice, followed by a 7-day acclimation period, at 9 weeks of age. It was used for the experiment. Bone marrow and spleen were collected at the expense of C57 / BL6 mouse (Donor), and 10 × 10 ⁶ bone marrow cells and 2 × 10 ⁶ splenic T cells were transferred to BDF1 (Recipient) [F1 of C57BL / 6 X DBA / 2]. Implantation caused acute graft-versus-host disease. Three days after induction, a solvent control or test substance was administered in a single vein into the microvein of the mouse model. Dosing timing and dosage for each group is shown in Figure 10 and Table 4 below. In the case of cytokine-treated stem cells, when the confluency of the cells in adherent culture reached about 50% -70%, 20ng / ml of IFN-γ and 5ng / ml of IL-1β were incubated for 24 hours. The cells were harvested and washed three times before administration.

group	Acute GVHD Induction	Number of test animals	Number of Cells Administered	Volume
Negative control group (PBS group)	Cause	N = 8		200 μl
General cell administration group (MSC 0.3)	Cause	N = 4	0.3 × 10 6 dogs	200 μl
Test cell administration group (MSC 0.3 + IFNγ / IL-1β)	Cause	N = 4	0.3 × 10 6 + IFN-γ / IL-1β	200 μl
General cell administration group (MSC 1)	Cause	N = 8	1 × 10 6 dogs	200 μl
Test cell administration group (MSC 1 + IFNγ / IL-1β)	Cause	N = 8	1 × 10 6 + IFN-γ / IL-1β	200 μl
General cell administration group (MSC 3)	Cause	N = 4	3 × 10 6 dogs	200 μl
Test cell administration group (MSC 3 + IFNγ / IL-1β)	Cause	N = 4	3 × 10 6 + IFN-γ / IL-1β	200 μl

9-2: IFN -γ and IL- 1β  Validate the therapeutic effect of treated stem cells at the gross lesion level

Survival of the mouse model was observed up to 30 days after inducing acute graft-versus-host disease as described above. Kaplan-Meier was used to compare survival rates and then the statistical significance between the control group and the test group was tested by the log-rank method.

In addition, the solvent control agent and the test substance were administered on the third day after inducing acute graft-versus-host disease, and GVHD scores were measured at two check-points per week. To compare the GVHD scores, Repeated measures ANOVA was performed and the statistical significance between the control group and the test group was tested by the Greenhouse-Geisser method (p <0.05). In addition, Tukey's method was additionally performed for post test.

On the other hand, GVHD score measurement criteria are shown in Table 5.

standard		Grade 0	Grade 1	Grade 2
Weight loss	Less than 10%	10-25% reduction	> 25% reduction
Posture	Normal	Hunching noted only at rest	Severe hunching impairs movement
Activity	Normal	Mild to moderate decreased	Stationary unless stimulated
Fur texture	Normal	Mild to moderate ruffling	Severe ruffling / poor grooming
Skin integrity	Normal	Scaling of paws / tail	Obvious area of denuded skin

As a result, the survival rate did not show a difference between the control group and the test group, and it was confirmed that the addition of the culture medium did not worsen the survival rate when compared between the groups (FIG. 11).

On the other hand, in the case of GVHD score, the total number of mesenchymal stem cells administered was 3 × 10 ⁶ or more, and the symptoms of acute GVHD were significantly reduced. It was confirmed that the number of 1 × 10 ⁶ significantly reduced the symptoms of acute GVHD (Fig. 12).

9-3: IFN -γ and IL- 1β  Therapeutic effect of treated stem cells is verified through histopathological examination

To determine the therapeutic effect of stem cells treated with IFN-γ and IL-1β on graft-versus-host disease, an autopsy was performed on the 30th day after the acute graft-versus-host disease was induced. The small intestine was collected and fixed in formalin and histopathologically evaluated by H & E staining.

