Anticancer composition containing stem cell-derived exosomes and method for producing the same

Abstract

Regarding an anticancer composition containing stem cell-derived exosomes and a method for producing the same, the composition according to the present invention has excellent anticancer effects and can be used as a therapeutic agent for cancer in combination with existing anticancer agents or independently.

Description

Technical Field

[0001] The present invention relates to a composition containing stem cell-derived exosomes and a method for producing the same, and more particularly, to a composition containing exosomes isolated from mesenchymal stem cells or their cultures, which have excellent anti-cancer effects.

Background Art

[0002] Extracellular Vesicles are vesicles composed of a spherical lipid bilayer with a size of 30 to 1000 nm, including Microvesicles, Exosomes, etc.

[0003] The lipid bilayer of exosomes has a phospholipid bilayer structure like the origin cell (donor cell), and is a constituent of substances secreted by cells outside the cell, and is known to play functional roles such as cell-cell communication and cellular immune arbitration.

[0004] Exosomes contain cell-specific components that reflect the biological functions unique to the origin cells, and in addition to phospholipids, mRNA, miRNA, also contain various water-soluble proteins, extrinsic proteins, and transmembrane protein components.

[0005] Such exosomes are discharged from all animal cells such as mast cells, lymphocytes, stellate cells, platelets, nerve cells, endothelial cells, epithelial cells, etc., and are found in various body fluids such as blood, urine, mucus, saliva, bile fluid, ascites fluid, cerebrospinal fluid, etc. Exosomes can also pass through the Blood-Brain Barrier (BBB), and because of their high selective permeability to the extent that they can permeate the cell membranes of epidermal cells and endothelial cells, they are also used in the development of DDS (drug delivery system), a nanocarrier for specific drugs.

[0006] Exosomes and microvesicles secreted from mesenchymal stem cells are known to be involved in cell-to-cell communication and exhibit the regenerative medical treatment efficacy of stem cells.

[0007] It is known that paracrine factors secreted from cells without long-term survival after transplantation of stem cells into the body bring about a trophic effect on these factors. Low-molecular substances such as growth factors, chemokines, and cytokines are secreted by extracellular vesicles such as exosomes, and such exosomes are derived from stem cells. Therefore, exosomes are utilized to identify the characteristics of stem cells and evaluate their therapeutic efficacy. Recently, various studies have been actively conducted on the therapeutic effects of exosomes secreted by mesenchymal stem cells without using mesenchymal stem cells themselves, and this is expected to be a new alternative that can overcome the limitations of existing stem cell therapies in the academic and industrial fields.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Therefore, the present inventors developed a composition separated from stem cells or their culture solution and confirmed that the composition according to the present invention has excellent anti-cancer effects.

[0009] Therefore, an object of the present invention is to provide a composition containing exosomes derived from stem cells.

[0010] Another object of the present invention is to provide a method for producing a composition containing exosomes derived from stem cells.

[0011] Yet another object of the present invention is to provide a composition for alleviating, suppressing, preventing, or treating cancer containing exosomes derived from stem cells.

[0012] Another object of the present invention is to provide a method for producing a composition for alleviating, suppressing, preventing or treating cancer.

[0013] Another object of the present invention is to provide a method for alleviating or treating cancer.

[0014] Another object of the present invention is to provide the use of a composition containing exosomes derived from stem cells for alleviating, suppressing, preventing or treating cancer.

Means for Solving the Problems

[0015] Regarding an anti-cancer composition containing exosomes derived from stem cells and a method for producing the same, the composition according to the present invention exhibits excellent effects in the treatment or prevention of cancer.

[0016] Therefore, the present inventors confirmed that the composition according to the present invention has excellent anti-cancer effects.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] One aspect of the present invention is a composition containing exosomes derived from stem cells.

[0019] The term "exosome" in this specification is a cellular vesicle, which exists in the body fluids of almost all eukaryotes, and means a vesicle having a diameter of about 30 to 100 nm, which is larger than LDL protein but much smaller than red blood cells. It is well known that exosomes can be released from cells when multivesicular bodies fuse with the cell membrane or can be released directly from the cell membrane, and have important but specialized functions such as coagulation and intercellular signal transmission.

[0020] The term "stem cell" in this specification is an undifferentiated cell, which means a cell having the ability of self-renewal and the ability to differentiate into two or more different types of cells.

[0021] In one embodiment of the present invention, the stem cells may be autologous or allogeneic stem cells, may be derived from any type of animal including humans and non-human mammals, may be stem cells derived from an adult, or may be stem cells derived from a germ. For example, the stem cells may be, but are not limited to, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells derived from induced pluripotent stem cells, BxC stem cells, mesenchymal stem cells derived from induced pluripotent stem cells pretreated with interferon gamma (IFN-γ), and BxC-117 stem cells.

[0022] The term "adult stem cell" as used herein refers to cells extracted from umbilical cord blood, adult bone marrow, blood, etc., and means cells immediately before differentiating into specific organ cells, and means undifferentiated cells having the ability to develop into body tissues when necessary.

