KR20160037113A - Pharmaceutical composition comprising stem cells treated with Interferon-gamma or Interleukin-1beta, or culture thereof for prevention and treatment of immune diseases and inflammatory diseases - Google Patents
Pharmaceutical composition comprising stem cells treated with Interferon-gamma or Interleukin-1beta, or culture thereof for prevention and treatment of immune diseases and inflammatory diseases Download PDFInfo
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Abstract
The present invention relates to a pharmaceutical composition for preventing or treating immunological diseases and inflammatory diseases including stem cells or cultures thereof cultured by adding interferon-gamma and inflammatory cytokines of interleukin-1 beta to stem cells.
Description
The present invention relates to a method for the prevention of immune diseases and inflammatory diseases including stem cells cultured by adding interferon-gamma (IFN-?) And / or interleukin-1 beta (IL-1?) Cytokines to stem cells or cultures thereof Or pharmaceutical compositions for therapeutic use.
In order to protect the human body from internal and external pathogenic substances, the immune system constructs a defense system consisting of various cells, which is accomplished through the specific function of each cell and signal transmission between the cells. The immune system of the human body can be largely divided into immunity (immune tolerance) which controls immune suppression and immunity which promotes immunity. These two immune functions are balanced to form immunological homeostasis . However, if for some reason its balance is broken and the immune response is greater than that of immune tolerance, immune and inflammatory diseases will result.
Immunosuppression for the prevention or treatment of an immunological disease or an inflammatory disease includes a specific inhibitor and a nonspecific inhibitor that inhibit the response only. Theoretically, a specific inhibitor should be excellent, but a nonspecific inhibitor is mainly used. Clinically, the most commonly used immunosuppressants are cyclosporine (Neoral, Cipol A), azathioprine (imuran), and prednisolone (a kind of steroid). The above three cases showed less side effects and immunosuppressive effects than when they were taken together. Recently, various immunosuppressants such as FK 506, RATG, OKT3, and Cellcept have been developed and used.
These immunosuppressants inhibit several processes such as antigen phagocytosis by macrophage, antigen recognition by lymphocyte, cell division, division of T cells and B cells, and antibody production during the course from antigen stimulation to antibody production, It causes. Most of them have antitumor activity because they inhibit cell division by mediating DNA disorder or DNA synthesis inhibition.
However, there have been problems such as hypertension and nephrotoxicity (lowered renal function), which are typical side effects, and that the incidence of this side effect is high, so it is necessary to observe the progress sufficiently during use. Other side effects include tremors, seizures, hepatitis, retention of fluid, increased blood uric acid, decreased muscle strength, hypertrichosis, and gingival hypertrophy. Azathioprine, a commonly used inhibitor, may inhibit bone marrow function such as decreased leukocyte levels, anemia, and thrombocytopenia. There may be complications such as pancreatitis, hepatitis, biliary retention, and rarely hair loss and fever. Prednisolone, one of the steroids, is the first of the immunosuppressants to be used and has the broadest inhibitory effect. It increases appetite, increases shoulders and back muscles, and temporarily brings happiness. However, these steroids not only promote atherosclerosis but also cause hypertension, gastric ulcer, diabetes, growth inhibition, osteoporosis, cataracts, glaucoma It is a drug to watch out for.
As a therapeutic agent for overcoming the above-mentioned side effects, a biopharmaceutical drug has become popular, and a typical example thereof is a stem cell treatment agent. Although mesenchymal stem cell research has been reported to utilize the differentiation ability of stem cells to treat damaged joint tissues and to treat inflammation and the like (U.S. Patent No. 6,835,377), the therapeutic mechanism has been clarified Therapeutics are a rare reality.
On the other hand, endotoxic substances such as lipopolysaccharide (LPS) are substances that induce the production of inflammatory factors in cells, and promote the production of inflammatory cytokines that cause an inflammatory reaction. That is, when an external stimulus that may cause an inflammatory reaction is applied, the expression of inflammatory cytokines such as TNF-a is induced, and the produced inflammatory cytokines stimulate the expression of genes encoding iNOS and COX-2, It produces NO and PGE 2 substances involved in the inflammatory reaction and causes an inflammatory reaction. However, to date, there has been no report on the mechanism of prophylactic or therapeutic treatment of inflammatory diseases or immune diseases by increasing the expression of inflammatory regulatory factors or cell migration-related factors secreted from stem cells when these inflammatory cytokines are administered to stem cells .
Accordingly, the present inventors have found that, when cultured (pre-treated) with the addition of cytokines 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 is remarkably increased The present inventors have completed the present invention by confirming that the stem cells can be used as a cell therapy agent for the prevention or treatment of immune diseases or inflammatory diseases.
One object of the present invention is to provide an economical, cost effective, and cost effective alternative to conventional immunosuppressants and antiinflammatories, such as 10-20 ng / ml interferon-gamma (IFN-?) And 2.5-10 ng / ml Of interleukin-1 beta (IL-1?) And cultured for 12 hours to 36 hours; or a culture thereof; Or a pharmaceutical composition for preventing or treating an immunological disease or inflammatory disease comprising the same.
Another object of the present invention is to provide a method for producing a stem cell or a culture thereof comprising the step of culturing with the addition of an inflammatory cytokine of interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?), To 20 ng / ml of IFN-y and 2.5 to 10 ng / ml of IL-1 [beta] are cultured for 12 hours to 36 hours, or a culture thereof.
Yet another object of the present invention is to provide a method for the treatment and prophylaxis of stem cell proliferation, which comprises culturing 1 x 10 6 to 1 x 10 9 stem cells with the addition of an interferon-gamma (IFN-?) And an interleukin-1 beta (IL-1?) Inflammatory cytokine; And 1 to 100 ml of a culture medium in which an inflammatory cytokine of interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?) Has been treated to cultivate stem cells. Which is characterized by exhibiting a stem cell treatment effect of 2 to 3 times the single dose of stem cells by the culture solution.
It is still another object of the present invention to provide a composition for single-dose administration of stem cells containing 1 x 10 < 6 > to 1 x 10 < 9 >
Another object of the present invention is to provide a composition for inhibiting the proliferative activity of mononuclear cells comprising said stem cells or a culture thereof; Or treating a stem cell or a culture thereof with a tissue to be transplanted, to thereby suppress the proliferative ability of the mononuclear cell.
Still another object of the present invention is to provide a composition for increasing the expression or activity of IDO (Indoleamine 2,3-dioxygenase) comprising the stem cell or a culture thereof; Or a method of increasing the expression or activity of IDO by treating a stem cell or a culture thereof cultured with the addition of an interferon-gamma (IFN-y) and an inflammatory cytokine of interleukin-1 beta (IL-1 beta) .
