Method for producing mesenchymal stem cell-like cells and mesenchymal stem cell-like cells produced by the method

Abstract

The present invention provides a method for producing mesenchymal stem cells-like from human pluripotent stem cells, and mesenchymal stem cells-like produced by the method. [Solution] The present invention relates to a method for producing mesenchymal stem cell mimics having anti-inflammatory and immunosuppressive properties, a therapeutic composition comprising said mesenchymal stem cell mimics, or a composition for preventing or treating a disease comprising a carrier, an active ingredient secreted from said mesenchymal stem cell mimics, or exosomes, by inducing differentiation of human pluripotent stem cells that have been subcultured 70 times or less after the establishment of a cell line and selecting cystic embryoid bodies, loading said cystic embryoid bodies onto a cell-permeable three-dimensional culture insert to isolate mesenchymal stem cell mimics, and isolating and homogenizing only the monolayer cell population from the cells that have permeated the cell-permeable three-dimensional culture insert.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing similar mesenchymal stem cells from human pluripotent stem cells and similar mesenchymal stem cells prepared by the method. Specifically, human pluripotent stem cells (HSTs) that have been passaged 70 times or less after the establishment of a pluripotent cell line are used. Cystic embryoid bodies are selected from various embryoid bodies obtained by inducing differentiation of these cells. Cystic embryoid bodies are then loaded onto a cell-permeable 3D culture unit to isolate similar mesenchymal stem cells. From the isolated similar mesenchymal stem cells, only monolayer-form cell populations are isolated and the size of the monolayer-form cell populations is homogenized to produce similar mesenchymal stem cells. The highly purified similar mesenchymal stem cells thus prepared have anti-inflammatory and immunosuppressive properties and express CD90 and SOX2 at 95% or higher. The present invention also relates to mesenchymal stem cell analogs produced by the above-mentioned production method, a therapeutic composition containing the mesenchymal stem cells, or a disease prevention or treatment composition or cosmetic composition containing a transmitter, an active ingredient or exosomes secreted from the mesenchymal stem cells. [Background technology]

[0002] Mesenchyme is the term given to the mesodermal tissue, which corresponds to the intermediate layer formed by the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Human embryonic stem cells and induced pluripotent stem cells are the only cells that can explain this early development in vitro. Because human embryonic stem cells and induced pluripotent stem cells have pluripotency, they can be used as a source of cells with a variety of functions.

[0003] Currently, research is being actively conducted on mesenchymal stem cells derived from human pluripotent stem cells, as well as mesenchymal progenitor cells. In adults, it was first discovered that bone-forming precursor cells, like mesenchymal stem cells, exist in the bone marrow. After that, it was reported that mesenchymal stem cells could be isolated from bone marrow, adipose tissue, peripheral blood, and fetal tissues such as umbilical cord blood and amniotic membrane.

[0004] These adult-derived mesenchymal stem cells have various properties that make them useful for cell therapy. Therefore, in actual clinical treatments, they are used in a wide range of areas, from the musculoskeletal system (such as bone, cartilage, and muscle), the cardiovascular system (such as myocardial infarction and vascular injury diseases), the respiratory system (such as acute and chronic lung injury), the autoimmune system (such as multiple sclerosis, lupus, and rheumatoid arthritis), and the nervous system (such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, and spinal cord injury). In other words, cell therapy is being attempted in a variety of fields.

[0005] However, unlike embryonic stem cells, typical adult mesenchymal stem cells exhibit limited proliferation ability when subcultured in vitro for long periods of time, with some cells known to divide no more than 40 times. Adult mesenchymal stem cells used in actual cell therapy are limited to those cultured for seven passages or less. Furthermore, there are no established cell lines, and repeated cell preparation from adults is required, limiting research.

[0006] To overcome these limitations, research is being conducted into isolating and culturing mesenchymal stem cells (MSCs) or mesenchymal progenitor cells (MSCs) from human pluripotent stem cells. Conventional methods for obtaining mesenchymal stem cells or progenitor cells from known human pluripotent stem cells include separating cells that express a desired marker using a fluorescent activated cell sorter (FACS) and treating cells with large amounts and a variety of cytokines and chemicals to induce differentiation.

[0007] However, the flow cytometer method uses lasers, which not only reduces cell viability but also results in fewer cells obtained after isolation and increases the culture period. Cytokine and chemical treatment methods have the problems of high cost due to continuous treatment and genetic stability after differentiation and proliferation. Furthermore, it is difficult to efficiently control the pluripotency of embryonic stem cells.

[0008] Because there are many different methods for isolating and expanding mesenchymal stem cells and approaches to characterizing them vary depending on the tissue and cell source, the International Society for Cell Therapy (ISCT) has proposed three criteria for defining human mesenchymal stem cells. First, human mesenchymal stem cells must be adherent when maintained under standard culture conditions. Second, when analyzing the surface antigens of human mesenchymal stem cells, CD90, CD44, CD73, and CD105 must be expressed in over 95% of cells, while CD45, CD34, CD14, and CD19 must be expressed in less than 2% of cells. Furthermore, the undifferentiated regulatory markers Oct3 / 4, Tra-1-60, and Tra-1-81, as well as the immune rejection antigen HLA-DR (Human Leukocyte Antigen-Antigen D Related), must not be expressed. Third, human mesenchymal stem cells must be differentiated in vitro into osteogenic cells, adipogenic cells, and chondrogenic cells.

[0009] CD90 (Cluster of Differentiation 90), a representative marker defining mesenchymal stem cells, is a key element involved in the immune regulation of mesenchymal stem cells. CD90-expressing mesenchymal stem cells upregulate sHLA-G and IL-10, and regulate the immune responses of peripheral blood mononuclear cells activated by phytohemagglutinin (PHA).

[0010] HLA-G (human leukocyte antigen G) is a non-classical MHC class I gene that exists in membrane-bound forms (HLA-G1, -G2, -G3, -G4) and soluble forms (sHLA-G5, -G6, and G7). It has been reported to exert tolerogenic functions, affecting innate and adaptive cellular responses, such as NK and CD81+ T cell cytotoxicity and CD41+ T cell activity. HLA-G is currently recognized as a molecule that plays an important role in immune tolerance. Furthermore, HLA-G is known to modulate decidual mononuclear cells (monocytes) to a Th2 (T helper type 2) cytokine profile through cytokine secretion modulation. The shift to a Th2 cytokine profile is associated with suppression of immune function. IL-10, a Th2 cytokine, has a wide range of biological functions, including anti-inflammatory and immunosuppressive effects. Numerous reports have shown that IL-10 induces HLA-G expression, and HLA-G also stimulates IL-10 expression.

[0011] SOX2 (Sex determining region Y-box2) is a core transcription factor known to be expressed in human embryonic stem cells and induced pluripotent stem cells, and plays an important role in maintaining cellular pluripotency. SOX2 is expressed in some adult stem cells and is known to have a significant effect on cell differentiation and proliferation through the regulation of DKK1, a lytic inhibitor of Wnt signaling, and cMyc. Therefore, the functions of SOX2 expressed in human pluripotent stem cells and SOX2 expressed in mesenchymal stem cells are considered to be separate and have different roles.

[0012] Currently, biological and clinical interest in stem cell therapeutics continues to grow, primarily due to cell characteristics such as immunosuppression and tolerance. The human pluripotent stem cell-derived similar mesenchymal stem cells of the present invention are mesenchymal stem cells characterized by expressing CD90 and SOX2 at 95% or more, which has never been reported before. This not only satisfies the interest in the relevant research field, but also has significant implications from a clinical perspective.

[0013] There is a need to develop a method for producing mesenchymal stem cells that can increase cell survival rates, produce highly purified mesenchymal stem cell-like cells, efficiently control human pluripotent stem cells, and maintain the genetic stability of the produced mesenchymal stem cell-like cells. Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention relates to a method for producing similar mesenchymal stem cells that meet the minimum standards for human mesenchymal stem cells established by the International Society for Stem Cell Therapy.

[0015] The present invention relates to a method for producing human pluripotent stem cells (HSTs) that have been subcultured 70 times or less since the establishment of a cell line; (b) selecting cystic embryoid bodies from among the embryoid bodies obtained by inducing differentiation of the HSTs; (c) isolating mesenchymal stem cell-like cells by loading the cystic embryoid bodies onto a cell-permeable three-dimensional culture insert; (d) isolating only a monolayer of cells from the cells that have permeated the cell-permeable three-dimensional culture insert; and (e) homogenizing the monolayer of cells to a size of 100 μm to 500 μm in length and width, and culturing the mesenchymal stem cell-like cells. The mesenchymal stem cell-like cells have anti-inflammatory and immunosuppressive properties and express CD90 and SOX2 at 95% or more. The present invention also relates to a method for producing HSTs and HST-derived HSTs produced by the method.

[0016] However, the problems to be solved by the present invention are not limited to those mentioned above. Further problems not mentioned above will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0017] (b) selecting cystic embryoid bodies from embryoid bodies obtained by inducing differentiation of the human pluripotent stem cells; (c) isolating the cystic embryoid bodies by loading the cystic embryoid bodies onto a cell-permeable three-dimensional culture insert; (d) isolating only monolayer cell populations from the cells that have permeated the cell-permeable three-dimensional culture insert; and (e) homogenizing the monolayer cell populations to a size of 100 μm to 500 μm in both length and width, and culturing the similar mesenchymal stem cells. The similar mesenchymal stem cells have anti-inflammatory and immunosuppressive properties and express CD90 and SOX2 at 95% or more.

[0018] In one aspect, the cell-permeable 3D culture insert may be made of one or more of nylon, fiber, polyethylene, polypropylene, graphene, titanium, copper, nickel, silver, gold, and platinum.

[0019] According to another embodiment of the present invention, there is provided a method for producing similar mesenchymal stem cells derived from human pluripotent stem cells, which are characterized in that the similar mesenchymal stem cells express MMP-1 protein and HGF protein at higher levels than bone marrow-derived mesenchymal stem cells, express CD95 at lower levels than bone marrow-derived mesenchymal stem cells, and express CD90 and SOX2 at 95% or more.