[0023] In one embodiment of the present invention, the adult stem cells may be, but are not limited to, adult stem cells of human, animal or animal tissue origin, mesenchymal stromal cells derived from human, animal or animal tissue, and mesenchymal stem cells derived from induced pluripotent stem cells of human, animal or animal tissue origin.

[0024] In the present invention, the human, animal or animal tissue may be, but is not limited to, selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta.

[0025] In the present invention, the stem cells of various tissue origins of human or animal may be, but are not limited to, selected from the group consisting of hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells, and testicular stem cells.

[0026] The term "embryonic stem cell" in this specification refers to cells extracted during the embryonic development process, which means cells obtained by extracting the inner cell mass from the embryo at the blastocyst stage, just before the fertilized egg implants into the mother's uterus, and culturing them in vitro.

[0027] Embryonic stem cells refer to cells with pluripotent or totipotent self-renewal ability that can differentiate into cells of all tissues of an individual. In a broad sense, it also means including embryoid bodies derived from embryonic stem cells.

[0028] In the present invention, the stem cells may include embryonic stem cells from any source such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., but are not limited thereto.

[0029] The term "induced pluripotent stem cell (iPSC)" in this specification refers to cells induced to have pluripotent differentiation ability through an artificial dedifferentiation process from differentiated cells, and may be used in the same meaning as "dedifferentiated stem cells".

[0030] The artificial dedifferentiation process is carried out by virus-mediated or non-viral vector utilization using retroviruses, lentiviruses, and Sendai viruses, or by introducing non-viral mediated dedifferentiation factors such as proteins and cell extracts, or may include a dedifferentiation process using stem cell extracts, compounds, etc.

[0031] Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells. Specifically, they have a similar cell shape, similar gene and protein expression, have the ability to differentiate completely in vitro and in vivo, form teratomas, form chimera mice when inserted into the blastocysts of mice, and are capable of germline transmission of genes.

[0032] In one embodiment of the present invention, the stem cells may be mesenchymal stem cells derived from induced pluripotent stem cells.

[0033] In one embodiment of the present invention, the composition may include exosomes derived from mesenchymal stem cells derived from induced pluripotent stem cells.

[0034] As used herein, the term "mesenchymal stem cell (MSC)" means a stem cell derived from mesenchyme. Mesenchymal stem cells may differentiate into one or more cells selected from the group consisting of osteoblasts, chondrocytes, adipocytes, or myocytes. Mesenchymal stem cells may be isolated from any type of adult tissue, for example, bone marrow adipose tissue, umbilical cord, or peripheral blood. A population of mesenchymal stem cells may be defined as exhibiting a specific phenotype. A population of mesenchymal stem cells differentiated from induced pluripotent stem cells may exhibit the same phenotypic characteristics as a population of normal mesenchymal stem cells, and a population of mesenchymal stem cells may be understood as a population of stem cells that express 95% or more of the CD105, CD73, and CD90 markers and 2% or less of the CD45, CD34, and SSEA-4 markers.

[0035] In one embodiment of the present invention, the stem cells may be BxC stem cells.

[0036] In one embodiment of the present invention, the composition may include exosomes derived from BxC stem cells.

[0037] The term "BxC stem cells" in this specification can mean stem cells produced by culturing induced pluripotent stem cells (iPSCs), separating clusters of induced pluripotent stem cells that do not express the SSEA-4 (stage-specific embryonic antigen 4) protein, and then further culturing them. BxC stem cells are cells at a stage just before complete differentiation from induced pluripotent stem cells into mesenchymal stem cells and can have the properties of complete mesenchymal stem cells through further culture. Therefore, the phenotype of the BxC stem cell cluster does not show exactly the same phenotype as the mesenchymal stem cell cluster, but can show a phenotype similar to that of the mesenchymal stem cell cluster in the range of 96% to 99.9%. For example, the induced pluripotent stem cell cluster expresses 0.3% of the CD90 protein, while the mesenchymal stem cell cluster expresses 99.7% of the CD90 protein, and the BxC stem cell cluster can express 96.9%, which is about 98% of the mesenchymal stem cells. Thus, BxC stem cells can be defined as stem cells that are differentiated 96% to 99.9% without complete differentiation into mesenchymal stem cells by further culturing induced pluripotent stem cells that do not express the SSEA-4 protein after culturing the induced pluripotent stem cells. BxC stem cells are superior in stemness compared to mesenchymal stem cells differentiated from the same induced pluripotent stem cells and can secrete a large amount of proteins related to functionality. Specifically, the BxC stem cells of the present invention show a difference in proliferative ability of more than 10 times compared to mesenchymal stem cells (MSCs) derived from the same tissue when subculturing is repeated 9 times or more, and no decrease in proliferative ability is observed even when subculturing is performed 12 times or more. Also, the BxC stem cells show an expression level of Ki67, a marker related to cell proliferative ability, that is more than 2 times higher than that of general mesenchymal stem cells.And BxC stem cells can express one or more genes selected from the group consisting of ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4, and ACKR3 at a higher level and one or more genes selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10, and KCNE4 at a lower level compared to mesenchymal stem cells differentiated from the same induced pluripotent stem cells.