Still another object of the present invention is to provide a composition for inhibiting the differentiation of CD4 + T cells into Th1 cells or Th17 cells, comprising the stem cells or cultures thereof; Or Th cells of CD4 + T cells comprising the step of treating CD4 + T cells with stem cells or cultures thereof cultured by the addition of an interferon-gamma (IFN-?) And an inflammatory cytokine of interleukin-1 beta (IL- 1 < / RTI > cells or Th17 cells.
It is another object of the present invention to provide a composition for increasing the migration or aggregation of CD4 + T cells comprising said stem cells or a culture thereof; Or CD4 + T cells comprising the step of treating CD4 + T cells with stem cells or cultures thereof cultured by adding an interferon-gamma (IFN-γ) and an inflammatory cytokine of interleukin-1 beta (IL-1β) thereby increasing migration or recruitment.
It is yet another object of the present invention to provide a method of targeting stem cells to secondary lymphoid organs (SLO), wherein the stem cells are selected from the group consisting of interferon-gamma (IFN-y) and inflammatory cytokines of interleukin-1 beta Adding and culturing; Wherein the stem cells targeted to the secondary lymphoid organs are selected from the group consisting of CCR7, CCR7, and CCR7, as compared to stem cells not treated with interferon-gamma (IFN-?) And interleukin-1 beta (CC-chemokine receptor type 7) gene expression is increased.
Another object of the present invention is to provide a method for the treatment and prophylaxis of inflammatory cytokines such as interferon-gamma (IFN-y) and interleukin-1 beta (IL-1?), Which are administered immediately after the onset of acute graft versus host disease, A pharmaceutical composition for the treatment of acute graft-versus-host disease comprising a stem cell or a culture thereof cultured by addition of a culture medium; Or a stem cell or culture thereof cultured by adding an inflammatory cytokine of interferon-gamma (IFN-γ) and interleukin-1 beta (IL-1β) within 5 days from the onset or onset of acute graft versus host disease Comprising administering to a patient in need thereof an effective amount of a compound of the invention.
It is still another object of the present invention to provide a method for preventing or treating Crohn's disease, which comprises culturing a stem cell cultured by adding an interferon-gamma (IFN-y) and an interleukin-1 beta (IL-1?) Inflammatory cytokine or a culture thereof To provide a pharmaceutical composition.
It is still another object of the present invention to provide a method for the treatment of IDO-mediated immune diseases or diseases, which comprises culturing stem cells cultured with the addition of an interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?) Inflammatory cytokine, And to provide a pharmaceutical composition for preventing or treating an inflammatory disease.
Another object of the present invention is to provide a method for producing an immunosuppressant or an anti-inflammatory agent comprising the step of culturing stem cells by adding interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?).
Another object of the present invention is to provide a method for producing stem cells comprising the steps of culturing stem cells in a medium supplemented with inflammatory cytokines such as COX-2 (Cyclooxygenase-2), Indoleamine 2,3-dioxygenase (IDO), PGE 2 (Prostaglandin E2) (CC-chemokine receptor type 7). The present invention also provides a method for mass production of at least one protein selected from the group consisting of CC-
In one aspect of the present invention, the present invention provides a stem cell or a culture thereof cultured by treating an stem cell with an inflammatory cytokine; Or a pharmaceutical composition for the prevention or treatment of an immunological disease or inflammatory disease comprising the same.
In the present invention, when stem cells are treated with an inflammatory cytokine, specifically, interferon-gamma (IFN-?), Interleukin-1 beta (IL-1?) Or the above factors, the stem cell- (COX-2, PGE 2 , IDO) and the factor associated with migration (CCR7), thereby controlling immunity and inflammation. In addition, it was confirmed that the stem cells treated with the two cytokines were administered to a graft-versus-host disease or Crohn's disease model. That is, the present inventors confirmed that stem cells treated with inflammatory cytokines and cultures thereof can be used as a cell therapy agent for immunomodulation and inflammation control, and thus 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 an immunological disease or an inflammatory disease including the culture.
The term " proinflammatory cytokine " in the present invention refers to a cytokine that induces an inflammatory reaction in the body. Typically, the cytokine includes tumor necrosis factor-alpha (TNF-a), interferons (IFNs) Interleukins (ILs), monocyte chemoattractant proteins, and the like. In the present invention, the inflammatory cytokine may be, but is not limited to, IFN-y and / or IL-l [beta].
In the present invention, the "addition and cultivation" of the inflammatory cytokine may be performed, for example, by adding the inflammatory cytokine to the stem cell culture medium. Specifically, the cytokine may be 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-y can be treated at 10-20 ng / ml and IL-1 beta at 2.5-10 ng / ml. When the concentration of the treated cytokine is lower than the above concentration, the secretion of immunoregulatory factors secreted from stem cells such as IDO and PGE 2 is not sufficient, so that the increase in immunoregulatory ability may be weak or not present. On the other hand, when the concentration of the treated cytokine is higher than the above-mentioned concentration, it affects the characteristics of the cell, which may promote the senescence of the stem cell, inhibit the proliferation and decrease the differentiation ability.
In addition, the cells can be cultured 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 in the treatment (culture) time of these cytokines. If the treatment time of cytokine is shorter than the above, secretion of immunoregulatory factors secreted from stem cells such as IDO and PGE 2 is not sufficient, so that the increase of immunoregulatory ability may be weak or not appear. On the other hand, when the treatment time of cytokine is longer than the above, there is a disadvantage that it affects the characteristics of cells and promotes senescence of stem cells, inhibition of proliferation and decrease of differentiation ability.
Preferably, the stem cells of the present invention may be cultured for 10 to 20 ng / ml of IFN-? And 2.5 to 10 ng / ml of IL-1? For 12 to 36 hours.
In addition, it may be cultured by adding an inflammatory cytokine, and then culturing by replacing the medium.
In the present invention, "stem cells" may be cells having the ability to differentiate into various tissues, i.e., undifferentiated cells.
In the present invention, the stem cells may be human adult stem cells, human pluripotent stem cells, human pluripotent stem cells, induced pluripotent stem cells, animal embryonic stem cells, or animal adult stem cells Lt; / RTI >
Multipotent stem cells are pluripotent stem cells derived from various adult cells such as bone marrow, umbilical cord blood, placenta (or placental tissue cells), and fat (or adipose tissue cells). For example, mesenchymal stem cells derived from bone marrow have undergone various studies for development as a cell therapy agent by the pluripotency that can be differentiated into adipose tissue, bone / cartilage tissue, and muscle tissue.
