High-purity and high-efficacy cell therapeutic agent comprising decidua-derived stromal cells

Abstract

The present invention relates to a population of decidua-derived stromal cells, a high-purity and high-efficacy therapeutic pharmaceutical composition comprising same, and use thereof. Placenta-derived by-products formed after being detached from the maternal uterus during childbirth can be isolated and cultured so as to be utilized as various therapeutic pharmaceutical compositions.

Description

High-purity, high-efficacy cell therapy containing decidua-derived stromal cells

[0001] The present invention relates to a population of stromal cells derived from the decidua, a high-purity, high-efficacy therapeutic pharmaceutical composition comprising the same, and uses thereof.

[0002] Recent biotechnology offers new solutions to food, environmental, and health issues, with human welfare as the ultimate goal. Among these, stem cell technology is emerging as a new frontier in the treatment of incurable diseases. Previously, organ transplantation and gene therapy were proposed to treat incurable diseases, but effective practical application was hindered by immune rejection, organ shortages, vector development, and a lack of knowledge about disease genes. This has led to a surge in interest in stem cells, leading to the recognition that pluripotent stem cells, capable of forming all organs through proliferation and differentiation, hold the potential to treat most diseases and fundamentally resolve organ damage. Furthermore, numerous scientists have proposed diverse applications of stem cells, ranging from the regeneration of virtually all organs in the human body to the treatment of previously incurable diseases such as Parkinson's disease, various cancers, diabetes, spinal cord injuries, and immune disorders.

[0003] Stem cells are undifferentiated cells that have the ability to self-replicate and differentiate into two or more different types of cells. Stem cells can be divided into embryonic stem cells and adult stem cells based on their cytological origin. Embryonic stem cells are derived from fertilized eggs or developing fetal tissues, whereas adult stem cells are derived from tissues of the individual after fetal growth is complete, such as bone marrow, umbilical cord blood, fat, placenta, muscle, synovium, brain, liver, and pancreas. On the other hand, embryonic stem cells have ethical restrictions, which limit their use as cell therapy. In contrast, adult stem cells can be obtained mainly from bone marrow, fat, umbilical cord blood, and placenta, and thus do not raise ethical issues.

[0004] Among these, placental-derived stem cells have the advantage of being easy to collect and readily obtain large quantities of stem cells by utilizing the placenta, which is discarded after birth. The placenta, umbilical cord, and umbilical cord blood are fetal tissues and stem cells that can be obtained during birth. In particular, the placenta is a byproduct of the mother's uterus falling off and serves as a link between the fetus and the mother. Until recently, the placenta, umbilical cord, and umbilical cord blood were considered temporary organs discarded after birth. However, recent research has revealed that various cells exist in different parts of the placenta, such as mesenchymal cells, decidua cells, amnion cells, and endothelial cells.

[0005] Against this backdrop, the inventors of the present invention have made extensive research efforts to isolate cell populations other than stem cells derived from the placenta and develop a technology for utilizing them.

[0006] As a result, the present inventors completed the present invention by identifying cells isolated from the decidua surrounding the placenta and developing a method for utilizing such cells.

[0007] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating an immune disease, which comprises cells isolated from stromal cells derived from the decidua surrounding the placenta.

[0008] Another object of the present invention is to provide a method for preventing or treating an immune disease, which comprises administering to a subject in need thereof a pharmaceutical composition for preventing or treating an immune disease, which comprises cells isolated from stromal cells derived from the decidua surrounding the placenta.

[0009] Another object of the present invention is to provide a use of stromal cells derived from the decidua surrounding the placenta for preparing a pharmaceutical composition for preventing or treating an immune disease.

[0010] Against this backdrop, the inventors of the present invention have made extensive research efforts to isolate cell populations other than stem cells derived from the placenta and develop a technology for utilizing them.

[0011] As a result, the inventors of the present invention completed the present invention by identifying stromal cells isolated from the decidua surrounding the placenta and developing a method for utilizing such cells.

[0012] Hereinafter, the configuration of the present invention will be described in detail.

[0013] One aspect of the present invention is a pharmaceutical composition for preventing or treating an immune disease, comprising cells isolated from stromal cells derived from the decidua surrounding the placenta.

[0014] In the present invention, the decidua-derived stromal cells may be substantially maternally derived. Substantially maternally derived may mean, but is not limited to, that the decidua-derived stromal cells are at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% maternally derived.

[0015] As used herein, “mother” means the body of a woman who is pregnant with an offspring.

[0016] In this specification, “placenta” refers to the internal tissue created for the fetus during pregnancy, and is a disc-shaped organ weighing 500 to 600 g, with a diameter of 15 to 20 cm, and a thickness of 2 to 3 cm. One side of the placenta is in contact with the mother, and the other side is in contact with the fetus, and between them, nutrients and oxygen are transferred between the mother’s blood and the fetal blood vessels. The membranes surrounding the placenta can be broadly divided into the amnion, amniotic epithelial cells, chorion, and decidua.