[0020] In one aspect, the analogous mesenchymal stem cells are provided that express the MMP-1 protein at a level 15 times or more higher and the HGF protein at a level two times higher than bone marrow-derived mesenchymal stem cells.

[0021] In one aspect, the mesenchymal stem cell analog has 15-fold or more lower expression of CD95 than bone marrow-derived mesenchymal stem cells.

[0022] According to one aspect, the analogous mesenchymal stem cells express inflammation-regulating genes at 2 to 100 times or more higher levels than bone marrow-derived mesenchymal stem cells, and the inflammation-regulating genes are any one or more of the group consisting of Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Acp5, Bcr, Socs5, and Mdk.

[0023] In one aspect, the analogous mesenchymal stem cells are provided as analogous mesenchymal stem cells that simultaneously express Gata3, Adora2a, and Gps2 genes.

[0024] According to one aspect, the mesenchymal stem cell analog has 2 to 100 times or more higher expression of immunosuppressive genes than bone marrow-derived mesenchymal stem cells, and the immunosuppressive genes are one or more of Gata3, Gps2, Psma1, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Bcr, Socs5, and Mdk.

[0025] In one aspect, the analogous mesenchymal stem cells are provided as analogous mesenchymal stem cells that simultaneously express Gata3, Gps2, and Psma1 genes.

[0026] According to a further embodiment of the present invention, there is provided a therapeutic composition comprising similar mesenchymal stem cells derived from human pluripotent stem cells produced by the method for producing similar mesenchymal stem cells, wherein the therapeutic composition is any one of a cell therapy composition, a cell gene therapy composition, a tissue engineering therapy composition, an anti-inflammatory therapy composition, an immunotherapy composition, and a composition for the prevention or treatment of cancer.

[0027] According to one aspect, the therapeutic composition further comprises an active ingredient or exosomes secreted from the similar mesenchymal stem cells.

[0028] In one aspect, the therapeutic composition is for preventing or treating any one or more of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic pulmonary disease, acute pulmonary disease, Crohn's disease, fecal incontinence, graft-versus-host disease, lower limb ischemia, thromboangiitis obliterans (Buerger's disease), foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injuries caused by various exercises, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage caused by burns, skin damage caused by ultraviolet rays, and inflammatory and immune system diseases including retinopathy; ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system diseases.

[0029] According to yet a further embodiment of the present invention, there is provided a delivery body comprising similar mesenchymal stem cells derived from human pluripotent stem cells produced by a method for producing similar mesenchymal stem cells, the delivery body being characterized in that it carries a pharmaceutical composition.

[0030] According to a further embodiment of the present invention, there is provided a composition for preventing or treating a disease, which comprises an active ingredient or exosomes secreted from mesenchymal stem cells derived from human pluripotent stem cells and produced by the method for producing mesenchymal stem cells.

[0031] In one aspect, the disease is one or more of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic pulmonary disease, acute pulmonary disease, Crohn's disease, fecal incontinence, graft-versus-host disease, lower limb ischemia, thromboangiitis obliterans, foot ulcers, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injuries caused by various exercises, knee cartilage injuries, tinnitus, atopic dermatitis, psoriasis, skin damage caused by burns, skin damage caused by ultraviolet rays, etc., prevention of inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system diseases.

[0032] According to a further embodiment of the present invention, there is provided a cosmetic composition comprising an active ingredient or exosomes secreted from mesenchymal stem cells derived from human pluripotent stem cells and produced by the method for producing mesenchymal stem cells. [Effects of the Invention]

[0033] The method for producing mesenchymal-like stem cells of the present invention enables the isolation, culture, and proliferation of mesenchymal-like stem cells with high efficiency and purity.Meanwhile, the method for producing mesenchymal-like stem cells of the present invention enables the production of novel human pluripotent stem cell-derived mesenchymal-like stem cells that have anti-inflammatory and immunosuppressive effects and express CD90 and SOX2 at levels of 95% or more from human pluripotent stem cells.

[0034] The mesenchymal stem cell-like cells produced by the production method of the present invention can exhibit anti-inflammatory effects and immunosuppressive abilities.

[0035] The mesenchymal stem cell-like cells produced by the production method of the present invention can exhibit anti-inflammatory effects and immunosuppressive abilities according to the expression patterns of specific markers.

[0036] The mesenchymal stem cells produced by the production method of the present invention express CD90 and SOX2 at 95% or more, and can simultaneously exhibit the minimum criteria for human mesenchymal stem cells and additional characteristics.

[0037] The method of the present invention can be applied to a therapeutic composition or a delivery medium containing similar mesenchymal stem cells.

[0038] The active ingredients or exosomes secreted from mesenchymal stem cell-like substances produced by the production method of the present invention can be applied to disease prevention or treatment compositions or cosmetic compositions containing the same.

[0039] However, the effects of the present invention are not limited to the above-mentioned effects, but should be understood as including all effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a method for producing human pluripotent stem cell-derived mesenchymal stem cells (Human pluripotent stem cells) according to one embodiment of the present invention. [Figure 2] FIG. 2 shows the morphology of cystic embryoid bodies selected from various types of embryoid bodies induced to differentiate from human pluripotent stem cells during the preparation of human pluripotent stem cell-derived mesenchymal stem cells according to one embodiment of the present invention. The embryoid body indicated by the white arrow is a cystic embryoid body. Among the various types of embryoid bodies, the cystic embryoid body refers to an embryoid body that is observed to contain a transparent, bright portion within the embryoid body. [Figure 3] According to one embodiment of the present invention, a cell-permeable 3D culture insert (3D culture unit) is used for isolating human pluripotent stem cell-derived similar mesenchymal stem cells. Cyst-like embryoid bodies can be loaded onto the cell-permeable 3D culture insert, and similar mesenchymal stem cells can be isolated. By using the cell-permeable 3D culture insert, similar mesenchymal stem cells can be isolated with high purity from the selected cyst-like embryoid bodies. [Figure 4]1 shows an image of a mesenchymal stem cell-like population initially separated by permeation through a cell-permeable 3D culture insert according to one embodiment of the present invention. The graph also shows a comparative measurement of cell surface antigen expression after each population adheres to a dish (P0) following initial separation. The mesenchymal stem cell-like population initially separated by permeation through a cell-permeable 3D culture insert may include one or more of a multilayer or monolayer morphology. However, in one embodiment of the present invention, only monolayer cell populations are separated and cultured. [Figure 5] 1 is a photographed image of the morphology of similar mesenchymal stem cells according to an embodiment of the present invention. [Figure 6] 1 is a graph showing the measurement of the expression of various surface antigens in mesenchymal stem cells (MSCs) according to one embodiment of the present invention. The mesenchymal stem cells according to the present invention highly express CD90 (98.2%), CD44 (98.8%), CD73 (96.77%), and CD105 (95.6%). The mesenchymal stem cells according to the present invention also express CD45 (0.15%), CD34 (1.46%), CD14 (0.17%), and CD19 (0.09%), and also express Oct34 (0.2%), Tra-1-60 (0.08%), and Tra-1-81 (0.03%). The mesenchymal stem cells according to the present invention barely express HLA-DR (0.08%). Therefore, the mesenchymal stem cells according to the present invention are novel mesenchymal stem cells that meet the minimum standards for human mesenchymal stem cells established by the International Society for Stem Cell Therapy. The mesenchymal stem cells of the present invention highly express CD90 and express SOX2 at 99.37%. [Figure 7] 1 shows images of staining based on Oil Red O staining, Alizarin Red S staining, Alcian Blue staining, and immunocytochemistry staining to demonstrate differentiation characteristics of mesenchymal stem cells similar to those of one embodiment of the present invention into adipogenic cells, osteoblasts, chondrogenic cells, and myogenic cells. [Figure 8] 1 is a graph showing the proliferation ability (cumulative PDL (cPDL)) and cell size of a similar mesenchymal stem cell according to one embodiment of the present invention, compared with the proliferation ability and cell size of bone marrow mesenchymal stem cells (BM-MSCs). [Figure 9] 1 is a graph showing that mesenchymal stem cell-like cells (MMSCs) were isolated with high purity according to an embodiment of the present invention. As a marker for regulating undifferentiation, Oct4 was removed by 99% or more. [Figure 10A] 1 shows the genetic stability of similar mesenchymal stem cells according to one embodiment of the present invention confirmed through GTG-banding and SNP (single nucleotide polymorphisms) analysis. [Figure 10B] 1 shows the genetic stability of similar mesenchymal stem cells according to one embodiment of the present invention confirmed through GTG-banding and SNP (single nucleotide polymorphisms) analysis. [Figure 11] 1 is a graph and an image showing the tissue regeneration ability of mesenchymal stem cells according to one embodiment of the present invention as a function of the degree of cell migration, compared with the tissue regeneration ability of bone marrow-derived mesenchymal stem cells (BM-MSCs). [Figure 12] 1 shows the material exchange ability of similar mesenchymal stem cells according to one embodiment of the present invention when co-cultured with human umbilical vein endothelial cells (HUVEC). [Figure 13] 1 shows the direct angiogenic potential of similar mesenchymal stem cells according to one embodiment of the present invention. [Figure 14] 1 is a graph comparing the immunosuppressive ability and anti-inflammatory effects of similar mesenchymal stem cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) according to one embodiment of the present invention. [Figure 15]1 is a graph comparing the cell survival, tissue regeneration, and secretion ability of a cell death-inhibiting functional substance between similar mesenchymal stem cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) according to one embodiment of the present invention. [Figure 16] 1 shows inflammation regulation-related genes that are highly expressed in similar mesenchymal stem cells according to one embodiment of the present invention compared to bone marrow-derived mesenchymal stem cells (BM-MSCs). [Figure 17] 1 shows genes related to the immunosuppressive ability of similar mesenchymal stem cells according to one embodiment of the present invention, which are more highly expressed than bone marrow-derived mesenchymal stem cells (BM-MSCs). [Figure 18] According to one embodiment of the present invention, the protein content of the cell lysate of the similar mesenchymal stem cells was analyzed, and the protein content was found to be more highly expressed than that of bone marrow-derived mesenchymal stem cells (BM-MSC). [Figure 19] According to one embodiment of the present invention, the protein content of the cell lysate of the similar mesenchymal stem cells was analyzed, and the protein content was found to be lower than that of bone marrow-derived mesenchymal stem cells (BM-MSC). [Figure 20] The proteins in the culture supernatant of similar mesenchymal stem cells according to one embodiment of the present invention were analyzed, and proteins that are more highly expressed than those in bone marrow-derived mesenchymal stem cells (BM-MSC) were shown. [Figure 21] According to one embodiment of the present invention, proteins in the culture supernatant of similar mesenchymal stem cells were analyzed, and proteins that are expressed at lower levels compared to bone marrow-derived mesenchymal stem cells (BM-MSCs) were shown. [Figure 22] When embryonic stem cells (hESCs) are passaged more than 70 times (P68+7 or P68+15), the expression of surface antigens similar to mesenchymal stem cells is reduced. In particular, the expression of CD90, a surface antigen similar to mesenchymal stem cells, is significantly reduced as the number of passages of embryonic stem cells exceeds 70. [Figure 23]The results of PCR electrophoresis of gene expression in adipocytes and osteoblasts differentiated from similar mesenchymal stem cells separated by the number of passages of human embryonic stem cells (hESCs) (a (P68), b (P68+7), c (P68+15)) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0041] Hereinafter, the embodiments will be described in detail with reference to the accompanying drawings. However, various modifications can be made to the embodiments, and the scope of the patent application is not limited to or restricted by the embodiments. All modifications, equivalents, or alternatives to the embodiments should be understood as being included in the scope of the patent.