[0038] As used herein, the term "stem cell ability" means pluripotency, the ability to generate any cell, and self-renewal, the ability to generate an unlimited number of cells similar to itself. For example, it refers to increasing the proliferation ability of stem cells while maintaining the undifferentiated state of undifferentiated cells, increasing telomerase activity, increasing the expression of stemness acting signals, or increasing cell migration activity, and may include the presence of one or more of these characteristics.

[0039] In one embodiment of the present invention, the stem cells may be mesenchymal stem cells derived from induced pluripotent stem cells pretreated with interferon gamma (IFN-γ).

[0040] In one embodiment of the present invention, the composition may contain exosomes derived from mesenchymal stem cells derived from induced pluripotent stem cells pretreated with interferon gamma (IFN-γ).

[0041] As used herein, the term "pretreatment" refers to the process of adding a specific substance to the medium of mesenchymal stem cells and culturing them. For example, pretreatment means culturing mesenchymal stem cells that have completed differentiation in induced pluripotent stem cells in a medium supplemented with interferon gamma (IFN-γ).

[0042] In the present invention, interferon gamma can increase the stemness and proliferation ability of stem cells, and increase the number of stem cell-derived exosomes and the content of proteins and RNAs in the exosomes.

[0043] In one embodiment of the present invention, the stem cells may be BxC-I17 stem cells.

[0044] In one embodiment of the present invention, the composition may contain exosomes derived from BxC-I17 stem cells.

[0045] As used herein, the term "BxC-I17 stem cells" refers to mesenchymal stem cells obtained by culturing (pretreating) mesenchymal stem cells derived from induced pluripotent stem cells in a medium containing interferon gamma. At this time, the mesenchymal stem cells derived from induced pluripotent stem cells may be stem cells obtained by culturing BxC stem cells and completely differentiating them into mesenchymal stem cells. That is, in the present specification, BxC-I17 stem cells can be defined as mesenchymal stem cells obtained by culturing BxC stem cells to completely differentiate them from mesenchymal stem cells and then culturing (pretreating) them in a medium containing interferon gamma. Therefore, BxC-I17 stem cells can be obtained by culturing BxC stem cells to completely differentiate them into mesenchymal stem cells and then culturing them in a medium containing 0.001 to 1000 ng, for example, 10 ng of interferon gamma for 12 to 48 hours. BxC-I17 stem cells have a cell proliferation rate increased by about 240%, an exosome production efficiency increased by about 6 times or more, and an amount of exosome-derived proteins increased by 6 times or more compared to mesenchymal stem cells that have not been pretreated with any substance.

[0046] In one embodiment of the present invention, interferon-gamma may be pretreated in a medium at 0.001 to 1000 ng, 0.005 to 500 ng, 0.01 to 100 ng, 0.05 to 50 ng, 0.1 to 25 ng, 0.5 to 15 ng, 1 to 10 ng, or 5 to 15 ng, for example, it may be pretreated at 10 ng, but is not limited thereto.

[0047] Another aspect of the present invention is a pharmaceutical composition containing exosomes derived from stem cells as an active ingredient.

[0048] In the present invention, the pharmaceutical composition may contain exosomes separated from induced pluripotent stem cell-derived mesenchymal stem cells or their cultures as an active ingredient.

[0049] In the present invention, the pharmaceutical composition may contain exosomes derived from stem cells pretreated with interferon gamma.

[0050] In the present invention, the pharmaceutical composition may contain exosomes separated from induced pluripotent stem cell-derived mesenchymal stem cells or their cultures pretreated with interferon-gamma.

[0051] In the present invention, the pharmaceutical composition may contain one or more exosomes selected from the group consisting of exosomes derived from BxC stem cells (BxC-e) and exosomes derived from BxC-I17 stem cells (BxC-I17e).

[0052] As used herein, the term "containing as an active ingredient" means containing an amount sufficient to achieve the alleviating, suppressing, preventing, or therapeutic activity of exosomes separated from stem cells or their cultures against a specific disease.

[0053] In the present invention, the pharmaceutical composition may contain a pharmaceutically acceptable carrier, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, etc., but is not limited thereto.

[0054] In the present invention, the pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc.

[0055] In the present invention, the pharmaceutical composition can be administered orally and parenterally, for example, by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, pulmonary administration, rectal administration, intrathecal administration, intraocular administration, skin administration, and transdermal administration, etc., but is not limited thereto.

[0056] In the present invention, the dosage of the pharmaceutical composition may be determined variously depending on factors such as the formulation method, the administration mode, the age, weight, gender, pathological condition, diet, administration time, administration route, excretion rate, and responsiveness of the patient, and may be determined or formulated as a dosage effective for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001 to 1000 mg / kg.