[0017] In this specification, the “membrane surrounding the placenta” can be classified into the placental region, which is the area where the placenta remains, and the reflected region, which is the area where the membrane surrounding the fetus (dedidua) remains.

[0018] In this specification, the “placental zone” may be divided into the peri-placental zone or the proximal amnion. The placental zone may also be divided into the placental chorion and the placental amnion.

[0019] As a result of observing the cell morphology in one embodiment of the present invention, the placental chorion showed a spindle shape, and the placental amniotic membrane showed a small, round shape.

[0020] In this specification, “placental zone and distal portion” may be defined as the portion proximal to the cervix, which may be classified as the cervical / apical zone or the distal amnion.

[0021] In the present invention, the decidua-derived stromal cells may be derived from the reflected region, but are not limited thereto.

[0022] In the present invention, the reflex zone may be a part close to or partially overlapping with the cervix / apical zone or the distal part of the amniotic membrane, but is not limited thereto.

[0023] In this specification, the “reflected region” can be classified as the area close to or partially overlapping the cervix / apex or the distal amniotic membrane. The chorion, amnion, and decidua of the reflected region can be classified as the reflected chorion, reflected amnion, and reflected decidua.

[0024] In one embodiment of the present invention, as a result of observing the cell morphology, the reflective chorion showed a branched shape, the reflective amnion showed a small, round shape, and the reflective decidua showed a cell morphology in which both branched and round shapes were mixed.

[0025] In the present invention, the reflection area includes a mid-zone.

[0026] The above midzone may be located between the point of contact between the reflex zone and the cervix / apical zone and the point of contact between the reflex zone and the peri-placental zone.

[0027] In one embodiment of the present invention, analysis of the short-term sequence of each cell revealed that both the placental region and the reflective region were composed of a mixture of maternal and fetal cells. However, the area where the reflective decidua and the midzone of the reflective region overlapped did not contain any fetal cells, confirming that the entire area consisted solely of maternal cells.

[0028] In this specification, cells in the area where the reflective decidua and the midzone overlap in the reflective area may be referred to as decidual stromal cells (DSCs) or human placenta derived decidual stromal cells (hP-DSCs).

[0029] In this specification, “stromal cells” is a concept distinct from mesenchymal stem cells (MSCs).

[0030] In the present invention, the decidua-derived stromal cells are cells without self-renewal ability in which the Oct-4 (POU5F1) gene is not substantially expressed, for example, less than 5%, for example, less than 4%, for example, less than 3%. The decidua-derived stromal cells exhibit a difference in self-renewal ability due to the difference in the Oct-4 expression rate from stem cells characterized in that the Oct-4 gene is substantially expressed, for example, more than 90%, for example, more than 95%, for example, more than 98%.

[0031] In one embodiment of the present invention, the expression patterns of immunoregulatory factors of cells derived from reflex decidua (R-Decidua) and bone marrow-derived mesenchymal stem cells (BM-MSC) were analyzed, and reflex decidua-derived stromal cells expressed PD-L1 and PD-L2 at higher levels than BM-MSC, indicating superior immunoregulatory capabilities. In addition, they expressed CD49d, VCAM-1, and ICAM-1 at higher levels than BM-MSC, demonstrating excellent homing effects to inflamed tissues.

[0032] In the present invention, the decidua-derived stromal cells may express one or more positive CD markers selected from the group consisting of CD20, CD44, CD73, CD90, and CD105, but are not limited thereto.

[0033] In one embodiment of the present invention, cell surface antigens of placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua were analyzed according to the standards of the International Society for Cellular Therapy (ISCT), and decidual-derived stromal cells expressed at least one CD marker selected from the group consisting of CD20, CD44, CD73, CD90, and CD105.

[0034] In the present invention, the decidual membrane-derived stromal cells may express one or more negative CD markers selected from the group consisting of CD45, CD34, CD31, HLA-DR, and CD86, but are not limited thereto.

[0035] In one embodiment of the present invention, cell surface antigens of placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua were analyzed according to the standards of the International Society for Cellular Therapy (ISCT), and decidual-derived stromal cells did not express one or more CD markers selected from the group consisting of CD45, CD34, CD31, HLA-DR, and CD86.

[0036] In the present invention, the decidua-derived stromal cells may have increased expression of one or more immunoregulatory factors selected from the group consisting of PD-L1, PD-L2, CD49d, and ICAM-1, but are not limited thereto.

[0037] In one embodiment of the present invention, the expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua were analyzed, and the expression of PD-L1, PD-L2, CD49d, and ICAM-1 was found to be higher in reflected decidua than in other cells. These results indicate that reflected decidua can be utilized as a pharmaceutical composition for the prevention or treatment of diseases related to immunoregulation.