On the other hand, adult stem cells can be mesenchymal stem cells, mesenchymal stromal cells derived from human tissue, mesenchymal stem cells derived from human tissues, multipotential stem cells or amniotic epithelial cells, , Mesenchymal stem cells selected from the group consisting of umbilical cord blood, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta and may be of human origin. (Human Umbilical Cord Blood Mesenchymal Stem Cell (hUCB-MSC)). In the present invention, it was confirmed that the expression levels of the immunity and inflammation-regulating factors (COX-2, PGE 2 ) were significantly higher in human-derived umbilical cord blood-derived mesenchymal stem cells than in other tissues (FIG. The method of obtaining stem cells from each of the strains can be performed 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, the term "culture product" may include a cell culture solution containing stem cells, a culture supernatant from which stem cells have been removed from the cell culture solution, and a dilution solution thereof. The composition of the culture may further include components necessary for culturing stem cells as well as components that act synergistically with stem cell proliferation. The composition may be easily prepared by those skilled in the art Can be selected.
In addition, in preparation of the stem cells or the culture thereof according to the present invention, IFN-? And IL-1? May be added simultaneously and cultured, or IL-1? May be added after addition of IFN-? -γ, and the addition order is not limited thereto.
For example, DMEM (Dulbecco's modified Eagle medium) or Keratinocyte-SFM (Keratinocyte serum free medium) may be used as a culture medium for culturing the stem cells. Can be used.
The stem cell medium may be supplemented with an additive. In general, it may contain neutral buffer (e.g., phosphate and / or high concentration bicarbonate) and protein nutrients (e.g., serum, such as FBS, serum replacement, albumin, or essential and nonessential amino acids such as glutamine) in the isotonic solution. Furthermore, it has been found that lipid (fatty acid, cholesterol, serum HDL or LDL extract) and other components found in most types of storage medium of this kind (such as insulin or transferrin, nucleoside or nucleotide, pyruvate, G., Glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as? -Mercaptoethanol).
According to the embodiment of the present invention, the adult stem cells treated with IFN-? And / or IL-1? Significantly increase the expression of COX-2, IDO, PGE 2 and CCR7, which are known as inflammatory or cell migration- Respectively. Particularly, in the present invention, it was confirmed that the immunosuppressive effect through treatment with IFN-y and / or IL-1? Is the main mechanism of inhibiting mixed lymphocyte by the increase of IDO expression (Fig.
Accordingly, the stem cell of the present invention or the culture thereof has a mononuclear cell proliferation inhibitory effect, and therefore, there is provided a composition for inhibiting mononuclear cell proliferative potency comprising the same, or a step of treating the stem cell of the present invention or a culture thereof with a transplantable tissue A method for suppressing the proliferative capacity of the mononuclear cells can be provided (Example 6).
In addition, since stem cells or cultures thereof of the present invention have an activity of inhibiting the differentiation of CD4 + T cells into
In addition, since stem cells or cultures thereof of the present invention have an activity of increasing the migration or recruitment of CD4 + T cells, interferon-gamma (IFN-y) and interleukin-1 beta (IL- ) In the form of a composition for increasing the migration or aggregation of CD4 + T cells, comprising the step of treating the stem cells or a culture thereof with CD4 + T cells Or a method of increasing the migration or recruitment of CD4 + T cells, including < RTI ID = 0.0 >
The present invention provides a method for targeting stem cells to a secondary lymphoid organs (SLO), wherein the stem cells are cultured by adding an inflammatory cytokine of interferon-gamma (IFN-?) And interleukin-1 beta (IL- Culturing; Wherein the stem cells targeted to the secondary lymphoid organs are selected from the group consisting of CCR7, CCR7, and CCR7, as compared to stem cells not treated with interferon-gamma (IFN-?) And interleukin-1 beta (CC-chemokine receptor type 7) gene expression is increased (Example 8). The stem cells to be targeted may also include artificially genetically engineered stem cells.
Meanwhile, the stem cells cultured by adding the inflammatory cytokines of interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?) Of the present invention or cultures thereof may be used for the increase of expression or activity of IDO In the form of a composition, or in the form of a method for increasing the expression or activity of IDO by treating the stem cell or a culture thereof with a cell (Example 5).
Accordingly, 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 immunological diseases or inflammatory diseases mediated by IDO. Herein, IDO-mediated disease refers to an immune disease or inflammatory disease that can be caused by the fact that the IDO gene is not normally expressed in the immune or inflammation-related mechanism or the protein is not secreted.
The term "inflammatory disease" in the present invention refers to a disease in which inflammation is the main lesion, and includes, but is not limited to, edema, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, Inflammatory bowel disease, inflammatory bowel disease, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, rhabdomyosarcoma, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, Sjogren ' s syndrome or multiple sclerosis.
In the present invention, the term "immune disease" is a disease that is a problem when a specific immune response occurs, but it may be an autoimmune disease, a graft rejection, or a graft versus host disease.
The autoimmune disease may be Crohn's disease, erythema nevi, atopic dermatitis, rheumatoid arthritis, Hashimoto's thyroiditis, autoimmune disease, autoimmune disease, inflammatory disease, autoimmune disease, graft rejection, graft versus host disease, arthritis, bacterial infection, sepsis or inflammation. , Meniere's syndrome (Meniere's syndrome), Crohn's disease, scleroderma, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, chronic myasthenia syndrome, fibromyalgia, hypothyroidism and hyperlipidemia, scleroderma, , Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma or ulcerative colitis, and the like.
In the present invention, "prevention" is any action that inhibits or delays the onset of an immunological disease or inflammatory disease upon administration of the composition, and "treatment" It is an act.
In addition, the stem cell or culture thereof according to the present invention may be used in an amount of 1.0 × 10 5 to 1.0 × 10 9 , particularly 1.0 × 10 6 to 1.0 × 10 8 , more specifically 1.0 × 10 7 Lt; / RTI > cells.
The stem cells or cultures thereof according to the present invention may be used without being frozen or frozen for future use. If required to be frozen, standard cryopreservatives (eg, DMSO, glycerol, Epilife cell freezing medium (Cascade Biologics)) may be added to the pre-freeze cell population.
In addition, the stem cells or cultures thereof according to the present invention can be formulated into a unit dosage form suitable for administration to a patient in the body according to a conventional method in the pharmaceutical field, and the preparation can be administered once or several times Lt; RTI ID = 0.0 > administration. ≪ / RTI > Suitable formulations for this purpose include injection agents such as ampoules injected for parenteral administration, injecting agents such as injection bags, and spray agents such as aerosol formulations. The injectable ampoule may be mixed with an 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 be made of polyvinyl chloride or polyethylene and may be manufactured by Baxter, Becton Dickinson, Medcep, National Hospital Products or Terumo, An injection bag of a yarn can be exemplified.