[0057] In the present invention, the pharmaceutical composition is manufactured in the form of a unit dosage or incorporated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient by a method that can be easily implemented by a person having ordinary knowledge in the technical field to which the present invention pertains. At this time, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further contain a dispersant or a stabilizer, but is not limited thereto.

[0058] The dosage of the pharmaceutical composition of the present invention may vary depending on the age, weight, gender, dosage form, health status, and degree of disease of the patient, and may be administered once or divided into several times a day at regular intervals according to the judgment of a doctor or pharmacist. For example, the daily dosage may be 1 to 1000 μg / ml based on the content of the active ingredient, but this is only an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0059] In the present invention, the pharmaceutical composition may be a pharmaceutical composition for treating, preventing, alleviating, or suppressing cancer.

[0060] The term "treatment of cancer" in this specification means all actions that suppress cancer. Specifically, it can mean the action of regulating the cell cycle of cancer cells or the action of inducing apoptosis of cancer cells, but is not limited thereto.

[0061] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, glioblastoma, or myeloid cancer, but is not limited thereto.

[0062] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of liver cancer, pancreatic cancer, glioblastoma, colon cancer, breast cancer, prostate cancer, and blood cancer, but is not limited thereto.

[0063] Another aspect of the present invention is a food composition containing stem cell-derived exosomes as an active ingredient.

[0064] Since the food composition according to the present invention contains the stem cell-derived exosomes according to the present invention in the same manner as the above-described pharmaceutical composition, the content common to both is omitted in the description in order to avoid excessive complexity of this specification.

[0065] The food composition according to the present invention may contain components generally added during food production, for example, it may contain, but is not limited to, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents.

[0066] Carbohydrates that may be included in the food composition according to the present invention may include, but are not limited to, monosaccharides such as glucose and fructose, disaccharides such as maltose, sucrose, and oligosaccharides, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0067] Flavoring agents that may be included in the food composition according to the present invention may include, but are not limited to, natural flavoring agents such as thaumatin and stevia extract, and synthetic flavoring agents such as saccharin and aspartame.

[0068] In the present invention, the food composition may be a food composition for relieving, suppressing, or improving cancer.

[0069] Still another aspect of the present invention is a cell therapy agent containing induced pluripotent stem cell-derived mesenchymal stem cells.

[0070] As used herein, the term "cell therapy agent" means a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention by a series of actions such as proliferating and selecting living autologous, allogenic, or xenogenic cells in vitro or changing the biological characteristics of cells by other methods to restore the functions of cells and tissues.

[0071] In one embodiment of the present invention, the cell therapy agent may be a stem cell therapy agent.

[0072] In one embodiment of the present invention, the induced pluripotent stem cell-derived mesenchymal stem cells may include BxC-I17 stem cells.

[0073] In the present invention, the cell therapy agent may be a cell therapy agent for alleviating, suppressing, preventing or treating cancer.

[0074] Still another aspect of the present invention is a method for producing a composition comprising exosomes isolated from stem cells or a culture thereof, comprising the following steps:

[0075] A separation step of separating exosomes from stem cells or a culture thereof.

[0076] In the present invention, the method may be a method for producing a composition comprising exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells or a culture thereof.

[0077] In one embodiment of the present invention, the separation step may be separating exosomes from induced pluripotent stem cell-derived mesenchymal stem cells or a culture thereof.

[0078] In one embodiment of the present invention, the stem cells may be autologous or allogeneic stem cells, may be derived from any type of animal including humans and non-human mammals, may be stem cells derived from adults, or may be stem cells derived from embryos. For example, the stem cells may be, but are not limited to, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), induced pluripotent stem cell-derived mesenchymal stem cells, BxC stem cells, and BxC-I17 stem cells.

[0079] In the separation step, after centrifuging the culture medium of the stem cells at 200 to 400 xg for 5 to 20 minutes to remove the remaining cells and cell residues, the supernatant is taken and centrifuged at 9,000 to 12,000 xg for 60 to 80 minutes, and then the supernatant is taken again and centrifuged at 90,000 to 120,000 xg for 80 to 100 minutes to remove the supernatant, whereby the exosomes remaining in the lower layer can be obtained.

[0080] In one embodiment of the present invention, the method may further comprise a pretreatment step of pretreating induced pluripotent stem cell-derived mesenchymal stem cells with interferon-gamma.

[0081] In one embodiment of the present invention, the method may further include a selection culture step of separating and culturing SSEA-4(-) cells among the cultured induced pluripotent stem cells and differentiating them into BxC stem cells.

[0082] In one embodiment of the present invention, the method may further include a cell exosome production step of culturing the stem cells in a cell culture medium.