[0038] In the present invention, the decidua-derived stromal cells may express at least one gene selected from the group consisting of FAM105A, VAT1L, ST6GAL2, and TRBV3-1 at a higher level than mesenchymal stem cells, but are not limited thereto.

[0039] In the present invention, high expression compared to mesenchymal stem cells means that decidua-derived stromal cells express at least 5 times, for example, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times higher than the expression of one or more genes selected from the group consisting of FAM105A, VAT1L, ST6GAL2, and TRBV3-1 of mesenchymal stem cells.

[0040] In the present invention, the mesenchymal stem cell may be a bone marrow-derived mesenchymal stem cell (BM-MSC), but is not limited thereto.

[0041] In one embodiment of the present invention, decidua-derived stromal cells were found to express FAM105A at a level of about 20% or more, VAT1L at a level of about 15% or more, ST6Gal2 at a level of about 11% or more, and TRBV3-1 at a level of about 10% or more compared to mesenchymal stem cells.

[0042] In this specification, “FAM105A” may be referred to as “OTULINL (OTU deubiquitinase with linear linkage specificity like)”, and in humans (Homo sapiens), it may be located at NC_000005.10 (14581792..14616180), NC_060929.1 (14520844..14555231) or NC_000005.9 (14581901..14616289) of chromosome 5, but is not limited thereto.

[0043] In this specification, “FAM105A” may be, but is not limited to, an mRNA having the unique number NM_019018.

[0044] In this specification, “VAT1L (vesicle amine transport 1 like)” may be located at NC_000016.10 (77788564..77980107), NC_060940.1 (83844682..84036081) or NC_000016.9 (77822461..78014004) of chromosome 16 in humans (Homo sapiens), but is not limited thereto.

[0045] In this specification, “VAT1L” may be, but is not limited to, mRNA having the unique number NM_020927.

[0046] In this specification, “ST6Gal2” may be located at NC_000002.12 (106801600..106887277, complement), NC_060926.1 (107263027..107350150, complement) or NC_000002.11 (107418056..107503565, complement) of chromosome 2 in humans (Homo sapiens), but is not limited thereto.

[0047] In this specification, “ST6Gal2” may be, but is not limited to, an mRNA having the unique number NM_001142351.

[0048] In the present specification, “TRBV3-1” may be located at NC_000007.14 (142308589..142309048), NC_060931.1 (143623672..143624131) or NC_000007.13 (142008412..142008871) of chromosome 7 in humans (Homo sapiens), but is not limited thereto.

[0049] In this specification, “TRBV3-1” may be, but is not limited to, mRNA having the unique number BC070387.

[0050] In this specification, a “preventive or therapeutic pharmaceutical composition” may include a cell therapy agent. “Cell therapy agent” refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogenic, and xenogenic cells in vitro or changing the biological characteristics of cells to restore the functions of cells and tissues. The United States has managed cell therapy agents as medicines since 1993, and Korea since 2002. These cell therapy agents can be broadly classified into two categories: the first is stem cell therapy agents for tissue regeneration or organ function recovery, and the second is immune cell therapy agents for regulating immune responses, such as suppressing or enhancing immune responses in vivo.

[0051] The pharmaceutical composition of the present invention may be administered via any common route as long as it can reach the target tissue. Examples of parenteral administration include, but are not limited to, intraperitoneal administration, intravenous therapy (iv), intramuscular administration, subcutaneous administration, intradermal administration, and direct administration.

[0052] The pharmaceutical composition of the present invention may include a therapeutically effective amount of decidua-derived stromal cells for the treatment of a disease. The term "therapeutically effective amount" refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human, as deemed by a researcher, veterinarian, physician, or other clinician, including an amount that induces alleviation of symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the cells included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell content can be readily determined by those skilled in the art and can be adjusted according to various factors, including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the excretion rate of the composition, the duration of treatment, and concurrently used drugs. It is important to consider all of the above factors and include an amount that achieves the maximum effect with the minimum amount without side effects.

[0053] It is obvious to those skilled in the art that the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell content can be easily determined by those skilled in the art and can be adjusted according to various factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the secretion rate of the composition, the treatment period, and concurrently used drugs. It is important to include an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration. For example, the decidua-derived stromal cells of the present invention are 1×10 per 1 kg of body weight. 5　 5×10 7 Number of cells, e.g., 1×10 6　 1×10 7Cell counts may be included.