In addition to the active ingredient, the pharmaceutical preparation may contain one or more pharmaceutically acceptable inert carriers such as a preservative, an anhydrous agent, a solubilizing agent or a stabilizer in the case of injection, May further comprise a base, an excipient, a lubricant or a preservative.
The stem cells, cultures or pharmaceutical preparations according to the present invention thus prepared may be used in combination with other stem cells used for transplantation and other uses or a mixture thereof with such stem cells , And specifically can be directly implanted or implanted in a disease site of a patient in need of treatment, or can be implanted or injected directly into the abdominal cavity, but the present invention is not limited thereto. In addition, the above-mentioned administration is both non-surgical administration using a catheter and surgical administration such as implantation or transplantation after dissection of a diseased site, but a non-surgical administration method using a catheter is more appropriate. In addition to parenteral administration, for example, to direct lesions according to conventional methods, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible.
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 6 cells / 8 cells / kg body weight can be administered once or several times. It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the body weight, age and sex of the patient, The scope of the present invention is not limited thereto.
Particularly, the cells containing 1.0 × 10 6 to 1.0 × 10 9 stem cells cultured with the interferon-gamma (IFN-γ) and interleukin-1-beta (IL-1β) inflammatory cytokines of the present invention Dose. ≪ / RTI >
In addition, when the stem cell as well as the culture medium thereof are contained together, a dose of 1.0 x (1 x 10 < 6 >), which is cultured by adding an interferon-gamma (IFN-?) And an interleukin- 10 6 to 1.0 x 10 9 stem cells; And 1 to 100 ml of a culture medium in which stem cells have been cultured by treating an inflammatory cytokine of interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?).
That is, when the culture medium of stem cells is contained in the administration means as a preparation for cell therapy, the number or amount of stem cells necessary for achieving an equivalent effect is remarkably reduced to 1/2 to 1/10, 1/2 to 1/3, , Resulting in a more economical effect in formulation (Example 9).
Also with respect to the time of administration, said stem cells or cultures thereof may be used for the treatment of diseases to be treated, for example, immune or inflammatory diseases, such as acute graft versus host disease or inflammatory bowel disease such as Crohn's disease Within 5 days, within 3 days, within 2 days or within 1 day. In the examples of the present invention, interferon-gamma (IFN-gamma) and interleukin-1 beta (IL-1 beta) were treated within 3 days and 1 day after induction of each disease in acute graft versus host disease and Crohn's disease model As a result of administration of stem cells or cultures thereof, it was confirmed that the symptoms of these lesions were improved, showing therapeutic activity (Examples 9-1 and 10-1). This is expected to be associated with a significant increase in the level of expression of inflammatory regulators after 12 to 24 hours of treatment with interferon-gamma (IFN-y) and interleukin-1 beta (IL-1 beta).
In another embodiment, the present invention provides a method for inhibiting an immune response or controlling inflammation by administering an inflammatory cytokine-treated stem cell and a culture thereof according to the present invention, There is provided a method for inhibiting an immune reaction or an inflammatory reaction of a subject or a method of preventing or treating an immunological disease or an inflammatory disease, comprising administering a stem cell or a culture thereof to a subject.
The term "subject" or "subject" as used herein includes, but is not limited to, mammals including cows, dogs, pigs, chickens, sheep, horses and humans. In addition, the administration of the stem cell or culture thereof cultured by adding the inflammatory cytokine specifically may be peritoneal or intravascular administration, direct administration to a lesion, or synovial cavity administration.
The inhibition of the immune response or the inflammatory response may be characterized by the prevention or treatment of an immune or inflammatory disease.
In another aspect, the present invention provides a method for producing an immunosuppressant or an anti-inflammatory agent comprising culturing stem cells by adding an inflammatory cytokine. The immunosuppressant or anti-inflammatory agent may be in the form of a cell therapy agent.
As described above, the term "immunosuppressive agent" as used herein refers to a stem cell obtained by culturing stem cells with an inflammatory cytokine added thereto, or a preparation containing the same, which is capable of treating immune diseases by inhibiting the immune response It can be the best.
As described above, the term "anti-inflammatory agent" as used herein refers to a stem cell obtained by culturing stem cells with an inflammatory cytokine added thereto or a culture containing the same, have.
In another aspect, the present invention relates to a method for producing stem cells comprising the step of culturing stem cells in a medium supplemented with inflammatory cytokine, wherein the expression of COX-2 (Cyclooxygenase- 2),
In the present invention, the protein can be recovered by collecting the medium in which the stem cells have been cultured, removing the cells and suspension by centrifugation and filtration, and recovering the remaining supernatant.
In another aspect, the present invention provides an implant comprising stem cells and an inflammatory cytokine.
In the present invention, an "implant" is a material that can be implanted in a human body or animal, isolating the damaged site from the outside or containing transplanted cells or secreted therapeutic materials. Such grafts may include a biocompatible synthetic polymer as a support for tissue engineering and a variety of materials used in the art such as natural materials. Since the graft of the present invention contains stem cells and inflammatory cytokines, there is an advantage that it does not cause an immune rejection reaction or an inflammation reaction due to the graft. Therefore, even if there is no need to administer an immunosuppressive agent or an anti-inflammatory agent to inhibit the immune rejection reaction or the inflammatory reaction that may occur when various grafts are transplanted into the body, the grafted graft is stable in vivo without immunodeficiency or inflammation .
In another embodiment, the present invention provides an implant having stem cells removed after culturing stem cells by adding an inflammatory cytokine on a support.
In another aspect, the present invention provides a method for preparing an implant comprising culturing stem cells on a support by adding an inflammatory cytokine.
Specifically, it may further include a step of removing stem cells after the culturing step.
In another aspect, the present invention provides a stem cell; And a cytokine of 10-20 ng / ml interferon-gamma (IFN-y) and 2.5-10 ng / ml interleukin-1 beta (IL-1 beta). The complex may be used as a cell therapy agent.
In another embodiment, the present invention provides a method for producing stem cells comprising the steps of: (i) adding 10 to 20 ng / ml of interferon-gamma (IFN-y) and 2.5 to 10 ng / ml of a cytokine of interleukin- Cultured stem cells or a culture thereof.
The culture may exhibit an effect of preventing or treating an immunological disease or an inflammatory disease, and inflammatory regulatory factors such as COX-2, IDO, PGE 2 , and CCR7 or cell migration-related factors may be present.