[0042] The terms used in this specification are merely used to describe particular embodiments and are not intended to limit the present invention. The singular expressions include the plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprise" or "have" indicate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0043] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Commonly used predefined terms should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and should not be interpreted as having an ideal or overly formal meaning unless expressly defined herein.

[0044] In addition, in the description with reference to the accompanying drawings, the same components are denoted by the same reference numerals regardless of the reference numerals, and redundant description thereof will be omitted. In the description of the embodiments, if a detailed description of related known technology is determined to unnecessarily obscure the gist of the embodiments, the detailed description thereof will be omitted.

[0045] In addition, in describing the components of the embodiments, terms such as first, second, A, B, (a), (b), etc. are used only to distinguish the components from other components, and the terms do not limit the essence, order, or sequence of the components.

[0046] Components having common functions with components included in one embodiment will be described using the same names in other embodiments. Unless otherwise specified, the description of one embodiment will be applied to other embodiments, and detailed description will be omitted to the extent that they overlap.

[0047] Production of mesenchymal stem cells with enhanced anti-inflammatory and immunosuppressive effects

[0048] FIG. 1 illustrates a method for producing human pluripotent stem cell-derived mesenchymal stem cells (MSCs) according to one embodiment of the present invention, comprising the steps of: (a) preparing human pluripotent stem cells that have been subcultured 70 or fewer times since the establishment of a cell line; (b) selecting cystic embryoid bodies from among the embryoid bodies obtained by inducing differentiation of the human pluripotent stem cells; (c) loading the cystic embryoid bodies onto a cell-permeable 3D culture insert to isolate mesenchymal stem cell-like cells; (d) isolating only a monolayer of cells from the cells that have permeated the cell-permeable 3D culture insert; and (e) homogenizing the monolayer of cells to a size of 100 μm to 500 μm in length and width, and culturing the mesenchymal stem cell-like cells, wherein the mesenchymal stem cell-like cells have anti-inflammatory and immunosuppressive properties and express CD90 and SOX2 at 95% or more.

[0049] The first image in Figure 1 shows the steps for culturing and maintaining human pluripotent stem cells. The human pluripotent stem cells were subcultured less than 70 times after the establishment of a cell line. The second image in Figure 1 shows the steps for selecting cystic embryoids after embryoid body (EB) formation and differentiating the selected cystic embryoids using a cell-permeable 3D culture unit. The selected cystic embryoids were loaded onto the cell-permeable 3D culture insert and passed through the insert. The third image in Figure 1 shows the cystic embryoids passing through the insert and migrating to the underside of the insert. The fourth image in Figure 1 shows the steps for collecting the cells that passed through the insert and homogenizing the population size. Multilayered clusters were mechanically removed from the collected cell population, and only the monolayered clusters were cut into uniformly sized pieces using a micropipette tip and selectively cultured. The fifth image in Figure 1 shows the establishment of similar mesenchymal stem cells expressing CD90+ and SOX2+.

[0050] As used herein, "human pluripotent stem cells" refers to undifferentiated cells that can differentiate into all cells that make up the human body. The human pluripotent stem cells include human embryonic stem cells (hESCs), human pluripotent stem cells via somatic cell nuclear transfer (SCNT), and somatic cell nuclear transfer (SCNT).

[0051] The stem cells may be one or more of human pluripotent stem cells (hPSCs) derived from human somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs).

[0052] As used herein, the term "mesenchymal stem cell analog" refers to a multipotent mesenchymal stem cell (MMSC) that can differentiate into various cells, including bone, cartilage, fat, and muscle cells. In other words, "mesenchymal stem cell analog" refers to a cell that has a function similar to that of mesenchymal stem cells (MSCs) that are differentiated from the mesoderm formed by the division of a fertilized egg and exist in bone marrow matrix, cartilage, bone tissue, adipose tissue, etc. In other words, the mesenchymal stem cell analog of the present invention refers to a novel mesenchymal stem cell that has improved immunosuppressive ability, anti-inflammatory effect, or ability to maintain pluripotency by overexpressing CD90 and SOX2. Therefore, the terms "mesenchymal stem cell analog" and "mesenchymal stem cell analog" will be used interchangeably herein.

[0053] As used herein, "n passages" or "passage n" (Pn) means that the parent cell line has been passaged n times. For example, "70 passages" or "P70" means that the parent cell line has been passaged 70 times. The "n" is an integer.

[0054] The human pluripotent stem cells used to produce mesenchymal stem cells-like in the present invention are characterized by having been passaged 70 times or less after the establishment of a pluripotent cell line. Typically, in the production of mesenchymal stem cells using pluripotent stem cells, the pluripotent stem cells are used as is without any restrictions on passage. When pluripotent stem cells are used as is without any restrictions on passage, most of the surface antigens of the produced mesenchymal stem cells are expressed at low levels and inconsistently, resulting in extremely low differentiation efficiency. Furthermore, the produced mesenchymal stem cells do not express CD90 or SOX2 at 95% or more. However, when human pluripotent stem cells passaged 70 times or less are used as in the present invention, all surface antigens of the produced mesenchymal stem cells are expressed at high levels and at a consistent rate of 95% or more. In particular, mesenchymal stem cells that express CD90 and SOX2 at 95% or more can be produced. Referring to Figure 22, when pluripotent stem cells (e.g., embryonic stem cells (hESCs)) are passaged more than 70 times (P68+7 or P68+15), the expression of CD90, one of the surface antigens of mesenchymal stem cells, decreases rapidly. That is, as the number of passages increases, based on passage 68, the expression of CD90 decreases to 25% or less, while the expression of CD44, CD73, and CD105 decreases to 90% or less. Furthermore, when pluripotent stem cells are passaged more than 70 times (P68+7 or P68+15), the expression of CD44, CD73, and CD105 of the mesenchymal stem cells becomes irregular. In contrast, mesenchymal stem cells derived from pluripotent stem cells subcultured 70 or fewer times express high levels of CD90 (98.2%), CD44 (98.8%), CD73 (96.77%), and CD105 (95.6%) (see Figure 6). As shown in Figure 23, a, b, and c are PCR electrophoresis results for gene expression of adipocytes and osteoblasts induced to differentiate from mesenchymal stem cells isolated from pluripotent stem cells (e.g., human embryonic stem cells (hESCs)) at P68, P68+7, and P68+15, respectively. As shown in Figure 23, a (P68), which has been subcultured 70 or fewer times, exhibits excellent differentiation potential into adipocytes and osteoblasts. On the other hand, b (P68+7) and c (P68+15), which have been subcultured more than 70 times, exhibit low differentiation efficiency into adipocytes and chondrocytes.That is, mesenchymal-like stem cells prepared by using pluripotent stem cells that have been passaged 70 times or less can retain superior proliferation ability, differentiation ability, genetic stability, tissue regeneration ability, material exchange ability, angiogenesis ability, immunosuppressive ability, and anti-inflammatory effect compared to bone marrow-derived mesenchymal stem cells. Furthermore, mesenchymal-like stem cells prepared by using pluripotent stem cells that have been passaged 70 times or less can retain superior cell survival, tissue regeneration, and the ability to secrete functional substances that inhibit apoptosis compared to bone marrow-derived mesenchymal stem cells.

[0055] More preferably, pluripotent stem cells that have been passaged 70 times or less are used, and differentiated cell populations that have permeated a cell-permeable 3D culture insert are removed from the multilayered populations, and only the monolayered populations are selected, mechanically homogenized to a certain size, and cultured to express 95% or more of the surface antigens of mesenchymal stem cells. In particular, it is possible to produce similar mesenchymal stem cells that express 95% or more of the surface antigens CD90 and SOX2 of mesenchymal stem cells.

[0056] Figure 2 shows the morphology of cystic embryoid bodies used in preparing human pluripotent stem cell-derived mesenchymal stem cells-like cells according to one embodiment of the present invention. In the present invention, "cystic embryoid bodies" refers to the morphology of embryoid bodies that are observed to contain transparent, bright areas within the embryoid body, among various embryoid body morphologies. Based on these characteristics, cystic embryoid bodies can be isolated with the naked eye.

[0057] Figure 3 shows the cell-permeable 3D culture insert used to load selected cystic embryoid bodies, which are embryoid bodies induced to differentiate human pluripotent stem cells, and to isolate similar mesenchymal stem cells.

[0058] As used herein, the term "cell-permeable 3D culture insert" refers to a cell-permeable mechanism. That is, it refers to a mechanism that allows epithelial-mesenchymal transition (EMT), which occurs during embryonic development, to occur naturally when embryonic stem cells, induced pluripotent stem cells, or embryoid bodies derived from somatic cell nuclear transfer stem cells are induced to differentiate into similar mesenchymal stem cells. That is, the cell-permeable 3D culture insert can be used to isolate, culture, and proliferate similar mesenchymal stem cells with high purity and efficiency.