[0054] The above pharmaceutical composition may be formulated into a suitable form together with a pharmaceutical carrier commonly used in cell therapy. “Pharmaceutically acceptable” refers to a composition that is physiologically tolerable and does not typically cause allergic reactions or similar reactions, such as gastrointestinal upset or dizziness, when administered to humans. Pharmaceutically acceptable carriers include, for example, parenteral carriers such as water, suitable oils, saline solutions, aqueous glucose, and glycols, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0055] In the present invention, a pharmaceutical composition containing decidua-derived stromal cells can be used for the purpose of treating or preventing various immune diseases. Specifically, immune diseases include graft-versus-host disease (GvHD), ulcerative colitis, bleeding cystitis, acute respiratory distress syndrome (ARDS), Crohn's disease, Behcet's disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, and pernicious anemia. (pernicious anemia), glomerulonephritisb pulmonary fibrosis, inflammatory bowel disease, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD), Graves disease,It may be one or more selected from the group consisting of, but not limited to, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, and small artery disease.

[0056] In addition, in the present invention, the immune disease may be an “inflammatory disease,” and the inflammatory disease means a disease caused by an inflammatory substance (inflammatory cytokine) such as TNF-a (Tumor necrosis factor-a), IL-1 (Interleukin-1), IL-6, IL-17, prostaglandin, leukotriene, or nitric oxide (NO) secreted from immune cells such as macrophages by excessively stimulating the human immune system due to harmful stimuli such as inflammatory factors or radiation exposure.

[0057] In the present invention, “graft-versus-host disease (GVHD)” refers to a disease in which the patient’s body recognizes T lymphocytes in the peripheral blood or bone marrow of the donor injected during allogeneic transplantation as those of another person, causing an immune response.

[0058] In one embodiment of the present invention, the disease-inhibiting effect of reflective decidua-derived stromal cells was confirmed in a mouse model of graft-versus-host disease (GVHD). The survival rate of mice treated with reflective decidua-derived stromal cells remained constant compared to the control group. Furthermore, it was confirmed that reflective decidua-derived stromal cells exhibited the same effect of suppressing GVHD even after freezing and thawing.

[0059] In the present invention, “inflammatory disease (IBD)” refers to chronic inflammation of unknown cause occurring in the intestines. It usually refers to idiopathic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, but Behcet's disease, which is relatively common in Korea, can also be said to fall into this category. In a broad sense, it refers to infectious enteritis such as bacterial, viral, amebic, and tuberculous enteritis, as well as inflammatory diseases occurring in the intestines such as ischemic enteropathy and radiation enteritis.

[0060] In one embodiment of the present invention, the disease-inhibiting effect of reflective decidua-derived stromal cells was confirmed in a mouse model of inflammatory bowel disease. The survival rate of mice treated with reflective decidua-derived stromal cells remained constant compared to the control group. In particular, the survival rate of mice administered reflective decidua-derived stromal cells was higher than that of mice administered bone marrow-derived mesenchymal stem cells, and histological findings confirmed that intestinal damage was protected, similar to normal findings.

[0061] In the present invention, it was confirmed that decidua-derived stromal cells can be transplanted into patients with immune rejection reactions to transplantation of skin, blood, cornea, liver, lung, intestine, pancreas, heart, kidney, bone marrow, stem cells, stromal cells or progenitor cells, graft-versus-host disease, inflammatory bowel disease, allergic disease, autoimmune disease or inflammatory disease, and can be widely used for the treatment and / or prevention of the above diseases.

[0062] In the present invention, “treatment” means reversing, alleviating, inhibiting the progression of, or preventing a disease or condition, or one or more symptoms of said disease or condition.

[0063] Another aspect of the present invention is a method for preventing or treating an immune disease, comprising administering to a subject in need thereof a pharmaceutical composition for preventing or treating an immune disease, the pharmaceutical composition comprising cells isolated from stromal cells derived from the decidua surrounding the placenta.

[0064] Another aspect of the present invention is the use of stromal cells derived from the decidua surrounding the placenta for preparing a pharmaceutical composition for preventing or treating an immune disease.

[0065] Another aspect of the present invention is a method for isolating stromal cells derived from the decidua, comprising the following steps:

[0066] a) a step of separating cells from the membrane surrounding the placenta; and

[0067] b) A step of confirming whether the cells isolated in step a) express at least one selected from the group consisting of FAM105A, VAT1L, ST6GAL2, and TRBV3-1.

[0068] In the present invention, the step of separating cells in step a) may include, but is not limited to, the step of separating stromal cells from among the cells in step c).

[0069] The step of isolating the stromal cells of the above step c) is a step d) of treating the cells with a cell lysing agent to remove cells other than the stromal cells, such as red blood cells (RBCs), white blood cells (WBCs), or epithelial cells; and

[0070] e) It may include a step of subculturing the cells obtained in step d) 3 to 10 times, 3 to 8 times, 3 to 6 times, for example, 3 to 5 times, and allowing them to attach, but is not limited thereto.

[0071] In the present invention, the step of removing cells other than the substrate cells in step d) may include, but is not limited to, a step of treating with a cell lysing agent to cause a reaction and then adding a xeno-free culture additive to stop the reaction.