In another aspect, the present invention provides a method for the preparation of a pharmaceutical composition for the prevention or treatment of an immunological disease or inflammatory disease, comprising administering to a
The 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, the stem cells according to the present invention can produce an inflammatory regulator or a cell migration-related factor with a simple, economical and chemical process and an improved yield. The stem cell or culture thereof according to the present invention is a cell therapy agent which can be used economically and has no side effect, replacing existing immunosuppressive agents and inflammation inhibitors known to have side effects, and can be used for immunization such as Clonus disease, rheumatoid arthritis, Can be used for the prevention or treatment of diseases and inflammatory diseases.
FIG. 1A is a graph showing the expression level of the immunoregulatory factor COX-2 mRNA in the case of IFN-y and IL-1β, TNF-α or IL-2, respectively, treated with cord blood-derived mesenchymal stem cells.
FIG. 1B is a graph showing the expression level of the immuno-regulatory factor PGE 2 when the umbilical cord blood-derived mesenchymal stem cells were treated with IFN-γ, IL-1β, TNF-α or IL-2, respectively.
FIG. 1C is a graph showing the level of IDO activity of the immunomodulatory factor in the case of IFN-y and IL-1β, TNF-α or IL-2, respectively, in the umbilical cord blood-derived mesenchymal stem cells.
FIG. 1D is a graph showing the level of proliferation of human mononuclear cells when a mixed lymphocyte reaction is performed after the combined treatment of IFN-γ, IL-1β, TNF-α or IL-2 with cord blood derived mesenchymal stem cells .
FIG. 2A is a graph showing the expression levels of IDO mRNAs at various times after treating 20 ng / ml IFN-y and 10 ng / ml IL-1 beta simultaneously for 3, 6, 12, and 24 hours.
FIG. 2B is a graph showing the expression levels of PGE 2 according to each time after simultaneously treating 20 ng / ml of IFN-γ and 10 ng / ml of IL-1β for 3, 6, 12 and 24 hours.
FIG. 3A is a graph showing expression levels of COX-2 mRNA according to respective concentrations after simultaneously treating IFN-
FIG. 3B is a graph showing the expression levels of IDO mRNA according to respective concentrations after simultaneously treating IFN-
FIG. 4 is a graph showing the results of measurement of expression levels of immunoregulatory factors PGE 2 and NOD2 of mesenchymal stem cells isolated from different tissues (bone marrow, fat, cord blood) of humans.
FIG. 5A is a graph showing the expression levels of PGE 2 after inhibiting 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE 2 inhibitor.
FIG. 5B is a graph showing the expression levels of IDO after inhibiting each factor using 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE 2 inhibitor.
FIG. 5c is a graph showing the level of mononuclear cell proliferation after inhibiting each factor using 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE 2 inhibitor.
FIG. 5d shows the expression levels of IL-17 after inhibiting 1-Methyltryptophan (1-MT) as an IDO inhibitor and Indomethacin as a PGE 2 inhibitor, respectively.
FIG. 6 is a graph showing the effect of IFN-γ secreted during Th1 cell differentiation and Th17 cell differentiation into Th1 cells or Th17 cells in the experiment of CD4 + T cells co-cultured with mesenchymal stem cells 17 (IL-17) secreted by the process of the present invention.
FIG. 7 is a diagram showing the result of co-culturing the mesenchymal stem cells cultured with IFN-y or / and IL-1? And CD4 + T cells by fluorescent staining.
FIG. 8 is a graph showing the mobility of CD4 + T cells relative to the culture medium secreted from the cells after 3 days of incubation after replacing the mesenchymal stem cells pretreated with IFN-y or / and IL-1?
FIG. 9 is a graph showing the expression levels of CCR7 according to each time after treatment with IFN-y and / or IL-1? For 6, 12, and 24 hours.
FIG. 10 is a diagram showing a process of producing an acute graft versus host animal model, that is, a process of inducing acute graft versus host disease, and a method of administering IFN-γ and / or IL-1β pre-treated stem cells.
11 is a graph showing a change in survival rate according to administration of mesenchymal stem cells pretreated with IFN-y and / or IL-1? In an acute graft versus host animal model.
FIG. 12 is a graph showing changes in GVHD score according to administration of IFN-y and / or IL-1? Pre-treated mesenchymal stem cells in an acute graft versus host animal model.
13a-13d show the expression of IFN-y and / or IL-l [beta] in the skin tissue (a), hepatic tissue (b), small intestine tissue (c) . The histopathological evaluation of mesenchymal stem cells treated with pre-treated mesenchymal stem cells is shown in FIG.
14 is a graph showing changes in survival rate according to administration of mesenchymal stem cells pretreated with IFN-y and / or IL-1? In an animal model of Crohn's disease.
FIG. 15 is a graph showing an improvement effect of weight loss upon administration of mesenchymal stem cells pretreated with IFN-y and / or IL-1? In an animal model of Crohn's disease.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.
Manufacturing example : From cord blood Of mesenchymal stem cells detach
The umbilical cord blood was mixed with the Hetasep ™ solution at a ratio of 5: 1 and allowed to stand at room temperature for 40 minutes. The supernatant was then carefully placed on a Ficoll and centrifuged at 2500 rpm for 20 minutes. After centrifugation, the mononuclear cells were collected, washed twice with PBS, and plated on fibronectin-coated plates at a concentration of 2 × 10 5 to 2 × 10 6 cells / cm 2 using EGM-2 medium containing 20% fetal bovine serum ≪ / RTI > After 3 days, the non-adherent cells were removed, and the medium was changed every 2-3 days to observe whether or not colonies were formed. The thus formed colonies were subcultured to obtain mesenchymal stem cells. As a result of observing the expression markers using the FACS in the mesenchymal stem cells obtained, 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: Inflammatory cytokine treatment Immunoregulatory Expression level analysis
In order to confirm the immunosuppressive effect or the anti-inflammatory effect of stem cells according to pretreatment of four inflammatory cytokines such as IFN-y, IL-1 beta, TNF-alpha and IL-2 on stem cells, Expression levels of COX-2 and IDO mRNA in the cord blood-derived mesenchymal stem cells were measured by real-time RT-PCR using IFN-γ, IL-1β, IL-2 or TNF- Were used for comparison analysis. Specifically, 20 ng / ml of IFN-γ and 5 ng / ml of IL-1β, 20 pg / ml of IL-2 or 20 ng / ml of TNF- α was added, followed by culturing for 24 hours.
The expression levels of COX-2 and IDO mRNA in cultured mesenchymal stem cells were then analyzed using real-time RT-PCR. Primer sequences for each gene used for this purpose are shown in Table 1 below.
In addition, the level of expression of PGE 2 was analyzed by ELISA. IDO converts L-tryptophan into N-formylkynurenine, which is then hydrolyzed by reacting with 30% (wt / vol) trichloroacetic acid. The amount of IDO secretion, i.e., the activity of IDO, was measured by measuring the amount of kynurenine.