As a result, in skin and liver tissue analysis, no significant difference was found between the groups (FIGS. 13A and 13B). However, intestinal tissue analysis showed the most damage in 1 × 10 ⁶ mesenchymal stem cells added with culture medium. Small (Figure 13C). The total GVHD scores measured in the skin, liver, and small intestine tissues also showed the lowest damage in 1 × 10 ⁶ mesenchymal stem cells added with the culture medium (FIG. 13D).

Seen through the test results, it was confirmed as acute GVHD is that the clinical indicators effect on GVHD symptoms improve in a mouse model and histological when using mesenchymal stem cells was added to the culture, 1 × 10 ⁶ of mesenchymal stem cells By adding IFN-γ and IL-1β to the culture medium, it was confirmed that the treatment effect was equivalent to 3 × 10 ⁶ mesenchymal stem cells.

Example  10: in animal models of Crohn's disease IFN -γ and IL- 1β  Confirmation of treatment effect of treated stem cells

10-1: Inducing Crohn's Disease and Injecting Stem Cells

Crohn's disease-induced model tested in the present invention is a dextran sulphate sodium (DSS) -induced colitis model, which is a chemically-induced colitis mouse model. It is the most commonly used model in that it is easy to control the degree of inflammation and duration of inflammation. Animals used in this experiment were purchased from 8 weeks old mouse C57 / B16 mice and after 7 days of acclimation, were used for the experiments at 9 weeks old. Inflammatory bowel disease (Crohn's disease) was 3% (w / v) DSS 1 Induced by feeding negatively over the week. One day after induction, a solvent control or test substance was administered intraperitoneally into the mouse model. Dosing time and dosage for each group is shown in Table 6 below. In the case of cytokine-treated stem cells, when the confluency of the cells in adherent culture reached about 50% -70%, 20ng / ml of IFN-γ and 5ng / ml of IL-1β were incubated for 24 hours. The cells were harvested and washed three times before administration.

group	DSS triggered	Number of test animals	Number of Cells Administered	Volume
Negative control group (PBS group)	-	N = 4	-	200 μl
Positive control group (PBS group)	Cause	N = 9	-	200 μl
General Cell Administered Group (MSC)	Cause	N = 6	2 x 10 6 pieces	200 μl
Test cell administration group (MSC + IFNγ)	Cause	N = 6	2 × 10 6 + IFNγ	200 μl
Test cell administration group (MSC + IL-1β)	Cause	N = 6	2 × 10 6 + IL-1β	200 μl
Test cell administration group (MSC + IFNγ / IL-1β)	Cause	N = 6	2 × 10 6 + IFNγ / IL-1β	200 μl

10-2: IFN -γ and IL- 1β  Validate the therapeutic effect of treated stem cells at the gross lesion level

Survival and weight change of the mouse model were observed until 8 days after inducing Crohn's disease as described above.

As a result, it was confirmed that the weight gain and the survival rate were increased when compared with cells treated with single material or cells not treated with material in the mesenchymal stem cells treated with the two substances (FIGS. 14 and 15). ).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Claims (25)

1. Stem cells cultured for 12 to 36 hours by adding 10 to 20 ng / ml of interferon-gamma (IFN-γ) and 2.5 to 10 ng / ml of interleukin-1 beta (IL-1β) to stem cells or their Culture.
2. According to claim 1, wherein the stem cells are stem cells or a culture thereof derived from umbilical cord blood.
3. A method of preparing a stem cell or a culture thereof, comprising the step of culturing by adding inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β),

   Method of producing a stem cell or a culture thereof characterized in that treated with 10 to 20 ng / ml of IFN-γ and 2.5 to 10 ng / ml of IL-1β and incubated for 12 to 36 hours.
4. 1 × 10 ⁶ to 1 × 10 ⁹ stem cells cultured with the addition of inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β); And