[0072] In the present invention, the cell lysing agent may be at least one selected from the group consisting of EDTA (ethylene-diamine-tetraacetic acid), trypsin, Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride; pH 7.0 to 7.5), NaCl, SDS 0.05 to 0.15, polyethylene glycol tert-octylphenyl ether, and CaCl2 0.05 to 0.15 (w / v)%, but is not limited thereto.

[0073] The present invention relates to a population of stromal cells derived from the decidua, a high-purity, high-efficacy therapeutic pharmaceutical composition comprising the same, and a use thereof. By-products derived from the placenta that are shed from the uterus of a mother during childbirth can be separated and cultured, and utilized as various therapeutic pharmaceutical compositions.

[0074] Figure 1 is a diagram showing the anatomical location and cellular morphology of the placenta and the amnion, chorion, and decidua surrounding it.

[0075] Figure 2 is a diagram showing the structure of the placenta and the membranes surrounding it.

[0076] Figure 3 is a diagram showing the location of the mid-zone, which is the area between the placental-peripheral area and the cervix / apex area.

[0077] Figure 4 is a diagram showing the anatomical locations of the placental region and reflected region of the membrane surrounding the fetus.

[0078] Figure 5 is a diagram showing the cellular structure and location of the placental region and reflected region of the membrane surrounding the fetus.

[0079] Figure 6 is a diagram showing the results of microscopic observation of specific cell morphologies of placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0080] Figure 7 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, the reflected region, and the midzone portion of the reflected region.

[0081] Figure 8 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0082] Figure 9 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0083] Figure 10 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0084] Figure 11 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0085] Figure 12 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0086] Figure 13 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0087] Figure 14 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0088] Figure 15 is a graph showing the results of short sequence (STR) analysis of cell origin in the placental region, reflected region, and midzone of the reflected region.

[0089] Figure 16 is a graph showing the results of analyzing cell surface antigens of placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0090] Figure 17 is a graph showing the results of analyzing the expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0091] Figure 18 is a graph showing the results of analyzing the expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0092] Figure 19 is a graph showing the results of analyzing the expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0093] Figure 20 is a graph showing the results of analyzing the expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0094] Figure 21 is a graph showing the results of analyzing the expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua.

[0095] Figure 22 is a diagram showing the results of analyzing the karyotype of R-Decidua.

[0096] Figure 23 is a graph showing the results of comparing the expression patterns of immunoregulatory factors in stromal cells derived from reflex decidua (R-Decidua) and mesenchymal stem cells derived from bone marrow (BM-MSC).

[0097] Figure 24 is a histogram showing the results of comparing the expressed genes of R-Decidua-derived stromal cells and bone marrow-derived mesenchymal stem cells (BM-MSC) using a microarray method.

[0098] Figure 25 is a graph showing the graft-versus-host disease inhibition effect in a group of mice administered R-Decidua-derived stromal cells.

[0099] Figure 26 is a diagram showing the histological protective effect of a group of mice administered R-Decidua-derived stromal cells.

[0100] Figure 27 is a graph showing the regulatory effect of helper T cells in a group of mice administered R-Decidua-derived stromal cells.

[0101] Figure 28 is a diagram and graph showing the inhibitory effect on inflammatory bowel disease in a group of mice administered R-Decidua-derived stromal cells.

[0102] Figure 29 is a diagram showing the histological protective effect of a group of mice administered R-Decidua-derived stromal cells.

[0103] Figure 30 is a photograph showing the therapeutic effect on inflammatory bowel disease in a group of mice administered R-Decidua-derived stromal cells.

[0104] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined solely by the scope of the claims.

[0105]

[0106] Manufacturing Example 1. Anatomical location and classification of the placenta

[0107] The placenta is an organ that develops in the uterus during pregnancy and is attached to the uterine wall. The umbilical cord emerges from the placenta. The membranes surrounding the fetus, including the placenta, can be divided into the amnion, chorion, and decidua. The amnion is the membrane that surrounds the fetus and amniotic fluid. The decidua is a membrane formed by the transformation of the mother's endometrium and is located in the area that contacts the uterus. The chorion is located between the decidua and the amniotic membrane (Figure 1).

[0108] Additionally, the structure of the placenta and the membrane surrounding it after delivery of the fetus can be classified into the placental region, which is the area where the placenta remains, and the reflected region, which is the area where the membrane surrounding the fetus (decidua) remains (Figure 2).

[0109] The placental zone and reflex zone can be further subdivided into the peri-placental zone or proximal amnion, the area closer to the placenta, and the cervix, the cervical / apical zone or distal amnion. The area between the peri-placental zone and the cervix / apical zone can be classified as the mid-zone, and the mid-zone may overlap with the reflex zone (Fig. 3).

[0110] When looking at the internal cross-section of the membrane surrounding the fetus, the inner side of the placental region is composed of the placental chorion and placental amnion, and the inner side of the reflected region is composed of the reflected decidua, reflected chorion, and reflected amnion (Figures 4 and 5).