Furthermore, mixed lymphocyte reaction was performed to confirm the level of proliferation of human mononuclear cells (hMNC) obtained through primary culture. The mixed lymphocyte reaction is described in Example 5 below.
As a result, COX-2, which is a typical immunomodulator, showed a higher expression level when IL-1β was combined with other cytokines (FIG. 1A), and COX-2-regulated PGE 2 (Fig. 1B), it was confirmed that IL-1β was also secreted to the highest level in the case of complex treatment with IL-1β. In addition, the activity of IDO, which is another immunomodulatory factor, was also highest in the complex treatment with IL-1β (FIG. 1c), and it was confirmed that IFN-γ and IL-1β Lt; RTI ID = 0.0 > RNA < / RTI > and protein levels. In addition, inhibition of mononuclear cell proliferation was confirmed by co-culturing with mixed lymphocytes, and the effect of further suppressing the proliferative activity was confirmed under the combined treatment conditions of IFN-y and IL-1 (Fig. 1d).
This can be used as a therapeutic agent for cells with enhanced immunosuppressive and anti-inflammatory effects by maximizing the expression of COX-2 and IDO, which are immunomodulators, from stem cells through pretreatment of IFN-γ and IL-1β with stem cells .
Example 2: IFN -γ and IL- 1β Depending on processing time Immunoregulatory Expression level analysis
In order to confirm the enhancement of stem cell immunosuppressive or antiinflammatory effect according to the treatment time of IFN-γ and IL-1β with inflammatory cytokines in the umbilical cord blood-derived mesenchymal stem cells obtained in the above-mentioned production examples, IFN-
As a result, it was confirmed that the expression level of IDO showed the highest expression level at 24 hours after the treatment (Fig. 2a). On the other hand, it was confirmed that the expression level of PGE 2 showed the highest expression level at 12 hours after treatment (Fig. 2B). These results suggest that the secretion of PGE 2 is secreted at 12 hrs in IFN-γ and IL-1β-pretreated stem cells, The longer the one hour, the more time-dependent increases in the amount of secretion within 24 hours.
Example 3: IFN -γ and IL- 1β Depending on treatment concentration Immunoregulatory Expression level analysis
In order to confirm the enhancement of stem cell immunosuppressive effect or anti-inflammatory effect according to the treatment concentration of IFN-γ and IL-1β in the cord blood derived mesenchymal stem cells obtained in the above Preparation Example, IFN- The expression levels of COX-2 and IDO mRNA were measured by real-time RT-PCR after 5, 10, 20 ng / ml and IL-1β 2.5, 5 and 10 ng / Respectively.
As a result, it was confirmed that the expression levels of COX-2 and IDO showed the highest expression levels in the combination treatment group of 20 ng / ml of IFN-y and 2.5 or 5 ng / ml (Figs. 3a and 3b).
Example 4: Depending on the origin of the stem cells Immunoregulatory Expression level analysis
In order to analyze the expression level of the immunoregulatory factor according to the stem tissues derived from the stem cells, mesenchymal stem cells were isolated from different tissues (bone marrow, fat, cord blood) of human.
The umbilical cord blood-derived mesenchymal stem cells were obtained by the method described in the above Preparation Example, and bone marrow-derived mesenchymal stem cells were used from the cell therapy center of Severance Hospital, Yonsei University College of Medicine. In addition, adipose-derived mesenchymal stem cells were obtained from Ji-Won Jung et al. , Cell Mol Life Sci. 2010 Apr; 67 (7): 1165-1176. . ≪ / RTI >
The levels of expression of immunoregulatory factors such as "PGE 2 " and "NOD 2 " in these mesenchymal stem cells were measured by ELISA and Western blotting, respectively, and compared with bone marrow and adipose derived stem cells And the expression of these factors was significantly higher in the cord blood-derived stem cells (Fig. 4). This suggests that stem cells derived from other tissues secrete immunoregulatory factors or inhibit immune cells when treated with cord blood derived stem cells compared with those treated with inflammatory cytokines.
Example 5: IFN -γ and IL- 1β Of stem cells by simultaneous (complex) treatment Immunosuppressive ability Enhancement analysis
In order to confirm whether any of IDO and PGE 2 , whose expression level is remarkably increased at the mRNA level through IFN-? And IL-1? Treatment, is involved in the immunoregulatory ability of stem cells, their inhibitors were treated in mesenchymal stem cells And mixed lymphocyte reaction after 20 ng / ml of IFN-γ and / or 10 ng / ml of IL-1β was added to the culture medium for 24 hours. In addition, 1-Methyltryptophan (1-MT) was used as an IDO inhibitor and Indomethacin was used as a PGE 2 inhibitor to inhibit the respective cytokines and confirm the mixed lymphocyte reaction (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 of each gene or protein, and the mixed lymphocyte reaction proceeded as follows.
The mesenchymal stem cells (hMSCs) obtained in this Preparation Example were seeded at a concentration of 3 × 10 5 cells / well in a 6-well plate and treated with 2 μM of 5-aza for 24 hours after 24 hours. For direct co-culture, the mesenchymal stem cells were treated with 25 mg / ml of mitomycin C, and the cells were plated at a concentration of 1 × 10 4 cells / well in a 96-well plate. Concanavalin A (ConA) was used to activate mononuclear cells (MNC) and seeded onto plates at a concentration of 5 x 10 4 cells / well.
For indirect co-cultivation, a transwell (24-well plate, Corning, Corning, NY) having a pore size of 0.4 μm was used. Sikyeotgo using ConA activated mononuclear cells (MNC), 1 × 10 was a plate 6 cells / well density in the lower chamber (lower chamber). 5-aza-treated mesenchymal stem cells were seeded in the upper chamber at a concentration of 1 × 10 5 cells / well. After 3 days, the proliferation rate of mononuclear cells was analyzed using BrdU assay (Bromodeoxyuridine kit, Roche, Upper Bavaria, Germany).
As a result, the IFN-γ-treated mesenchymal stem cells were more effective in inhibiting the mixed lymphocyte reaction regardless of the simultaneous treatment of IL-1β (FIG. 5C). In addition, the inhibitory effect of IDO inhibitor 1-MT was decreased to the control level, whereas the inhibitory effect of PGE 2 inhibitor Indomethacin was partially reduced (FIG. 5c).
This suggests that mixed lymphocyte inhibitory effect through IFN-? And / or IL-1? Treatment is involved in both of IDO and PGE 2 , but mixed lymphocyte inhibition effect by IDO is the main mechanism (FIG. 5c). This tendency was confirmed to be the same in the inhibitory effect on Th17 cells (Fig. 5d).