[0083] The exosome production step according to the present invention is a process of inducing the secretion or production of exosomes from stem cells. In the present invention, the cell culture medium may include any stem cell culture medium commonly used in the art. For example, commercially manufactured media such as DMEM (Dulbecco’s Modified Eagle’s Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco’s Modified Eagle’s Medium:Nutrient Mixture F-10), DMEM / F-12 (Dulbecco’s Modified Eagle’s Medium:Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow’s Minimal Essential Medium), IMDM (Isocove’s Modified Dulbecco’s Medium), KnockOut DMEM, E8 (Essential 8 Medium) or artificially synthesized media may be used, but it is not limited thereto.

[0084] In one embodiment of the present invention, the cell culture medium may further include components such as a carbon source, a nitrogen source, trace element components, amino acids, and antibiotics.

[0085] In one embodiment of the present invention, the exosome production stage may include an additional culture stage of culturing stem cells with fetal bovine serum (FBS) from which exosomes have been removed.

[0086] In the cell culture medium, FBS from which exosomes have been removed, unlike general FBS that contains a large amount of exosomes derived from bovine serum, has exosomes removed, so it is possible to prevent contamination of exosomes derived from FBS in addition to exosomes secreted by stem cells.

[0087] Still another aspect of the present invention is a method for producing a composition containing exosomes, including the following steps:

[0088] A first culture stage of culturing induced pluripotent stem cells in a medium;

[0089] A selection culture stage of separating SSEA-4(-) cells from the cultured induced pluripotent stem cells, culturing them for 1 to 10 days in a medium containing FBS and bFGF, and differentiating them into BxC stem cells;

[0090] A second culture stage of culturing BxC stem cells to differentiate them into mesenchymal stem cells;

[0091] A pretreatment stage of pretreating mesenchymal stem cells with interferon-gamma;

[0092] A production stage of culturing the pretreated mesenchymal stem cells to produce exosomes; and

[0093] A separation stage of separating exosomes from mesenchymal stem cells or their culture.

[0094] In one embodiment of the present invention, the first culture stage may be culturing induced pluripotent stem cells in a medium containing FBS and bFGF for 1 to 10 days.

[0095] In one embodiment of the present invention, the selection culture stage may be separating SSEA-4(-) cells from induced pluripotent stem cells, culturing them for 1 to 10 days in a medium containing FBS and bFGF, and differentiating them into BxC stem cells.

[0096] In one embodiment of the present invention, the pretreatment step may include culturing mesenchymal stem cells in a medium containing interferon-gamma at 0.001 to 1000 ng, 0.005 to 500 ng, 0.01 to 100 ng, 0.05 to 50 ng, 0.1 to 25 μM, 0.5 to 15 ng, 1 to 10 μM, or 5 to 15 ng.

[0097] In one embodiment of the present invention, the production step may include an additional culturing step of culturing mesenchymal stem cells with fetal bovine serum (FBS) from which exosomes have been removed.

[0098] In the present invention, the method may be a method for producing a pharmaceutical composition for alleviating, suppressing, preventing, or treating cancer, comprising an exosome isolated from induced pluripotent stem cell-derived mesenchymal stem cells or a culture thereof as an active ingredient.

[0099] Still another aspect of the present invention is a method for alleviating or treating cancer, comprising the following steps:

[0100] Administering to a subject a pharmaceutical composition comprising an exosome isolated from stem cells or a culture thereof as an active ingredient.

[0101] In one embodiment of the present invention, the exosome isolated from stem cells or a culture thereof may be one or more exosomes selected from the group consisting of exosomes derived from BxC stem cells (BxC-e) and exosomes derived from BxC-I17 stem cells (BxC-I17e).

[0102] Since the treatment method according to the present invention, like the above-described composition, contains an exosome isolated from stem cells or a culture thereof, the content common to both is omitted from the description herein in order to avoid excessive complexity of the present specification.

[0103] As used herein, the term "treatment" refers to all acts by which a disease is improved or beneficially changed by administration of a composition according to the present invention.

[0104] As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention may be oral or parenteral administration by any general route as long as the target tissue can be reached. Further, the composition of the present invention may be administered using any device capable of delivering the active ingredient to the target cells.

[0105] As used herein, the term "subject" is not particularly limited, and includes, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, and may be, for example, a human, but is not limited thereto.

[0106] In one embodiment of the present invention, the pharmaceutical composition of the present invention may be administered alone, but generally may be administered in admixture with a pharmaceutical carrier selected in consideration of the administration method and standard pharmaceutical practice.

[0107] Still another aspect of the present invention is the use of a composition containing exosomes derived from stem cells as an active ingredient for the alleviation, suppression, prevention or treatment of cancer.

Advantages of the Invention

[0108] Regarding an anticancer composition containing exosomes derived from stem cells and a method for producing the same, the composition according to the present invention has excellent anticancer effects and can be used as a therapeutic agent for cancer in combination with or independently of existing anticancer agents.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0118] A pharmaceutical composition for the treatment, prevention, alleviation or suppression of cancer, comprising exosomes isolated from induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSC) as an active ingredient.