[0111] In this specification, the reflected decidua portion is also called the maternal stromal cell population.

[0112]

[0113] Manufacturing Example 2. Isolation of placental-derived cells

[0114] Normal placentas were obtained with the informed consent of healthy mothers who had delivered normally at 37 weeks or more of pregnancy. The placentas and membranes isolated from the mothers were quickly transferred to a sterile container and washed with saline solution containing 1% penicillin-streptomycin antibiotics to remove contaminants. Next, a solution containing 2 mg / mL trypsin was added and the enzyme reaction was performed for 10 minutes in a shaking incubator at 37°C, after which a xeno-free culture supplement was added to stop the enzyme reaction. Then, after removing red blood cells, epithelial cells, and culture medium, only the obtained tissue was placed in a solution containing trypsin and centrifuged at 400 × g for 9 minutes to recover the cells. The recovered cells were cultured and grown three times in a medium containing the culture supplement.

[0115]

[0116] Example 1. Observation of cell morphology

[0117] The specific cell morphology of the placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua was observed under a microscope (Fig. 6).

[0118] The placental chorion (Figure 6, ① P-Chorion) and the reflective chorion (Figure 6, ③ R-Chorion) showed a spindle-shaped cell morphology. The placental amnion (Figure 6, ② P-Amnion) and the reflective amnion (Figure 6, ④ R-Amnion) showed a small, round-shaped cell morphology. And the reflective decidua (Figure 6, ⑤ R-Decidua) showed a cell morphology in which both the branched and round-shaped cells were mixed.

[0119] In the case of the reflective chorion, numerous granules were observed at the cell ends, which extended widely in both directions, showing a typical whirlpool pattern. The placental amnion and reflective amniotic membrane showed relatively small fibroblastic morphology, with cells extending only in one direction. The reflective decidua showed a morphology similar to the reflective chorion and showed a whirlpool pattern.

[0120]

[0121] Example 2. Short-term sequence analysis of cells

[0122] The cell origin of the placental region, reflected region, and midzone of the reflected region was confirmed by short sequence (STR) analysis.

[0123] It was confirmed that both the placental region and the reflective region were composed of a mixture of maternal and fetal cells. However, the area where the reflective decidua and the midzone of the reflective region overlapped did not contain any fetal cells, and it was confirmed that it was composed entirely of maternal cells (Fig. 7).

[0124] Short-stranded sequence (STR) analysis was performed on cells isolated from each tissue sample. Intercellular contamination was detected using personally identifiable DNA from maternal blood, umbilical cord blood, and amplified cells.

[0125] [Table 1]

[0126]

[0127] As a result, it was confirmed that only the reflective chorion (R-Chorion) and reflective decidua (R-Decidua) cell groups were 100% identical to maternal cells (Table 1, Figures 8 to 15).

[0128]

[0129] Example 3. Flow cytometry analysis of cells

[0130] Cell surface antigens of placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua were analyzed according to the criteria of the International Society for Cellular Therapy (ISCT).

[0131] As a result, it was confirmed as positive for CD markers such as CD29, CD44, CD73, CD90, and CD105, and negative for CD markers such as CD45, CD34, CD31, HLA-DR, and CD86 (Fig. 16).

[0132]

[0133] Example 4. Expression pattern of immunomodulatory factors

[0134] The expression patterns of immunoregulatory factors in placental chorion, placental amnion, reflected chorion, reflected amnion, and reflected decidua were analyzed.

[0135] As a result, it was confirmed that the expression of PD-L1, PD-L2, CD49d, and ICAM-1 was higher in the reflective decidua (R-Decidua) compared to other cells (Figs. 17 to 21).

[0136]

[0137] Example 5. Chromosome karyotype analysis

[0138] The karyotype of R-Decidua was analyzed. The chromosome count was determined from 20 metaphase cells, and at least two karyotype analyses were performed.

[0139] As a result, it was confirmed that the stromal cells derived from the reflective decidua had a normal karyotype 46, XX

[0020] with no numerical or structural abnormalities (Fig. 22).

[0140]

[0141] Example 6. Comparative evaluation with mesenchymal stem cells

[0142] The expression patterns of immunoregulatory factors in reflex decidua-derived cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) were compared by flow cytometry.

[0143] As a result, it was confirmed that the stromal cells derived from the reflective decidua had superior immunoregulatory ability by expressing PD-L1 and PD-L2 at higher levels than BM-MSCs, and had superior homing ability to inflamed tissues by expressing CD49d, VCAM-1, and ICAM-1 at higher levels than BM-MSCs (Figure 23).