Example 6: IFN -γ and IL- 1β Of stem cells by simultaneous (complex) treatment Th1 / Th17 Immunosuppressive ability analysis
In order to confirm the immunosuppressive ability of the stem cells treated with IFN-y and / or IL-1? By inhibiting the differentiation of CD4 + T cells into Th1 cells or Th17 cells, 20 ng / ml of IFN-γ and 5 ng / ml of IL-1β were incubated for 24 hours at 37 ° C and 5% CO 2 incubation. CD4 + T cells were isolated from the umbilical cord blood-derived mesenchymal stem cells and mononuclear cells (MNCs) Respectively.
1 × 10 6 sorted CD4 + T cells were placed in the lower chamber of the 24-well plate, and the mesenchymal stem cells of each culture condition were placed in the upper chamber. At this time, after the composition of the medium suitable for differentiating CD4 + T cells into Th1 cells or Th17 cells in the lower chamber, Th1 cells were cultured for 4 days, Th17 cells were cultured for 7 days, and the culture medium was recovered to measure secreted factors, T cell differentiation. The composition of each culture medium is shown in Table 2 below.
rIL-2 (2.78 pg / ml)
rIL-12 (2.5 ng / ml)
anti-IL-4 (5 [mu] g / ml)
IL-1? (20 ng / ml),
IL-6 (30 ng / ml),
IL-23 (30 ng / ml),
TGF-β1 (2.25 ng / ml),
anti-IFN-y (1 [mu] g / ml)
anti-IL-4 (2.5 μg / ml)
In order to confirm the effect of inhibiting the differentiation of CD4 + T cells into Th1 cells (after
As a result, IFN- [gamma] and IL-1 [beta] -mediated mesenchymal stem cells and CD4 + T cells were more proliferated than IFN- [gamma] and / or IL-1 beta untreated mesenchymal stem cells and CD4 + When co-cultured, it was confirmed that the secretion amount of IFN-y and IL-17 was further reduced, thereby confirming that the effect of suppressing the differentiation of CD4 + T cells into Th1 and Th17 cells was more excellent (Fig. 6).
Example 7: IFN -γ and IL- 1β Changes in T cell mobility by treated stem cells
To evaluate the influence of IFN-y or / and IL-1? Treated stem cells on the migration of CD4 + T cells, mesenchymal stem cells and mononuclear cells cultured in the culture medium containing the two cytokines MNC) were fluorescently stained and co-cultured for 4 hours.
As a result, it was confirmed that the mesenchymal stem cells cultured by pre-treatment with IFN-y and / or IL-1? Recruit more CD4 + T cells than the mesenchymal stem cells cultured in the medium without treatment (Fig. 7).
In addition, MSCs pretreated with both cytokines were replaced with fresh medium containing no cytokine, and the mobility of CD4 + T cells to the culture medium secreted from the cells was evaluated by the same method as above for 3 days. Mobility was obtained by obtaining three or more images taken at random and counting the number of cells with fluorescence.
As a result, it was confirmed that the number of CD4 + T cells in which the two cytokines migrated into the culture medium of the pretreated cells was large (FIG. 8).
This suggests that factors affecting T cell mobility are secreted in IFN-γ and IL-1β-treated mesenchymal stem cells.
Example 8: Treatment of inflammatory cytokines in stem cells CCR7 Expression level increase
Recently, expression of the CC-chemokine receptor 7 (CCR7) factor has been suggested as a direction of development of Graft-versus-host disease (GVHD) therapy (Coghill JM et al ., Blood. 2010
In order to examine whether the expression of CC-chemokine receptor 7 (CCR7) in stem cells is increased by pretreatment of IFN-γ and IL-1β, IFN-γ and IL-1β alone or in combination And the expression level of CCR7 was confirmed by RT-PCR over time. The primer sequences used therein are shown in Table 3 below.
As a result, it was confirmed that expression of CCR7 gene was increased at the RNA level when interleukin-1 beta (IL-1?) Was treated (FIG. 9).
Example 9: Acute Graft-versus-host disease (Acute GVHD) In animal models induced IFN -γ and IL-1β-treated stem cells
9-1: Acute Graft-versus-host disease Induction and injection of stem cells
Acute GVHD induction model The animals used in the experiment were mice (C57 / BL6 mouse (Donor), BDF1 (Recipient)), 8-week-old mice were purchased, and after 7 days of purifying period, Was used for the experiment. Bone marrow and spleen were sacrificed by C57 / BL6 mouse (donor) and then bone marrow cells (10 × 10 6 ) and spleen T cells (2 × 10 6 ) were administered to BDF1 (Recipient) [F1 of C57BL / 6X DBA / Transplantation resulted in acute graft versus host disease. On the third day after induction, the solvent control substance or the test substance was administered in a single dose into the subcutaneous vein of the mouse model. The administration timing and dosage for each group are shown in FIG. 10 and Table 4 below. In the case of cytokine-treated stem cells, 20 ng / ml of IFN-γ and 5 ng / ml of IL-1β were cultured for 24 hours when the confluency of cells in the adherent culture reached about 50% -70% The posterior cells were collected, washed three times, and then administered.
(PBS group)
(MSC 0.3)
Exclusive
(MSC 0.3 + IFN? / IL-1?)
+ IFN-? / IL-1?
(MSC 1)
Exclusive
(MSC1 + IFN? / IL-1?)
+ IFN-? / IL-1?
(MSC 3)
Exclusive
(MSC3 + IFN? / IL-1?)
+ IFN-? / IL-1?
9-2: IFN -γ and IL- 1β The treatment effect of treated stem cells was verified at the level of gross lesion
The survival rate of the mouse model was observed until 30 days after induction of acute graft versus host disease as described above. Kaplan-Meier was used to compare survival rates, and statistical significance was assessed by log-rank method between control and test groups.
In addition, the solvent control substance and the test substance were administered on the third day after inducing acute graft versus host disease, and the GVHD score was measured at two check points per week. To compare the GVHD score changes, the repeated measures ANOVA was performed and the statistical significance between the control group and the test group was examined by the Greenhouse-Geisser method (p <0.05). In addition, the Tukey method was additionally performed for the post test.
On the other hand, the criteria for measuring the GVHD score are shown in Table 5 below.
(Weight loss)
decrease
(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, there was no difference in the survival rate between the control and the test groups, and it was confirmed that the addition of the culture medium did not deteriorate the survival rate when the groups were compared (FIG. 11).