Examples

[0119] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are merely for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0120] Example 1: Culture of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

[0121] Induced pluripotent stem cells (iPSC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 10 ng / ml basic fibroblast growth factor (bFGF) for 7 days. Then, by FACS analysis, among the cultured induced pluripotent stem cells, SSEA-4(-) cells that do not express stage-specific embryonic antigen 4 (SSEA-4) protein on the cell surface were isolated to obtain precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells. Next, the isolated SSEA-4(-) cells were passaged and further cultured in DMEM medium supplemented with 10% FBS and 10 ng / ml bFGF for 7 days to prepare BxC stem cells.

[0122] Thereafter, the BxC stem cells were further cultured in a culture medium containing high glucose DMEM (Gibco, USA), 10% FBS (HyClone, USA), and 1% MEM non-essential amino acid solution (100X, Gibco, USA) to fully differentiate them into induced pluripotent stem cell-derived mesenchymal stem cells.

[0123] Example 2: Isolation and Characterization of Exosomes Derived from Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells (BxC-e)

[0124] 2-1. Isolation of BxC-e Exosomes

[0125] The culture medium of induced pluripotent stem cell-derived mesenchymal stem cells cultured in Example 1 was collected and centrifuged at 300 xg for 10 minutes to remove the remaining cells and cell residues. After centrifugation, the supernatant was taken and filtered using a 0.22 μm filter, and then centrifuged at 10,000 xg at 4°C for 70 minutes using a high-speed centrifuge.

[0126] Next, the centrifuged supernatant was taken again and centrifuged at 100,000 xg at 4°C for 90 minutes using an ultracentrifuge. Thereafter, the supernatant was removed, and the exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) to isolate exosomes derived from induced pluripotent stem cell-derived mesenchymal stem cells (hereinafter referred to as BxC-e exosomes), which were used in the following experiments.

[0127] 2-2. Characterization of BxC-e Exosomes

[0128] The size distribution of the BxC-e exosomes isolated in Example 2-1 was confirmed using a nanoparticle tracking assay (NanoSight NS300, Malvern Panalytical), and the morphology of the exosomes was confirmed using an electron microscope.

[0129] As a result, as can be confirmed from FIG. 1, it was found that the BxC-e exosome has characteristics as an exosome.

[0130] Example 3: Isolation of exosomes (BxC-I17e) derived from induced pluripotent stem cell-derived mesenchymal stem cells (BxC-I17) pretreated with interferon-gamma

[0131] The induced pluripotent stem cell-derived mesenchymal stem cells produced in Example 1 above were cultured in high-glucose DMEM culture medium containing 10% fetal bovine serum, 1% MEM non-essential amino acid solution, and 20 ng / ml of IFNγ for 24 hours to prepare induced pluripotent stem cell-derived mesenchymal stem cells (BxC-I17 stem cells) pretreated with IFNγ.

[0132] After completion of the culture, the BxC-I17 stem cells were washed and further cultured for 72 hours in a culture medium supplemented with 10% FBS from which exosomes had been removed.

[0133] After culturing for 72 hours, the culture medium treated with the pretreatment substance was collected and centrifuged at 300 xg for 10 minutes to remove the remaining cells and cell residues. Next, the supernatant was taken, filtered using a 0.22 μm filter, and then centrifuged at 10,000 xg at 4°C for 70 minutes using a high speed centrifuge. Thereafter, the centrifuged supernatant was taken again and centrifuged at 100,000 xg at 4°C for 90 minutes using an ultracentrifuge to remove the supernatant, and the exosomes remaining in the lower layer were diluted in PBS to isolate IFNγ-pretreated exosomes (hereinafter, BxC-I17e exosomes), which were used in the following experiments.

[0134] Example 4: Cancer cell survival test by treatment with exosomes (BxC-I17e) derived from induced pluripotent stem cell-derived mesenchymal stem cells (BxC-I17) pretreated with interferon-gamma

[0135] 4-1. Hepatocellular carcinoma cell line

[0136] 3×10³ cells were seeded on 96-well culture plates. After removing the culture medium 16 hours later, the cells were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing it with serum-free DMEM culture medium. Then, the CCK (Cell Counting Kit)-8 assay solution was added to the culture medium at a 1 / 10 dilution factor, and the viability of the liver cancer cell lines SNU449 and Huh7 was observed 3 hours later and shown in Figure 1 and Table 1.

[0137]

Table 1

[0138] As a result of the experiment, as can be confirmed from Figure 1 and Table 1, when BxC-I17e exosomes were administered to the liver cancer cell lines SNU449 and Huh7, the viability of the cancer cell lines could be significantly decreased compared to the control group.

[0139] 4-2. Pancreatic cancer cell lines

[0140] 3×10³ cells were seeded on 96-well culture plates. After removing the culture medium 16 hours later, the cells were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing it with serum-free DMEM culture medium. Then, the CCK (Cell Counting Kit)-8 assay solution was added to the culture medium at a 1 / 10 dilution factor, and the viability of the pancreatic cancer cell line MIA-PaCa-2 was observed 3 hours later and shown in Figure 2 and Table 2.