   A composition for single administration of stem cells containing 1 to 100 ml of a culture medium in which stem cells are cultured by treating inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). Characterized in that the culture medium exhibits two to three times the stem cell treatment effect compared to a single dose of stem cells, the composition for stem cell first administration.
5. A composition for single administration of stem cells containing 1 × 10 ⁶ to 1 × 10 ⁹ stem cells of claim 1.
6. According to claim 4 or 5, wherein the composition is a pharmaceutical composition, characterized in that for the prevention or treatment of immune diseases or inflammatory diseases.
7. According to claim 4 or 5, wherein the stem cells are human adult stem cells, human pluripotent stem cells, induced pluripotent stem cells (induced pluripotent stem cells), embryonic stem cells of animals other than human or adult stem of the animal Pharmaceutical composition, characterized in that the cell.
8. The method of claim 7, wherein the adult stem cells are mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells or amniotic epithelial cells, characterized in that Pharmaceutical composition.
9. According to claim 8, wherein the adult stem cells are cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal Pharmaceutical composition, characterized in that the mesenchymal stem cells selected from the group consisting of stem cells, skin-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells and placental-derived mesenchymal stem cells.
10. The pharmaceutical composition according to claim 6, wherein the autoimmune disease or inflammatory disease is autoimmune disease, transplant rejection, arthritis, graft versus host disease, bacterial infection, sepsis or inflammation.
11. 11. The method of claim 10, wherein the autoimmune disease is Crohn's disease, erythema disease, atopic dermatitis, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism Hyperhidrosis, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis , Vitiligo, ocular scleroderma, asthma and ulcerative colitis, the pharmaceutical composition characterized in that it is selected from the group consisting of.
12. A method of inhibiting proliferation of mononuclear cells, comprising the step of treating the stem cells of claim 1 or a culture thereof to the tissue to be transplanted.
13. Composition for inhibiting proliferation of mononuclear cells containing a stem cell or a culture thereof.
14. Stem cells cultured with the addition of inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) or cultures thereof were treated with cells to express IDO (Indoleamine 2,3-dioxygenase) or How to increase activity.
15. A composition for increasing the expression or activity of IDO, comprising stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β).
16. Th1 cells of CD4 + T cells comprising treating CD4 + T cells with stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) Or inhibiting differentiation into Th17 cells.
17. Inhibition of differentiation of CD4 + T cells into Th1 cells or Th17 cells, including stem cells cultured with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) or their cultures Composition.
18. Migration of CD4 + T cells comprising treating CD4 + T cells with stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) How to increase migration or recruitment.
19. A composition for increasing the migration or recruitment of CD4 + T cells, including stem cells or cultures thereof added with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β).
20. A method for targeting stem cells to secondary lymphoid organs (SLO),

    Culturing the stem cells by adding inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β); And including the step of administering the stem cells,

    Stem cells targeted to the secondary lymphoid organs have increased expression of CC-chemokine receptor type 7 (CCR7) genes compared to stem cells not treated with interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). It is characterized by being.
21. Immediately after the onset of acute graft-versus-host disease, or within 5 days from the onset, administration of stem cells or cultures thereof cultured with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). A method for treating acute graft-versus-host disease, comprising:
22. Stem cells cultured with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β), which are administered immediately after onset or within 5 days of onset of acute graft-versus-host disease. Or a pharmaceutical composition for treating acute graft-versus-host disease containing a culture thereof.
23. A pharmaceutical composition for preventing or treating Crohn's disease, comprising a stem cell cultured by adding inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) or a culture thereof.
24. A pharmaceutical for preventing or treating IDO-mediated immune diseases or inflammatory diseases, which contains stem cells or cultures thereof which are cultured with inflammatory cytokines of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β). Composition.
25. COX-2 (Cyclooxygenase-2), IDO (Indoleamine 2,3-dioxygenase), comprising culturing stem cells in a medium to which interferon-gamma (IFN-γ) and interleukin-1beta (IL-1β) are added. ), PGE ₂ (Prostaglandin E2) and CCR7 (CC-chemokine receptor type 7) mass production method of one or more proteins selected from the group consisting of.

Images