On the other hand, in the case of GVHD score, it was confirmed that the symptom of acute GVHD was alleviated significantly when the total number of mesenchymal stem cells was 3 × 10 6 or more, and in the case of mesenchymal stem cells supplemented with culture medium, And the number of acute GVHD symptoms was significantly reduced even at 1 × 10 6 (FIG. 12).
9-3: IFN -γ and IL- 1β Histopathological examination of the therapeutic effect of treated stem cells
In order to confirm the therapeutic effect of IFN-y and IL-1 beta on graft-versus-host disease, autopsy was performed on
As a result, there was no significant difference between the groups in terms of skin tissue and liver tissue analysis (Fig. 13A and Fig. 13B), but in the small intestine tissue analysis, 1 x 10 6 of mesenchymal stem cells (Fig. 13C). When all of the GVHD scores measured in the skin, liver, and small intestine were summed, the number of mesenchymal stem cells to which the culture medium was added was the smallest at 1 × 10 6 cells (FIG. 13d).
The results of this study confirmed the clinical indications and histologic findings of the effects of the mesenchymal stem cells supplemented with the culture medium on the improvement of GVHD symptoms in the acute GVHD mouse model, and 1 × 10 6 mesenchymal stem cells IFN-γ and IL-1β were added to the culture medium, which showed the same therapeutic effect as that of 3 × 10 6 mesenchymal stem cells.
Example 10: In an animal model that induced Crohn's disease IFN -γ and IL- 1β Identification of therapeutic effect of treated stem cells
10-1: Induction of Crohn's disease and injection of stem cells
The Crohn's disease-induced model of the present invention is a dextran sulfate-induced colitis model (DSS) -induced colitis model, which is a chemo-induced colitis mouse model, , And the degree of inflammation of the period is easy to control. The animals used in this experiment were mice fed C57 / B16 8-week-old mice. After 7 days of purification, mice were used at 9 weeks of age. Inflammatory bowel disease (Crohn's disease) was treated with 3% (w / v) And was induced by supplying water in a week. On the first day after induction, a solvent control substance or test substance was administered into the abdominal cavity of the mouse model in a single dose. The administration timing and dosage for each group are shown in Table 6 below. In the case of cytokine-treated stem cells, 20 ng / ml of IFN-γ and 5 ng / ml of IL-1β were cultured for 24 hours when the confluency of cells in the adherent culture reached about 50% -70% The posterior cells were collected, washed three times, and then administered.
(PBS group)
(PBS group)
(MSC)
(MSC + IFN gamma)
+ IFN?
(MSC + IL-1?)
+ IL-1?
(MSC + IFN? / IL-1?)
+ IFN? / IL-1?
10-2: IFN -γ and IL- 1β The treatment effect of treated stem cells was verified at the level of gross lesion
The survival rate and body 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 improvement in weight loss and the survival rate were increased when compared with untreated cells or untreated cells in mesenchymal stem cells in which the two substances were combined (Figs. 14 and 15 ).
Unless otherwise defined, 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.
<110> Kangstem Biotech CO., LTD. <120> Pharmaceutical composition comprising stem cells treated with Interferon-gamma or Interleukin-1beta, or culture thereof for prevention and treatment of immune diseases and inflammatory diseases <130> KPA141006-KR-P1 <150> KR 10-2014-0128178 <151> 2014-09-25 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward primer <400> 1 tgagcatcta cggtttgctg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse primer <400> 2 tgcttgtctg gaacaactgc 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IDO forward primer <400> 3 actgtgtcct ggcaaactgg aag 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IDO reverse primer <400> 4 aagctgcgat ttccaccaat agag 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCR7 forward primer <400> 5 gtggttttac cgcccagaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCR7 reverse primer <400> 6 cactgtggtg ttgtctccga 20
Claims (25)
Wherein the cells are treated with 10-20 ng / ml IFN-? And 2.5-10 ng / ml IL-1? And cultured for 12-36 hours.
A composition for once administration of stem cells, comprising 1 to 100 ml of a culture medium obtained by culturing stem cells by treating an inflammatory cytokine of interferon-gamma (IFN-y) and interleukin-1 beta (IL-1?). Wherein the stem cell-1 administration composition has a stem cell treatment effect of 2 to 3 times the stem cell single dose.
Culturing the stem cells by adding an inflammatory cytokine of interferon-gamma (IFN-?) And interleukin-1 beta (IL-1?); And administering said stem cells,
The stem cells targeted to the secondary lymphoid organs are more likely to express CCR7 (CC-chemokine receptor type 7) gene expression than stem cells not treated with interferon-gamma (IFN-?) And interleukin-1 beta ≪ / RTI >
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| WO2019216450A1 (en) * | 2018-05-08 | 2019-11-14 | 주식회사 휴코드 | Method for culturing stem cells secreting anti-inflammatory components, and anti-inflammatory composition containing said stem cell culture medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101512171B1 (en) * | 2012-02-22 | 2015-04-16 | 주식회사 강스템바이오텍 | A composition comprising stem cell for preventing or treating of immune or inflammatory disease |
-
2015
- 2015-09-25 KR KR1020150136755A patent/KR20160037113A/en not_active Application Discontinuation
- 2015-09-25 WO PCT/KR2015/010253 patent/WO2016048107A1/en active Application Filing
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| WO2019135645A1 (en) * | 2018-01-05 | 2019-07-11 | 재단법인 아산사회복지재단 | Composition for improving, preventing or treating skin disease comprising induced pluripotency stem cell-derived mesenchymal stem cells pretreated with interferon gamma and exosomes derived therefrom |
| KR20190083933A (en) * | 2018-01-05 | 2019-07-15 | 재단법인 아산사회복지재단 | Composition for Improving Skin Conditions, or Preventing or Treating Skin Diseases comprising Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells pre-treated with Interferon-gamma, and Exosomes derived therefrom |
| US11865141B2 (en) | 2018-01-05 | 2024-01-09 | The Asan Foundation | Composition for improving, preventing or treating skin disease comprising induced pluripotency stem cell-derived mesenchymal stem cells pretreated with interferon gamma and exosomes derived therefrom |
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| JP2021526360A (en) * | 2018-05-08 | 2021-10-07 | ヒューコードラボ カンパニー リミテッド | An anti-inflammatory composition containing a method for culturing stem cells secreting an anti-inflammatory component and a stem cell culture medium thereof. |
| KR102260243B1 (en) * | 2020-07-22 | 2021-06-03 | (주)세렌라이프 | Composition for anticancer comprising stem cell having enhanced efficacy |
| CN113832099A (en) * | 2021-10-13 | 2021-12-24 | 浙江领蔚生物技术有限公司 | Mesenchymal stem cell preparation for preparing medicine for treating rheumatoid arthritis |
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