[0144]

[0145] Example 7. Comparative evaluation of expressed genes

[0146] The expression of genes in stromal cells derived from reflex decidua (R-Decidua) and bone marrow-derived mesenchymal stem cells (BM-MSCs) was compared using a microarray method. The gene names, numbers, and mRNA identifiers of highly expressed genes, as well as the expression ratios in reflex decidua-derived stromal cells compared to bone marrow-derived mesenchymal stem cells, are shown in Table 2.

[0147] [Table 2]

[0148]

[0149] As a result, compared to bone marrow-derived mesenchymal stem cells, reflective decidua-derived stromal cells were found to express FAM105A by about 20% or more, VAT1L by about 15% or more, ST6Gal2 by about 11% or more, and TRBV3-1 by about 10% or more (Fig. 24).

[0150]

[0151] Example 8. Evaluation of inhibitory effects in a mouse model of immune disease

[0152] The immune disease suppression effect of stromal cells derived from the reflective decidua was evaluated.

[0153] A graft-versus-host disease (GVHD) model was created using BALB / c (H-2kd) mice as recipients and C57BL / 6 (H-2kb) mice as donors. The recipient mice were irradiated with 800 cGy of total body irradiation (TBI) as bone marrow ablation conditioning on the day of bone marrow transplantation, and 5 X 10 bone marrow cells (BM) isolated from the donor C57BL / 6 (H-2kb) mice were injected into the recipient mice. 6 and 5 X 10 spleen cells 6 Bone marrow transplantation was performed by injecting the donor mice into the tail vein.

[0154] Mice with graft-versus-host disease were induced and 5x10 stromal cells derived from the reflex decidua were injected into the venous region of the treated mice. 5 / 200ul was administered 3 times / on the day of bone marrow transplantation, 5 days after bone marrow transplantation, and 10 days after bone marrow transplantation, and the survival rate, weight loss rate, and clinical score were observed and evaluated.

[0155] Mice were divided into the following groups and evaluated.

[0156] A: Syn: BALB / c-> BALB / c autologous transplantation does not cause GVHD.

[0157] B: Allo: Group with GVHD development through C57BL / 6 -> BALB / c allogeneic transplantation

[0158] C: Fresh R-decidua: Group administered immediately after passage (p-3) of reflective decidua-derived stromal cells to mice with GVHD.

[0159] D: Cryo-Thawed R-decidua: A group in which cells derived from the reflective decidua were thawed from cells stored frozen in an LN2 tank and administered immediately within 1 hour to GVHD-inventing mice.

[0160] As a result, mice treated with decidua-derived stromal cells maintained a constant survival rate over time compared to the control group, showed suppressed body weight loss, and showed lower clinical scores. In addition, the graft-versus-host disease inhibitory effect of decidua-derived stromal cells was found to exhibit the same inhibitory effect in cells thawed after freezing (D) (Fig. 25).

[0161]

[0162] Example 9. Histological evaluation in a mouse model of graft-versus-host disease

[0163] In a mouse model of graft-versus-host disease, administration of reflective decidual-derived stromal cells demonstrated histologically protective effects. To confirm this, mouse small intestine, liver, and skin tissues were obtained, fixed in 4% formaldehyde, embedded in paraffin blocks, and stained with H&E for histological analysis.

[0164] As a result, infiltration of inflammatory cells was observed in the liver and skin, but normal histological findings were observed in the group administered stromal cells derived from the reflective decidua (Fig. 26).

[0165]

[0166] Example 10. Regulatory effect of helper T cells in a mouse model of graft-versus-host disease.

[0167] To determine the mechanism of action of reflective decidua-derived stromal cells in suppressing graft-versus-host disease in a mouse model of graft-versus-host disease, we analyzed subtypes of helper T cells.

[0168] As a control group, a graft-versus-host disease-induced group that was not administered reflective decidual-derived stromal cells was selected, and the group that was administered reflective decidual-derived stromal cells was compared. After isolating the spleen of each group, single cells were separated and the expression of CD4+IL-17+, which can be considered a Th1 phenotype, and CD4+CD25_Foxp3_, which is a regulatory T cell phenotype, was examined.

[0169] As a result, it was confirmed that the group administered with stromal cells derived from the reflective decidua had a superior effect in reducing the pro-inflammatory CD4 subtypes Th1 and Th17 compared to the control group, and increased the anti-inflammatory subtype Treg (Fig. 27).

[0170]

[0171] Example 11. Evaluation of survival rate, histology, and improvement effects in a mouse model of inflammatory bowel disease.

[0172] The effects of reflective decidua-derived stromal cells and bone marrow-derived mesenchymal stem cells on improving or treating inflammatory bowel disease were compared and evaluated using a mouse model in which enteritis was induced with dextran sulfate sodium (DSS).