[0141]

Table 2

[0142] As a result of the experiment, as can be confirmed from Figure 2 and Table 2, when BxC-I17e exosomes were administered to the pancreatic cancer cell line MIA-PaCa-2, the viability of the cancer cell line could be significantly decreased compared to the control group.

[0143] 4-3. Glioblastoma cell lines

[0144] 3×10³ cells were seeded onto 96-well culture plates. After removing the culture medium 16 hours later, the cells were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing it with serum-free DMEM culture medium. Subsequently, the CCK (Cell Counting Kit)-8 assay solution was added to the culture medium at a 1 / 10 dilution factor, and the survival rates of the glioblastoma cell lines HS683, U87MG, and A172 were observed 3 hours later and shown in Figure 3 and Table 3.

[0145]

Table 3

[0146] As a result of the experiment, as can be confirmed from Figure 3 and Table 3, when BxC-I17e exosomes were administered to the glioblastoma cell lines HS683, U87MG, and A172, the survival rate of the cancer cell lines could be significantly decreased compared to the control group.

[0147] 4-4. Colorectal cancer cell lines

[0148] 3×10³ cells were seeded onto 96-well culture plates. After removing the culture medium 16 hours later, the cells were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing it with serum-free DMEM culture medium. Subsequently, the CCK (Cell Counting Kit)-8 assay solution was added to the culture medium at a 1 / 10 dilution factor, and the survival rates of the colorectal cancer cell lines HCT116 and COLO205 were observed 3 hours later and shown in Figure 4 and Table 4.

[0149]

Table 4

[0150] As a result of the experiment, as can be confirmed from Figure 4 and Table 4, when BxC-I17e exosomes were administered to the colorectal cancer cell lines HCT116 and COLO205, the survival rate of the cancer cell lines could be significantly decreased compared to the control group.

[0151] 4-5. Breast cancer cell lines

[0152] 3×10³ cells were seeded on 96-well culture plates. After removing the culture medium 16 hours later, they were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing it with serum-free DMEM culture medium. Then, after adding the CCK (Cell Counting Kit)-8 assay solution to the culture medium at a 1 / 10 dilution factor, the viability of the breast cancer cell lines T47D and MCF7 was observed 3 hours later and is shown in Fig. 5 and Table 5.

[0153]

Table 5

[0154] As a result of the experiment, as can be confirmed from Fig. 5 and Table 5, when BxC-I17e exosomes were administered to the breast cancer cell lines T47D and MCF7, the viability of the cancer cell lines could be significantly decreased compared to the control group.

[0155] 4 - 6. Prostate cancer cell lines

[0156] 3×10³ cells were seeded on 96-well culture plates. After removing the culture medium 16 hours later, they were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing it with serum-free DMEM culture medium. Then, after adding the CCK (Cell Counting Kit)-8 assay solution to the culture medium at a 1 / 10 dilution factor, the viability of the prostate cancer cell line PC3 was observed 3 hours later.

[0157]

Table 6

[0158] As a result of the experiment, as can be confirmed from Fig. 6 and Table 6, when BxC-I17e exosomes were administered to the prostate cancer cell line PC3, the viability of the cancer cell line could be significantly decreased compared to the control group.

[0159] 4 - 7. Hematological cancer cell lines

[0160] 3×10³ cells were seeded on 96-well culture plates. After removing the culture medium 16 hours later, the cells were treated with 200 μg / ml of BxC-I17e for 48 hours while replacing the medium with serum-free DMEM medium. Then, the CCK (Cell Counting Kit)-8 assay solution was added to the culture medium at a dilution factor of 1 / 10. After 3 hours, the survival rates of the blood cancer cell lines K-562 and U266B1 were observed and shown in Figure 7 and Table 7.

[0161]

Table 7

[0162] As can be confirmed from Figure 7 and Table 7 in the experimental results, when BxC-I17e exosomes were administered to the blood cancer cell lines K-562 and U266B1, the survival rate of the cancer cell lines could be significantly decreased compared to the control group.

[0163] Example 5: Confirmation of the tumor size reduction effect in a xenograft tumor model by treatment with exosomes (BxC-I17e) derived from interferon-gamma-pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC-I17)

[0164] 5-1. Prostate cancer xenograft tumor model

[0165] The prostate cancer cell line (PC-3) was administered to 6-week-old male SCID mice (NOD.CB17-Prkdcscid / Jcl). For cell line transplantation, after confirming the viability of the cultured cell line using a microscope, the cell line was prepared at a concentration of 1.0×108 cells / mL. Subsequently, the back of the animal was disinfected with 70% alcohol, the skin on the nape of the neck was pulled with the thumb and index finger to create a space between the skin and muscle, and then an injection syringe with a 26-gauge needle was inserted into the subcutaneous space between the thumb and index finger from the front of the animal, and gradually subcutaneously administered at a dose of 1.0×107 cells / 0.1 mL / head. When the tumor size at the site where the cell line was transplanted reached approximately 100 - 150 mm3, the animals were assigned so that the tumor sizes of each group were distributed as uniformly as possible according to the ranked tumor sizes. BxC-I17e was administered into the tumors using a syringe with a 31-gauge needle according to each group. The tumor sizes were observed three times a week for one week starting from the initiation of the test substance administration (Day 0) and are shown in Figure 8 and Table 8.