[0059] The cell-permeable 3D culture insert may be a culture plate for cell culture, a 3D cell culture insert, or an artificial insert with guaranteed cell permeability, such as a mesh made of nylon or a fibrous material. The cell-permeable 3D culture insert may be manufactured by bioprinting technology. The cell-permeable 3D culture insert may be made of one or more of nylon, fiber, polyethylene, polypropylene, graphene, titanium, copper, nickel, silver, gold, and platinum. However, the cell-permeable 3D culture insert is not limited to the above materials as long as cell permeability is guaranteed.

[0060] Furthermore, the culture medium for human pluripotent stem cell-derived embryoid bodies using the cell-permeable 3D culture insert may be EGM2-MV, MCDB, DMEM, MEM-α, STEMPRO-MSC, or MesenCult-MSC medium. Preferably, the culture medium is EGM2-MV, MCDB, DMEM, or MEM-α, but is not limited to these. The culture medium for human pluripotent stem cell-derived embryoid bodies using the cell-permeable 3D culture insert may contain one or more additives selected from the group consisting of fetal bovine serum (FBS), serum replacement (SR), human serum, and human platelet lysate (HPL). Specifically, the additive may be 1-20% FBS, 1-20% SR, 1-20% human serum, or 1-20% HPL. Preferably, the additive may be 5% FBS or 2.5 to 5% HPL, but is not limited thereto.

[0061] Figure 4 shows images and graphs illustrating the characteristics of each cell population collected from beneath the cell-permeable 3D culture insert after penetrating the insert during the isolation of mesenchymal stem cells derived from human pluripotent stem cells. As shown in Figure 4, the cell population collected from beneath the cell-permeable 3D culture insert may be a multilayer or monolayer population. By culturing the multilayer and monolayer populations together or by culturing the multilayer or monolayer populations separately, it was confirmed that the monolayer populations isolated and cultured alone consistently expressed high levels of CD90, CD73, and CD105, which are major surface antigens indicative of mesenchymal stem cells.

[0062] FIG. 5 is a photographed image of the morphology of similar mesenchymal stem cells according to one embodiment of the present invention.

[0063] Figure 6 is a graph showing the expression of surface antigens of mesenchymal stem cells similar to those of one embodiment of the present invention. Referring to Figure 6, the surface antigens CD90, CD44, CD73, CD105, and SOX2 are expressed at 95% or more of the mesenchymal stem cells. Meanwhile, the surface antigens CD45, CD34, CD14, and CD19 are expressed at 2% or less of the mesenchymal stem cells. Furthermore, the undifferentiation-regulating markers Oct3 / 4, Tra-1-60, and Tra-1-81, and the immune rejection antigen HLA-DR are not expressed. Therefore, the mesenchymal stem cells similar to those of the present invention are characterized by high expression of CD90, an immunoregulatory marker, and SOX2, a marker associated with the maintenance of pluripotency, at 95% or more. The mesenchymal stem cells similar to those of the present invention can be confirmed to be novel mesenchymal stem cells with improved immunosuppressive ability and pluripotency maintenance.

[0064] Figure 7 shows the differentiation potential of mesenchymal stem cells (MSCs) into various mesodermal cells according to one embodiment of the present invention. Referring to Figure 7, the MSCs can differentiate into adipogenic cells, osteogenic cells, chondrogenic cells, and myogenic cells in an appropriate differentiation environment. This satisfies the differentiation potential criteria for mesenchymal stem cells as defined by the International Society for Stem Cell Therapy.

[0065] FIG. 8 is a graph comparing the proliferation ability and cell size of mesenchymal stem cells (MMSCs) according to one embodiment of the present invention with bone marrow-derived mesenchymal stem cells (BM-MSCs). In FIG. 8, cumulative PDL (cPDL) indicates cell proliferation ability. MMSCs divide an average of 16.13 times over four passages. The size of the MMSCs is 13.6 μm. It has been confirmed that MMSCs have approximately 1.6 times faster cell proliferation and approximately 0.8 times smaller cell size than bone marrow-derived mesenchymal stem cells. Because MMSCs have rapid cell proliferation ability and small cell size, when used in stem cell therapeutic compositions, they can reduce the possibility of side effects in the body and improve therapeutic effects.

[0066] 9 is a graph showing the isolation of highly pure mesenchymal stem cells according to one embodiment of the present invention. Referring to FIG. 9, it can be seen that the similar mesenchymal stem cells have been depleted by 99% or more of the undifferentiated marker Oct4. Therefore, when similar mesenchymal stem cells derived from human pluripotent stem cells that have been subcultured 70 times or less after the establishment of the cell line are isolated and cultured by the manufacturing method of the present invention, it can be confirmed that they are isolated and cultured with high purity without being contaminated with undifferentiated cells other than the similar mesenchymal stem cells.

[0067] Figure 10 shows the genetic stability of the mesenchymal stem cell analogs according to one embodiment of the present invention, as confirmed through GTG-banding and SNP analysis. Referring to Figure 10, it was confirmed that the chromosomes of all the mesenchymal stem cells analogs produced in the three batches, i.e., MSP-0005, MSP-0006, and MSP-0007, were normal, and no SNP abnormalities were observed.

[0068] 11 is a graph showing the tissue regeneration ability of mesenchymal stem cell-like cells according to one embodiment of the present invention as a function of their cell migration level. Referring to FIG. 11, in vitro wound healing analysis confirmed that the tissue regeneration ability of mesenchymal stem cell-like cells due to cell migration was approximately 3.5 times greater than that of bone marrow-derived mesenchymal stem cells, which are adult stem cells.

[0069] Figure 12 shows the material exchange capacity of similar mesenchymal stem cells according to one embodiment of the present invention. Referring to Figure 12, human umbilical vein endothelial cells (HUVECs) and similar mesenchymal stem cells were co-cultured to confirm the material exchange capacity between the cells.

[0070] 13 shows the angiogenic ability of similar mesenchymal stem cells according to one embodiment of the present invention. Referring to FIG. 13, similar mesenchymal stem cells were cultured on Matrigel to confirm their spontaneous angiogenic ability.

[0071] Figure 14 shows the immune cell proliferation inhibitory ability and anti-inflammatory effect of mesenchymal stem cell analogs according to one embodiment of the present invention. Referring to Figure 14, mesenchymal stem cells analogs were co-cultured with mononuclear cells, which are immune cells, and a strong immune cell proliferation inhibitory effect was confirmed. This indicates that mesenchymal stem cells analogs have a higher immunosuppressive ability and anti-inflammatory effect than bone marrow-derived mesenchymal stem cells.

[0072] FIG. 15 is a graph showing the cell survival, tissue regeneration, and secretion ability of a functional substance inhibiting cell death of mesenchymal stem cells similar to those according to one embodiment of the present invention.

[0073] As shown in Figure 15, an embodiment of the present invention provides human pluripotent stem cell-derived similar mesenchymal stem cells, characterized by higher expression of MMP-1 and HGF compared to bone marrow-derived mesenchymal stem cells, lower expression of CD95 compared to bone marrow-derived mesenchymal stem cells, and expression of CD90 and SOX2 at 95% or more. According to another embodiment of the present invention, the similar mesenchymal stem cells can express MMP-1 at a level 15-fold or more higher and HGF at a level 2-fold higher compared to bone marrow-derived mesenchymal stem cells. According to another embodiment of the present invention, the similar mesenchymal stem cells can express CD95 at a level 15-fold or more lower compared to bone marrow-derived mesenchymal stem cells.

[0074] As shown in Figure 15, human pluripotent-derived mesenchymal stem cells prepared according to one embodiment of the present invention secrete about 16.9 times more MMP-1 (Matrix Metalloproteinase-1) protein, which is related to tissue regeneration, than bone marrow-derived mesenchymal stem cells. Furthermore, hepatocyte growth factor (HGF) protein, which is related to cell survival and proliferation, than bone marrow-derived mesenchymal stem cells. Meanwhile, CD95 (Cluster Differentiation 95), which is related to cell apoptosis, than bone marrow-derived mesenchymal stem cells, was found to be expressed about 15 times less than bone marrow-derived mesenchymal stem cells.

[0075] CD95 (Cluster Differentiation 95) is a death receptor also known as FasR (Fas Receptor), APO-1 (Apoptosis antigen 1), or TNFRSF6 (Tumor Necrosis Factor Receptor Superfamily Member 6). CD95 is located on the cell surface and induces cell death through interaction with a ligand known as CD95L (CD95 Ligand). Specifically, binding of a ligand known as FasL (Fas Ligand) or CD95L (CD95 Ligand) to CD95 promotes cell death via the death-inducing signaling complex (DISC). That is, when CD95 is overexpressed in stem or progenitor cells, cell death signaling mediated by CD95L is activated. On the other hand, stem cells with relatively low expression of CD95 exhibit excellent cell survival rates due to suppression of cell suicide signaling.

[0076] FIG. 16 shows inflammation regulation-related genes that are highly expressed in human pluripotent-derived similar mesenchymal stem cells prepared according to one embodiment of the present invention compared to bone marrow-derived mesenchymal stem cells.

[0077] As shown in FIG. 16 , human pluripotent-derived mesenchymal stem cells prepared according to one embodiment of the present invention are provided that express inflammation-regulating genes at levels 2 to 100 times higher than bone marrow-derived mesenchymal stem cells, and the inflammation-regulating genes are at least one of the group consisting of Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Acp5, Bcr, Socs5, and Mdk. Furthermore, according to one embodiment of the present invention, there is provided a mesenchymal stem cell analog that simultaneously expresses Gata3, Adora2a, and Gps2 genes. The inflammation-regulating genes are genes related to inflammation suppression, and the mesenchymal stem cells are analogous mesenchymal stem cells that highly express the inflammation-regulating genes and have anti-inflammatory effects that are 2 to 100 times higher than bone marrow-derived mesenchymal stem cells. As shown in FIG. 16 ,

[0078] In the mesenchymal stem cell-like cells, the Gata3 gene was expressed at approximately 100-fold higher levels than in bone marrow-derived mesenchymal stem cells. Furthermore, the Adora2a and Gps2 genes were expressed at approximately 10-fold higher levels than in bone marrow-derived mesenchymal stem cells. Furthermore, the Psma1, Pbk, Lrfn5m, Cdh5, and Apoe genes were expressed at approximately 5-fold higher levels than in bone marrow-derived mesenchymal stem cells. Furthermore, the mesenchymal stem cell-like cells can simultaneously express the inflammation-regulating genes Gata3, Adora2a, and Gps2.