[0173] Enteritis was induced in 6- to 8-week-old C57BL / 6 mice by continuously feeding water containing 2.5% dextran sulfate sodium instead of water for 6 days. Decidua-derived stromal cells and bone marrow-derived mesenchymal stem cells were administered intravenously on days 1 and 3. From days 5 to 10, the mice were returned to regular water and allowed to drink water ad libitum. The survival rate of the mice was assessed until day 18 (Fig. 28). On day 10, the mice were euthanized and dissected, and the colon was removed for histological analysis. For histological analysis, small intestinal tissue was obtained, fixed in 4% formaldehyde, embedded in paraffin blocks, and stained with H&E.

[0174] Mice were divided into the following groups and evaluated.

[0175] A: Normal: A group of normal mice that did not develop IBD.

[0176] B: IBD Control DSS: IBD onset group through negative numbers

[0177] C: IBD + Cryo R-decidua IBD: Group in which cells derived from the reflective decidua were thawed and administered to mice with IBD within 1 hour after being frozen in an LN2 tank.

[0178] D: IBD + Cryo BM-MSC IBD: A group in which bone marrow-derived mesenchymal stem cells were thawed from cells stored frozen in an LN2 tank and administered immediately within 1 hour to mice with IBD.

[0179] As a result, the mouse group treated with reflective decidua-derived stromal cells (IBD + Cryo R-decidua IBD) showed a higher histological protective effect than the mouse group treated with bone marrow-derived mesenchymal stem cells (IBD + Cryo BM-MSC IBD) (Fig. 29). In particular, both the IBD control group and the Cryo BM-MSC administration group showed infiltration of inflammatory cells and damaged intestinal villi, but in the Cryo R-decidua administration group, it was confirmed that intestinal damage was protected similar to normal histological findings. In addition, diarrhea and bloody stool were observed in the IBD control group and the Cryo BM-MSC administration group, whereas diarrhea and bloody stool were not observed in the Cryo R-decidua administration group, confirming that it exhibits an excellent effect in the treatment of inflammatory bowel disease (Fig. 30).

Claims

1. A pharmaceutical composition for preventing or treating an immune disease, comprising cells isolated from stromal cells derived from the decidua surrounding the placenta.

2. In paragraph 1, A pharmaceutical composition for preventing or treating an immune disease, wherein the decidua-derived stromal cells are at least about 90% maternally derived.

3. In paragraph 1, A pharmaceutical composition for preventing or treating an immune disease, wherein the above-mentioned decidual membrane-derived stromal cells are derived from the reflected region.

4. In paragraph 3, A pharmaceutical composition for preventing or treating an immune disease, wherein the above reflex zone is a part close to or partially overlapping with the cervix / apical zone or the distal part of the amniotic membrane.

5. In paragraph 3, The above reflection area includes the mid-zone, A pharmaceutical composition for preventing or treating an immune disease, wherein the midzone is located between the contact point of the reflex zone and the cervix / apical zone and the contact point of the reflex zone and the peri-placental zone.

6. In paragraph 1, A pharmaceutical composition for preventing or treating an immune disease, wherein the above decidual membrane-derived stromal cells express at least one positive CD marker selected from the group consisting of CD20, CD44, CD73, CD90, and CD105.

7. In paragraph 1, A pharmaceutical composition for preventing or treating an immune disease, wherein the above decidual membrane-derived stromal cells express at least one negative CD marker selected from the group consisting of CD45, CD34, CD31, HLA-DR, and CD86.

8. In paragraph 1, A pharmaceutical composition for preventing or treating an immune disease, wherein the above decidual membrane-derived stromal cells have increased expression of one or more immunoregulatory factors selected from the group consisting of PD-L1, PD-L2, CD49d, and ICAM-1.

9. In paragraph 1, A pharmaceutical composition for preventing or treating an immune disease, wherein the decidua-derived stromal cells express at least one gene selected from the group consisting of FAM105A, VAT1L, ST6GAL2, and TRBV3-1 at a higher level than mesenchymal stem cells.

10. In paragraph 1, The above immune diseases include graft-versus-host disease (GvHD), ulcerative colitis, bleeding cystitis, acute respiratory distress syndrome (ARDS), Crohn's disease, Behcet's disease, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, polymyositis, scleroderma, Addison's disease, vitiligo, and pernicious anemia. (pernicious anemia), glomerulonephritisb pulmonary fibrosis, inflammatory bowel disease, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD), Graves disease, gastrointestinal allergies, conjunctivitis, atherosclerosis,A method for preventing or treating an immune disease, wherein the immune disease is at least one selected from the group consisting of coronary artery disease, angina, cancer metastasis, and small artery disease.

11. A method for preventing or treating an immune disease, comprising administering to a subject in need thereof a pharmaceutical composition for preventing or treating an immune disease, the pharmaceutical composition comprising cells isolated from stromal cells derived from the decidua surrounding the placenta.

12. Use of stromal cells derived from the decidua surrounding the placenta for producing a pharmaceutical composition for preventing or treating an immune disease according to any one of claims 1 to 11.

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