[0079] FIG. 17 shows genes related to immunosuppressive ability that are highly expressed in human pluripotent-derived similar mesenchymal stem cells prepared according to one embodiment of the present invention compared to bone marrow-derived mesenchymal stem cells.

[0080] As shown in Figure 17, the mesenchymal stem cell analog has 2- to 100-fold higher expression of immunosuppressive genes than bone marrow-derived mesenchymal stem cells, and the immunosuppressive genes are one or more of Gata3, Gps2, Psma1, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Bcr, Socs5, and Mdk. Furthermore, the mesenchymal stem cell analog simultaneously expresses the Gata3, Gps2, and Psma1 genes.

[0081] As shown in Figure 17, in the mesenchymal stem cell-like cells, the Gata3 gene was expressed at a level approximately 50 to 100 times higher than in the bone marrow-derived mesenchymal stem cells. Furthermore, in the mesenchymal stem cell-like cells, the Gps2 gene was expressed at a level approximately 5 to 10 times higher than in the bone marrow-derived mesenchymal stem cells. Furthermore, in the mesenchymal stem cell-like cells, the Psma1 and Apoe genes were expressed at a level approximately 5 times higher than in the bone marrow-derived mesenchymal stem cells. Furthermore, the mesenchymal stem cell-like cells can simultaneously express the immunosuppressive genes Gata3, Gps2, and Psma1.

[0082] Figure 18 shows proteins that are more highly expressed than bone marrow-derived mesenchymal stem cells, as determined by analyzing proteins in cell lysates of mesenchymal stem cells similar to those derived from bone marrow-derived mesenchymal stem cells according to one embodiment of the present invention. The mesenchymal stem cells express Angiopoietin-4 (ANGPT4), bone morphogenic protein receptor 1B (BMPR-1B), Activin B, Activin RII, CCR1, human chemokine receptor CRAM-A isoform (HCR), and intracellular adhesion molecule 2 (ICAM-2) at approximately two-fold or more higher levels than bone marrow-derived mesenchymal stem cells. The mesenchymal stem cells can simultaneously express Angiopoietin-4 and BMPR-1B. Preferably, the mesenchymal stem cells can express Angiopoietin-4 and BMPR-1B at approximately three-fold or more higher levels than bone marrow-derived mesenchymal stem cells. More preferably, the similar mesenchymal stem cells can express one or more of the proteins ANGPT4, BMPR-1B, Activin B, Activin RII, CCR1, HCR, and ICAM-2 at approximately twice or more levels compared to bone marrow-derived mesenchymal stem cells.

[0083] Figure 19 shows proteins that are less expressed than bone marrow-derived mesenchymal stem cells, as determined by analyzing proteins in cell lysates of mesenchymal stem cells similar to those of one embodiment of the present invention. Referring to Figure 19, the mesenchymal stem cells secrete angiogenin, angiopoietin-2, CCR8, EDA-A2, and IL-20 at levels approximately two-fold lower than those of bone marrow-derived mesenchymal stem cells. The mesenchymal stem cells secrete angiogenin and angiopoietin-2 simultaneously, and secrete angiogenin and angiopoietin-2 at levels approximately two-fold lower than those of bone marrow-derived stem cells.

[0084] Figure 20 shows proteins that are more highly expressed than bone marrow-derived mesenchymal stem cells when analyzed for proteins in the culture supernatant of mesenchymal stem cell-like cells according to one embodiment of the present invention. Referring to Figure 20, mesenchymal stem cells-like cells secrete GRO-a, IL-15R alpha, FasL, Activin RII, BMP-2, CCR2, CXCL14, FGFR4, uPA, and MMP-20 at levels approximately two-fold higher than bone marrow-derived stem cells. The mesenchymal stem cells secrete GRO-a and IL-15R alpha simultaneously and at levels approximately two-fold higher than bone marrow-derived stem cells. The mesenchymal stem cells secrete at least one of GRO-a, IL-15R alpha, FasL, Activin RII, BMP-2, CCR2, CXCL14, and FGFR4 at levels approximately two-fold higher than bone marrow-derived stem cells.

[0085] Figure 21 shows proteins that are less expressed than bone marrow-derived mesenchymal stem cells, as determined by analyzing proteins in the culture supernatant of mesenchymal stem cell-like cells according to one embodiment of the present invention. As shown in Figure 21, the mesenchymal stem cell-like cells secrete TIMP-2, Activin A, VEGF A, Follistantin-like 1, ErbB4m, and Thrombospondin-1 at levels about four times lower than bone marrow-derived mesenchymal stem cells. The mesenchymal stem cell-like cells can secrete TIMP-2 and Activin A simultaneously, and secrete TIMP-2 and Activin A at levels about five times lower than bone marrow-derived mesenchymal stem cells.

[0086] The mesenchymal stem cells of the present invention can suppress proliferation when co-cultured with human peripheral blood-derived mononuclear cells. More specifically, the mesenchymal stem cells of the present invention can suppress proliferation by about 5 to 8 times or more compared to bone marrow-derived mesenchymal stem cells when co-cultured with human peripheral blood-derived mononuclear cells.

[0087] The analogous mesenchymal stem cells of the present invention exhibit a repair ability that is approximately 3.5 times higher than that of bone marrow-derived mesenchymal stem cells in evaluation of cell migration and repair ability.

[0088] The mesenchymal stem cell-like cells of the present invention can secrete MMP-1 protein, a gene related to cell survival, at about 15 to 16 times higher levels than bone marrow-derived mesenchymal stem cells. In addition, the mesenchymal stem cell-like cells can secrete HGF protein, a gene related to cell growth and tissue regeneration, at about 2 times higher levels than bone marrow-derived mesenchymal stem cells.

[0089] The analogous mesenchymal stem cells of the present invention can express the cell death receptor CD95 at a level about 15 times lower than that of bone marrow-derived mesenchymal stem cells.

[0090] A therapeutic composition is provided comprising mesenchymal stem cell-like stem cells produced by a method according to one embodiment of the present invention, the therapeutic composition being any one of a cell therapy composition, a cell and gene therapy composition, a tissue engineering therapy composition, an anti-inflammatory therapy composition, an immunotherapy composition, and a composition for the prevention or treatment of cancer. The therapeutic composition further comprises an active ingredient or exosomes secreted from the mesenchymal stem cell-like stem cells. The therapeutic composition may be for the prevention or treatment of one or more of the following diseases: multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic pulmonary disease, acute pulmonary disease, Crohn's disease, fecal incontinence, graft-versus-host disease, lower limb ischemia, thromboangiitis obliterans, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injuries caused by various exercises, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage due to burns, skin damage due to ultraviolet rays, and inflammatory and immune system diseases including retinopathy; ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system diseases.

[0091] The active ingredient or exosome secreted from the similar mesenchymal stem cells includes all functional substances related to immunosuppressive ability, anti-inflammatory effect, cell survival, tissue regeneration, or inhibition of cell death, etc. However, the active ingredient or exosome is not limited to these as long as it has functionality as the therapeutic composition.

[0092] A delivery vehicle containing mesenchymal stem cells similar thereto, produced by a production method according to one embodiment of the present invention, is provided, characterized in that the delivery vehicle carries a pharmaceutical composition.

[0093] A composition for preventing or treating a disease is provided, comprising an active ingredient or exosomes secreted from mesenchymal stem cell-like stem cells produced by a method according to one embodiment of the present invention. The disease may be one or more of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic pulmonary disease, acute pulmonary disease, Crohn's disease, fecal incontinence, graft-versus-host disease, lower limb ischemia, thromboangiitis obliterans, foot ulcers, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injuries caused by various exercises, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin injury caused by burns, skin injury caused by ultraviolet rays, and prevention of inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system diseases.

[0094] A cosmetic composition is provided that contains an active ingredient or exosomes secreted from mesenchymal stem cell-like cells produced by a production method according to one embodiment of the present invention.

[0095] The human pluripotent stem cell-derived similar mesenchymal stem cells of the present invention have improved anti-inflammatory effects, immunosuppressive ability, and tissue regeneration ability compared to bone marrow-derived mesenchymal stem cells. [Example]

[0096] Embodiment 1: Isolation and culture of mesenchymal stem cells with enhanced anti-inflammatory and immunosuppressive effects

[0097] FIG. 1 is a schematic diagram illustrating the process of isolating and culturing similar mesenchymal stem cells from human pluripotent stem cells according to one embodiment of the present invention. Human pluripotent stem cells were used after passages of 70 or fewer times following the establishment of the cell line. Human pluripotent stem cells that had been passaged 70 or fewer times following the establishment of the cell line were maintained in a cell incubator at 37°C and 5% CO2. Maintained human pluripotent stem cells were isolated from the culture plate by simple enzymatic treatment. Cystic embryoid bodies were then selected by embryoid body formation. Before combining the cell-permeable 3D culture insert with a new culture plate (6-well plate), 2 ml of culture medium was added to the new culture plate for the first culture cycle only. The cell-permeable 3D culture insert was then combined with the culture plate containing 2 ml of culture medium. An additional 2 ml of culture medium was then added to the cell-permeable 3D culture insert, and the selected cystic embryoid bodies were loaded onto the combined cell-permeable 3D culture insert. The culture medium in the new culture plate and the culture medium in the cell-permeable 3D culture inserts were EGM-2MV. The embryoid bodies were cultured in the cell-permeable 3D culture inserts loaded with cystic embryoid bodies for two days without changing the culture medium. After that, the culture medium was removed and an additional 4 ml of culture medium was added to the cell-permeable 3D culture inserts alone. The culture medium was changed daily to induce differentiation into similar mesenchymal stem cells.

[0098] Embodiment 2: Isolation and proliferation of similar mesenchymal stem cells with enhanced anti-inflammatory and immunosuppressive effects

[0099] From the mesenchymal stem cell-like cells isolated in Example 1 above, cells in the form of clusters that had permeated the cell-permeable 3D culture insert and migrated downward were isolated. Among the isolated clusters, those in the form of multilayers were mechanically removed. From the isolated clusters, only the monolayer-like clusters were homogenized using a microfiber tip to a size of 500 μm or less in width and width, and selectively cultured. More preferably, the monolayer-like clusters were homogenized to a size of 100 μm to 500 μm in length and width, and the similar mesenchymal stem cells were cultured. After culturing for 5 to 7 days, only the similar mesenchymal stem cells that had protruded into single cells were isolated from the clusters and subcultured for proliferation. Before isolating single cells, clusters that were not in the form of single cells and cells with epithelial cell morphology were completely removed using a microfiber tip. The isolated single cells were transferred to a new culture plate and induced to proliferate through subculture. EGM-2MV was used as the culture medium. Specifically, as shown in Figure 4, when the similar mesenchymal stem cells in a cluster form migrated to the bottom of the cell-permeable 3D culture insert, the cell-permeable 3D culture insert was separated from the culture plate. After harvesting the similar mesenchymal stem cells in a cluster form that migrated to the bottom of the separated cell-permeable 3D culture insert, the multilayered clusters were mechanically removed, and the size of the monolayered clusters was homogenized for selective culture. The similar mesenchymal stem cells grown from the monolayered clusters maintained their cell morphology and proliferation ability even after continuous subculture, and no negative changes were observed in the cell morphology, proliferation, etc. Example 3: Analysis of surface antigen expression of similar mesenchymal stem cells

[0100] To analyze the characteristics of the mesenchymal stem cells similar to those prepared in Examples 1 and 2 above, the expression of stem cell-specific markers was analyzed using flow cytometry (Fluorescence Activated Cell Sorting, FACS).

[0101] Specifically, the proliferation-induced mesenchymal stem cells were lysed into single cells using TrypLE, and 5 × 10 5The cells were suspended in phosphate-buffered saline (PBS) at a concentration of 1000 cells / ml. Antibodies against Sox2, CD44, CD73, CD90, CD105, CD146, NG2, HLA-ABC, CD11b, CD11c, CD14, CD19, CD34, CD45, CD40, CD40L, CD80, CD86, CD95, CD133, KDR, Flt-1, Tie-2, HLA-DR, Oct3 / 4, Tra-1-81, and Tra-1-60 were added to each cell and incubated at room temperature for 45 minutes. Flow cytometry analysis was then performed on each cell. The nuclear proteins SOX2 and Oct3 / 4 were permeabilized by treatment with 0.1% Triton X-100 for 5 minutes at room temperature. Antibodies were then added and flow cytometry analysis was performed. The results are shown in Table 1 and Figure 6.

[0102] [Table 1]

[0103] As shown in Table 1 and Figure 6, the mesenchymal stem cell-like stem cells prepared by the method of the present invention expressed mesenchymal stem cell-specific markers CD44, CD73, CD90, and CD105 at over 95%. Meanwhile, CD45, CD34, CD14, and CD19 were expressed at less than 2%. Furthermore, expression of undifferentiation-regulating markers Oct3 / 4, Tra-1-60, and Tra-1-81, and the immune rejection antigen HLA-DR, were barely observed. Therefore, it was confirmed that the mesenchymal stem cells prepared by the method of the present invention possess all the characteristics of mesenchymal stem cells as defined by the International Society for Stem Cell Therapy. Furthermore, SOX2 and CD146, which are not defined as characteristics of adult mesenchymal stem cells by the International Society for Stem Cell Therapy, were expressed at over 90%, while CD95 expression was significantly lower. Because the mesenchymal stem cell-like stem cells prepared by the present invention are differentiated from human pluripotent stem cells that have been passaged 70 times or less, they are expected to have the surface antigen expression profile shown in Table 1. Furthermore, among the embryoid bodies obtained by inducing differentiation of human pluripotent stem cells, only selected cystic embryoid bodies are isolated using cell-permeable 3D culture inserts, and the monolayer cell population is homogenized and cultured, thereby maintaining a consistent expression profile of the surface antigens shown in Table 1. In particular, the mesenchymal stem cell-like cells produced by the production method of the present invention express SOX2 at 90% or more, which is different from ordinary mesenchymal stem cells.

[0104] Example 4: Analysis of differentiation potential of similar mesenchymal stem cells

[0105] To confirm the pluripotency of the similar mesenchymal stem cells prepared through the above-mentioned embodiments 1 and 2, differentiation into osteogenic, adipocyte, chondrocyte, and muscle cells, which are defined as characteristics of mesenchymal stem cells by the International Stem Cell Therapy Society, was induced.

[0106] Specifically, the prepared mesenchymal stem cell-like cells were cultured in low-glucose DMEM medium containing 10% FBS, 5 μg / ml insulin, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and 60 μM indomethacin. They were also cultured in low-glucose DMEM medium containing 10% FBS, 1 μM dexamethasone, 10 mM β-glycerophosphate, and 60 μM ascorbic acid-2-phosphate. Differentiation into osteoblasts, adipocytes, and chondrocytes was confirmed. Differentiation of osteoblasts generated by the differentiation was confirmed by Alizarin Red S staining. Differentiation of adipocytes generated by the differentiation was confirmed by Oil Red O staining. Differentiation of chondrocytes generated by the differentiation was confirmed by Alcian blue staining. To confirm differentiation into muscle cells, the similar mesenchymal stem cells were differentiated in DMEM medium containing 20% ​​FBS, 1% non-essential amino acids, 1% penicillin sulfate, and 0.1 mM β-mercaptoethanol. The differentiated muscle cells were confirmed to express αSMA, a smooth muscle-specific marker.

[0107] Therefore, as shown in Figure 7, the mesenchymal stem cells produced by the production method of the present invention exhibit a certain differentiation ability into osteoblasts, fat cells, cartilage cells, and muscle cells, which satisfies the characteristics of mesenchymal stem cells presented at the World Stem Cell Therapy Conference.

[0108] Embodiment 5: Proliferation potential and cell size analysis of similar mesenchymal stem cells

[0109] To confirm the proliferation ability of the similar mesenchymal stem cells produced in Examples 1 and 2, the total number of cell divisions from the 3rd to 7th passages was measured. The total number of cell divisions was also compared with that of bone marrow-derived mesenchymal stem cells, which are adult stem cells. The results are shown in Table 2 and Figure 8 below.

[0110] [Table 2]

[0111] As shown in Table 2 and Figure 8, the mesenchymal stem cell-like cells divided an average of 16.13 times between passages 3 and 7. In contrast, bone marrow-derived mesenchymal stem cells divided an average of 10.17 times. This demonstrates that mesenchymal stem cell-like cells have superior proliferation capacity.

[0112] Meanwhile, when the cell size of the expanded similar mesenchymal stem cells was investigated, the average cell size of the similar mesenchymal stem cells was 13.6±0.3 μm, as shown in Table 3 and FIG. 6. In contrast, the average cell size of bone marrow-derived mesenchymal stem cells was 17.7±0.4 μm. Therefore, it was confirmed that the size of the similar mesenchymal stem cells was relatively smaller than that of bone marrow-derived mesenchymal stem cells. Because the size of the similar mesenchymal stem cells is relatively small, when they are injected into a blood vessel during cell therapy, the possibility of pulmonary embolism due to vascular occlusion can be reduced. Therefore, the similar mesenchymal stem cells according to the present invention can be provided as therapeutic compositions, such as cell therapy compositions, cell gene therapy compositions, tissue engineering therapeutic compositions, anti-inflammatory therapeutic compositions, immunotherapeutic compositions, and compositions for the prevention or treatment of cancer, and can improve the efficacy of treatment and reduce the possibility of side effects.

[0113] [Table 3]

[0114] Embodiment 6: High-purity isolation of similar mesenchymal stem cells

[0115] The mesenchymal stem cell analogs produced by the above-mentioned embodiments 1 and 2 were confirmed to be highly pure cells free from the presence of undifferentiated cells. That is, the presence or absence of expression of Oct4, a marker for regulating undifferentiation, was confirmed using qPCR for the mesenchymal stem cells maintained and proliferating. Then, a human embryonic stem cell line, which is an undifferentiated pluripotent stem cell, and human dermal fibroblasts (hFF) were comparatively analyzed. As a result, as shown in Figure 9, Oct4, a marker for regulating undifferentiation, was expressed only in human embryonic stem cells. It was confirmed that the mesenchymal stem cells produced by the production method of the present invention were free from the presence of undifferentiated cells, as were human dermal fibroblasts (hFF). The mesenchymal stem cell analogs produced by the production method of the present invention were confirmed to be highly pure, isolated cells with no detectable expression of undifferentiated cells. Embodiment 7: Analysis of Genetic Safety of Mesenchymal Stem Cell Analogs

[0116] Meanwhile, chromosome analysis and SNP (Single Nucleotide Polymorphism) analysis were performed to confirm the genetic safety of the similar mesenchymal stem cells.

[0117] Specifically, for karyotyping, mesenchymal stem cells were added to 10 ml of medium containing 20 μl of 100 μg / μl colchicine solution and incubated at 37°C for 2 hours. The mixture was then centrifuged at 500 rpm for 5 minutes and the supernatant was removed. The cells were then suspended in 0.075 M KCl stock solution and incubated in a thermostatic water bath at 37°C for 25 minutes. Five drops of fixative (methanol:glacial acetic acid = 3:1, v / v) were added and the mixture was centrifuged at 1,200 rpm for 8 minutes. The supernatant was then removed, and additional fixative was added. This process was repeated twice, with each subsequent 10-minute incubation at room temperature. One drop of the cell suspension was then placed on a slide immersed in cold 70% ethanol. The slide was then dried using an alcohol lamp. The slide was then stained in 5% Gimesa solution for 12 minutes. The stain was then removed using distillation, and the slide was allowed to dry in air before being observed under a light microscope. SNP analysis was outsourced to a specialized testing institution, and the presence or absence of chromosomal abnormalities was confirmed using Illumina SNP chips. As shown in Figure 10, the chromosomes of all mesenchymal stem cell-like cells produced in the three batch numbers were normal, and no SNP abnormalities were observed. This demonstrates that the mesenchymal stem cell-like cells produced by the production method of the present invention are genetically stable.

[0118] Example 8: Analysis of tissue regeneration potential of similar mesenchymal stem cells

[0119] Various analyses were performed to evaluate the functionality of the analogous mesenchymal stem cells prepared according to the above-mentioned Examples 1 and 2. An in vitro cell migration assay was performed to analyze the tissue regeneration ability. Bone marrow-derived mesenchymal stem cells were used as a control cell in the tissue regeneration ability analysis.

[0120] Specifically, 3 × 10 human-derived mesenchymal stem cells and 3 × 10 bone marrow-derived mesenchymal stem cells were added to each 35 mm μ-dish. 4The cells were then dispensed at a concentration of 70 μl per well. The cells were then cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture insert was then removed, and 2 ml of 10% DMEM medium was added. The number of migrating cells was measured under a microscope. As shown in FIG. 11, the migratory capacity of the prepared mesenchymal stem cells was 67.51% on average. The migratory capacity of the mesenchymal stem cells prepared by the method of the present invention was 350% or more higher than that of bone marrow-derived mesenchymal stem cells. This confirms that the mesenchymal stem cells prepared according to one embodiment of the present invention are capable of rapid cell migration and proliferation when administered in vivo. Therefore, it can be predicted that the prepared mesenchymal stem cells have a significantly high tissue regeneration capacity.

[0121] Example 9: Analysis of the material exchange and angiogenesis capabilities of similar mesenchymal stem cells

[0122] As different methods for evaluating the functionality of the similar mesenchymal stem cells prepared according to the above embodiments 1 and 2, their material exchange ability and angiogenesis ability were analyzed.

[0123] Specifically, we analyzed the ability of material exchange using calcein, a fluorescent substance that can move through gap junctions, which are used to analyze cell-cell interactions. First, human umbilical vein endothelial cells (HUVECs), a type of vascular cell, were stained with calcein. Then, they were co-cultured with Dil-labeled analogous mesenchymal stem cells in an incubator at 37°C for 1 hour. The cells were then analyzed using a fluorescence microscope. As shown in Figure 12, HUVEC cells, shown in fluorescent light, and Dil-analogous mesenchymal stem cells, shown in red, demonstrated rapid material transfer after co-culture. After 48 hours, flow cytometry analysis confirmed that the number of cells that had undergone substantial exchange was 46.15%. This suggests that the analogous mesenchymal stem cells produced by the method of the present invention may promote intercellular interactions, including the supply of useful substances, through rapid gap junction formation with vascular cells.

[0124] To analyze angiogenesis, 50 μL of Matrigel thawed at room temperature was dispensed into a 96-well plate, and the plate was then left at 37°C for 30 minutes. Then, 1-2×10 single-cell-like mesenchymal stem cells were added. 4 The cells were seeded onto Matrigel and cultured at 37°C for 24 hours. Then, 10% formaldehyde fixative was added and the cells were further cultured for 10 minutes. The cells were then observed under a 10x microscope at 3-5 random locations to determine whether spontaneous tubule formation had occurred. As shown in Figure 13, all of the three batches of mesenchymal stem cell-like cells underwent spontaneous angiogenesis on Matrigel. This indicates that the mesenchymal stem cell-like cells produced according to Examples 1 and 2 retain the ability to form spontaneous angiogenesis. These characteristics indicate that the mesenchymal stem cell-like cells produced by the method of the present invention are capable of promoting neovascularization and material exchange at the site of injury.

[0125] Example 10: Analysis of immune cell proliferation inhibition and anti-inflammatory effects of similar mesenchymal stem cells

[0126] As one of the analyses of the functions of the similar mesenchymal stem cells produced according to the first and second embodiments, the expression of cytostatic and anti-inflammatory related proteins was analyzed through co-culture with immune cells.

[0127] Specifically, 1,000, 2,000, 5,000, and 10,000 cells of similar mesenchymal stem cells and bone marrow-derived mesenchymal stem cells prepared according to Examples 1 and 2 were prepared, respectively. 2 × 10 peripheral blood mononuclear cells (PBMCs) were also prepared. 5 Cells / well were co-cultured in a 96-well plate for 5 days. Each test group was labeled with CFSE (Carboxy Fluorecein Succinimidyl Ester) and the PBMC proliferation inhibition rate (%) was measured. The results are shown in Table 4 below and Figure 14.

[0128] [Table 4]

[0129] As shown in Table 4 and Figure 14, it can be confirmed that the mesenchymal stem cells of the present invention inhibit the proliferation of peripheral blood mononuclear cells in a concentration-dependent manner. That is, the mesenchymal stem cells of the present invention have a much stronger effect of inhibiting proliferation than bone marrow-derived mesenchymal stem cells. Therefore, the mesenchymal stem cells of the present invention have excellent immunosuppressive ability.

[0130] Meanwhile, the expression of genes related to anti-inflammatory and immunosuppressive effects was analyzed through analysis of the dielectric and protein bodies of mesenchymal stem cell-like cells. As a result, it was confirmed that Gata3, Adora2a, and Gps2, which show representative anti-inflammatory effects, were expressed at approximately 11 to 100 times higher levels than bone marrow-derived mesenchymal stem cells. Therefore, mesenchymal stem cell-like cells have superior anti-inflammatory and immunosuppressive effects compared to bone marrow-derived mesenchymal stem cells.

[0131] Embodiment 11: Secretion ability of functionally useful substances from similar mesenchymal stem cells

[0132] To analyze the functionality of the mesenchymal stem cell-like cells prepared in Examples 1 and 2, the concentrations of useful substances secreted from the cells were measured. Specifically, the mesenchymal stem cell-like cells prepared were cultured in serum-free DMEM medium for 24 hours. Then, the cells were centrifuged at 500 rpm, and the upper layer of medium was collected. The collected medium was quantitatively analyzed for MMP-1, HGF, and CD95 using an enzyme-linked immunosorbent assay (ELISA). Bone marrow-derived mesenchymal stem cells were also analyzed for comparison with adult stem cells, and the results are shown in Figure 15.

[0133] As shown in Figure 15, the concentration of MMP-1 (Matrix metalloproteinase-1), a representative tissue regeneration-related protein, was 20,953 pg / ml in the mesenchymal stem cell-like cells and 1,237 pg / ml in the bone marrow-derived mesenchymal stem cells. This means that the mesenchymal stem cells secreted approximately 16-fold more MMP-1 than the bone marrow-derived mesenchymal stem cells. Meanwhile, the concentration of HGF (Hepatic Growth Factor), a cell growth and proliferation-related protein, was 1,360 pg / ml in the mesenchymal stem cell-like cells and 534 pg / ml in the bone marrow-derived mesenchymal stem cells. This means that the mesenchymal stem cells secreted approximately 2.5-fold more HGF than the bone marrow-derived mesenchymal stem cells. Meanwhile, the concentration of CD95, which is known to play an important role in inducing cell death, was 53 pg / ml in the mesenchymal stem cell-like cells and 800 pg / ml in the bone marrow-derived mesenchymal stem cells. Therefore, CD95 expression was about 15 times lower in the mesenchymal stem cells compared to bone marrow-derived mesenchymal stem cells. Therefore, the mesenchymal stem cells prepared according to the present invention have superior secretion capabilities for proteins related to tissue regeneration and cell proliferation compared to existing bone marrow-derived mesenchymal stem cells. Furthermore, the expression of cell death-inducing proteins is extremely low, resulting in excellent tissue regeneration and cell survival rates.

[0134] Embodiment 12: Confirmation of the inflammatory regulation and immunosuppression-related gene expression characteristics of isolated and cultured similar mesenchymal stem cells

[0135] Next Generation Sequencing (NGS) was performed on the similar mesenchymal stem cells and bone marrow-derived mesenchymal stem cells isolated and cultured in Examples 1 and 2, and the differences in gene expression were analyzed based on the database published in NCBI GEO.

[0136] Specifically, we performed RNA sequencing (RNA-seq) to compare gene expression between similar mesenchymal stem cells and bone marrow-derived mesenchymal stem cells. The two types of cells were cultured at 3 × 10 in a 100 mm dish. 5The cells were aliquoted at a density of 1 / 2 a dish and used for analysis at 80% confluence. For RNA-seq, total RNA was extracted from the cells, and all RNA samples were confirmed to be of uniform quality above the standard. cDNA libraries were prepared using the Truseq Stranded mRNA LTSample Prep Kit (Illumina) according to standard procedures. The cDNA libraries were sequenced and analyzed on a NovaSeq6000 System (Illumina) according to the Truseq Stranded mRNA Sample Preparation Guide (Part #15031047Rev.E). Each RNA-seq data set was generated using three samples per cell (n=3). Phred quality scores were calculated from the acquired Illumina read data using BBDuk (BBtools), and only data with an average Q30 or higher were used. The filtered read data were mapped to the reference sequence (hg19; genome database: USCS) using Bowtie2 (Langmead & Salzberg, 2012). Read data were calculated using Bedtools (https: / / bedtools.readthedocs.io / en / latest / ). Mapping and quantification were performed for each sample. Quantified gene expression information was quantile-normalized using edgeR (Robinson, McCarthy, & Smyth, 2010). All data were analyzed to compare similar mesenchymal stem cells and bone marrow-derived mesenchymal stem cells. The results are shown in Figures 16 and 17.

[0137] As shown in Figure 16, analysis of inflammation-regulating genes revealed that 16 genes were expressed at approximately two-fold higher levels compared to bone marrow-derived mesenchymal stem cells: Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cxcl1, Ptger4, Acp5, Bcr, Socs5, and Mdk. In particular, the Gata3 gene was expressed at approximately 108-fold higher levels, the Adora2a gene at approximately 27.97-fold higher levels, and the Gps2 gene at approximately 11.02-fold higher levels. Therefore, it was confirmed that inflammation-regulating genes were expressed at approximately 10-fold higher levels in similar mesenchymal stem cells compared to bone marrow-derived mesenchymal stem cells.

[0138] Meanwhile, the results of analyzing gene expression related to immunosuppressive ability using RNA-seq are shown in Figure 15. There are 11 genes that are expressed at more than two-fold higher than in bone marrow-derived mesenchymal stem cells: Gata3, Gps2, Psma1, Apoe, Foxf1, Tek, Cxcl1, Ptger4, Bcr, Socs5, and Mdk. In particular, the Gata3 gene was expressed at 108-fold higher and the Adora2a gene was expressed at 11.02-fold higher, confirming that their expression is more than 10-fold higher than in bone marrow-derived mesenchymal stem cells.

[0139] Embodiment 13: Analysis of protein in cell lysates and culture supernatants of isolated and cultured similar mesenchymal stem cells

[0140] The cell lysate and medium of the similar mesenchymal stem cells prepared in Examples 1 and 2 were analyzed for enhanced proteins using the L507 antibody array.

[0141] Specifically, to compare the protein composition of cell lysates and culture supernatants from analogous mesenchymal stem cells and bone marrow-derived mesenchymal stem cells, we used the L507 (RayBiotech) quantitative protein antibody array chip. Cell lysates and culture supernatants were collected at the same time as total RNA was harvested. The L507 tip, which analyzes 507 proteins based on biotin labeling, was selected for analyzing analogous mesenchymal stem cell proteins. Proteins were extracted from cell lysates and culture supernatants for analysis. Proteins were then quantified (50–200 μg range) using a BCA protein assay kit (Abcam). Equal amounts of quantified proteins were biotin-labeled and then subjected to protein hybridization. Fluorescent images from hybridization were visualized using a streptavidin-cyanine3 conjugate and scanned using a GenePix 4100A Microarray Scanner (Molecular Devices). All data were quantitated using edgeR. The data was then analyzed using GenePix Pro 7.0 software (Molecular Devices). All data was analyzed to compare the similar mesenchymal stem cells with bone marrow-derived mesenchymal stem cells, and the results are shown in Figures 18, 19, 20, and 21.

[0142] Lysate proteins of the mesenchymal stem cell lysates were analyzed, as shown in Figure 18. The mesenchymal stem cell lysates expressed Angiopoietin-4 (ANGPT4), BMPR-1B (bone morphogenic protein receptor 1B), HCR (human chemokine receptor), Activin B, Activin RII, CCR1, and ICAM-2 (intracellular adhesion molecule 2) at approximately two-fold higher levels than bone marrow-derived stem cells. On the other hand, as shown in Figure 19, the expression levels of 15 proteins, including Angiogenin (ANG), Angiopoetin-1 (ANG-1), Angiopoetin-2 (ANG-2), Bone morphogenic proteins receptor 1A (BMPR1A), and CXCR6 (CXC-chemokine receptor 6), were approximately two-fold lower in the mesenchymal stem cells than in bone marrow-derived mesenchymal stem cells.

[0143] Meanwhile, the proteins in the culture supernatant were analyzed, as shown in Figure 20. The mesenchymal stem cell-like cells secreted approximately twice as much GRO-α, IL-15R alpha, FasL, Activin RII, BMP-2, CCR2, CXCL14, and FGFR4 as compared to bone marrow-derived stem cells. As shown in Figure 21, the mesenchymal stem cell-like cells expressed low levels of TIMP-2 (Tissue inhibitor of methalloproteinases-2), Activin A, VEGFA (Vascular endothelial factor), FSTL1 (Follistantin-like 1), ErB4, and Thrombospondin-1.

[0144] The present disclosure also includes the following aspects. <1> A method for producing mesenchymal stem cells similar to mesenchymal stem cells, comprising: (a) preparing human pluripotent stem cells that have been passaged 70 times or less after establishment of a cell line; (b) selecting cystic embryoid bodies from the embryoid bodies obtained by inducing differentiation of the human pluripotent stem cells; (c) loading the cystic embryoid bodies onto a cell-permeable three-dimensional culture insert to isolate similar mesenchymal stem cells; (d) separating only the monolayer cell population from the cells that have permeated the cell-permeable three-dimensional culture insert; (e) homogenizing the monolayer cell population to a size of 100 μm to 500 μm in length and width, and culturing similar mesenchymal stem cells; Including, The mesenchymal stem cell-like cells have anti-inflammatory and immunosuppressive effects, and express CD90 and SOX2 at 95% or more. <2> The cell-permeable three-dimensional culture insert is made of one or more of nylon, fiber, polyethylene, polypropylene, graphene, titanium, copper, nickel, silver, gold, and platinum. <1> A method for producing mesenchymal stem cells similar to those described in . <3> The aforementioned <1> or <2> In the method for producing human pluripotent stem cell-derived mesenchymal stem cells, The mesenchymal stem cells are characterized by having higher expression of MMP-1 protein and HGF protein than bone marrow-derived mesenchymal stem cells, lower expression of CD95 than bone marrow-derived mesenchymal stem cells, and expressing 95% or more of CD90 and SOX2. <4> The similar mesenchymal stem cells express the MMP-1 protein at a level 15 times or more higher and the HGF protein at a level 2 times or more higher than that of bone marrow-derived mesenchymal stem cells. <3> Similar mesenchymal stem cells as described above. <5> The similar mesenchymal stem cells express CD95 at a level 15 times lower than that of bone marrow-derived mesenchymal stem cells. <3> or <4> Similar mesenchymal stem cells as described above. <6> The similar mesenchymal stem cells express inflammation-regulating genes at 2 to 100 times higher levels than bone marrow-derived mesenchymal stem cells, the inflammation-regulating gene is any one or more of the group consisting of Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Acp5, Bcr, Socs5, and Mdk; <3> ~ <5> The similar mesenchymal stem cell described in any one of the above. <7> The analogous mesenchymal stem cells simultaneously express Gata3, Adora2a, and Gps2 genes. <3> ~ <6> The similar mesenchymal stem cell described in any one of the above. <8> The mesenchymal stem cells have 2 to 100 times higher expression of immunosuppressive genes than bone marrow-derived mesenchymal stem cells, the immunosuppressive gene is one or more of Gata3, Gps2, Psma1, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Bcr, Socs5, and Mdk; <3> ~ <7> The similar mesenchymal stem cell described in any one of the above. <9> The analogous mesenchymal stem cells simultaneously express Gata3, Gps2 and Psma1 genes. <3> ~ <8> The similar mesenchymal stem cell described in any one of the above. <10> The aforementioned <3> ~ <9> A therapeutic composition comprising the similar mesenchymal stem cells according to any one of the preceding claims, The therapeutic composition is any one of a cell therapy composition, a cell gene therapy composition, a tissue engineering therapy composition, an anti-inflammatory therapy composition, an immunotherapy composition, and a composition for the prevention or treatment of cancer. <11> The therapeutic composition further comprises an active ingredient or exosomes secreted from the similar mesenchymal stem cells. <10> The therapeutic composition according to claim 1. <12> The therapeutic composition is for preventing or treating one or more of the following diseases: multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic pulmonary disease, acute pulmonary disease, Crohn's disease, fecal incontinence, graft-versus-host disease, lower limb ischemia, thromboangiitis obliterans (Buerger's disease), foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injuries caused by various exercises, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage caused by burns, skin damage caused by ultraviolet rays, and inflammatory and immune system diseases including retinopathy; ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system diseases. <10> or <11> The therapeutic composition according to claim 1. <13> The aforementioned <3> ~ <9> A delivery body comprising the similar mesenchymal stem cells according to any one of the preceding claims, The vehicle is characterized in that it carries a pharmaceutical composition. <14> The aforementioned <3> ~ <9> A composition for preventing or treating a disease, comprising an active ingredient or exosomes secreted from the similar mesenchymal stem cells described in any one of the above. <15> The disease is one or more of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft-versus-host disease, lower limb ischemia, thromboangiitis obliterans, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injuries caused by various exercises, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage caused by burns, skin damage caused by ultraviolet rays, etc., prevention of inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system diseases. <14> A composition for preventing or treating the diseases described above. <16> The aforementioned <3> ~ <9> A cosmetic composition comprising an active ingredient or exosomes secreted from the similar mesenchymal stem cells described in any one of claims 1 to 4.

[0145] Although the embodiments have been described above with reference to limited examples and drawings, those skilled in the art will appreciate that various modifications and variations may be made from the above description. For example, the techniques described may be performed in a different order than described, and / or the components described may be combined or substituted in a different manner than described, or other components or equivalents may be substituted or replaced, and still achieve suitable results.

[0146] Accordingly, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

Claims

1. A mesenchymal stem cell analog obtained from an embryoid body of human pluripotent stem cells that has been subcultured 70 times or less after the establishment of the cell line, More than 95% of the cells express CD90, CD44, CD73, CD105, and SOX2; less than 2% of the cells express CD45, CD34, CD14, and CD19; More than 90% of cells express CD146, The similar mesenchymal stem cells express Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cx3cl1, Ptger4, Acp5, Bcr, Socs5 and Mdk at levels two times or more higher than bone marrow-derived mesenchymal stem cells. Similar mesenchymal stem cells with anti-inflammatory and immunosuppressive properties.

2. The mesenchymal stem cell analog of claim 1, wherein the mesenchymal stem cell analog expresses MMP-1 protein and HGF protein at a higher level than bone marrow-derived mesenchymal stem cells, and expresses CD95 at a lower level than bone marrow-derived mesenchymal stem cells.

3. The mesenchymal stem cell analog of claim 1, wherein the mesenchymal stem cell analog expresses MMP-1 protein at a level 15 times or more higher and HGF protein at a level 2 times or more higher than bone marrow-derived mesenchymal stem cells.

4. The mesenchymal stem cell-like stem cell according to claim 1 , wherein the expression of CD95 in the mesenchymal stem cell-like stem cell is 15 times or more lower than that in the bone marrow-derived mesenchymal stem cell.

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