[0166] [Table 8]

[0167] As a result of the experiment, as can be confirmed from Figure 8 and Table 8, in the case of BxC-I17e exosomes, unlike the control group, the growth of tumors induced from the prostate cancer cell line could be effectively suppressed even as time passed.

[0168] 5-2. Hepatocellular carcinoma xenograft tumor model

[0169] The hepatocellular carcinoma cell line (Huh7) was administered to 6-week-old male nude mice (CAnN.Cg-Foxn1nu / CrljOri). For cell line transplantation, after confirming the viability of the cultured cell line using a microscope, a cell suspension of 2.0×106 cells / 0.1 mL was mixed with an equal volume of Matrigel (1:1, v / v). Then, the back of the animal was disinfected with 70% alcohol, and the skin on the nape of the neck was pulled with the thumb and index finger to create a space between the skin and muscle. After that, an injection needle with a 26-gauge needle was inserted into the subcutaneous space between the thumb and index finger from the front of the animal, and the cells were gradually administered subcutaneously at a dose of 2.0×106 cells / 0.2 mL / head. When the tumor size at the site where the cell line was transplanted reached approximately 100 - 150 mm3, the animals were assigned so that the tumor sizes of each group were distributed as uniformly as possible according to the ranked tumor sizes. BxC-I17e was administered into the tumors using a syringe with a 31-gauge needle according to each group. The tumor sizes were observed three times a week for two weeks starting from the initiation of the test substance administration (Day 0) and are shown in Figure 9 and Table 9.

[0170]

Table 9

[0171] As a result of the experiment, as can be confirmed from Figure 9 and Table 9, in the case of BxC-I17e exosomes, unlike the control group, the growth of tumors induced from the hepatocellular carcinoma cell line could be effectively suppressed even as time passed.

Industrial Applicability

[0172] The present invention relates to a composition containing stem cell-derived exosomes and a method for producing the same, and more particularly, to a composition containing exosomes isolated from mesenchymal stem cells or their cultures, which has excellent anti-cancer effects.

Claims

1. A pharmaceutical composition for treating, preventing, alleviating or suppressing cancer, comprising exosomes isolated from mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) as an active ingredient.

2. The pharmaceutical composition for treating, preventing, alleviating or suppressing cancer according to claim 1, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are differentiated from precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells that do not express the SSEA-4 (stage-specific embryonic antigen 4) protein.

3. The pharmaceutical composition for treating, preventing, alleviating or suppressing cancer according to claim 1, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

4. The pharmaceutical composition for treating, preventing, alleviating or suppressing cancer according to claim 1, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are pretreated with a pretreatment substance.

5. The pharmaceutical composition for treating, preventing, alleviating or suppressing cancer according to claim 4, wherein the pretreatment substance is interferon-gamma (IFN-γ).

6. The cancer according to claim 1 is one or more selected from the group consisting of breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, glioblastoma, or myeloid cancer. The pharmaceutical composition for treating, preventing, alleviating or suppressing cancer according to claim 1.

7. A food composition for alleviating, suppressing or improving cancer, comprising exosomes isolated from mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) as an active ingredient.

8. The food composition for alleviating, suppressing or improving cancer according to claim 7, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are differentiated from precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells that do not express the SSEA-4 (stage-specific embryonic antigen 4) protein.

9. The food composition for alleviating, suppressing or improving cancer according to claim 7, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

10. The food composition for alleviating, suppressing or improving cancer according to claim 7, wherein the induced pluripotent stem cell-derived mesenchymal stem cells are pretreated with a pretreatment substance.

11. The food composition for alleviating, suppressing or improving cancer according to claim 10, wherein the pretreatment substance is interferon-gamma (IFN-γ).

12. The cancer is one or more selected from the group consisting of breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eyeball, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, glioblastoma, or myeloid cancer. The food composition for alleviating, suppressing or improving cancer according to claim 7.

13. The following steps: A first culture step of culturing induced pluripotent stem cells in a medium; A selection culture step of separating and culturing SSEA-4(-) cells among the cultured induced pluripotent stem cells and differentiating them into BxC stem cells; A second culture step of culturing BxC stem cells and differentiating them into mesenchymal stem cells; A pretreatment step of pretreating mesenchymal stem cells with interferon-gamma (IFN-γ); A production step of culturing the pretreated mesenchymal stem cells to produce exosomes; and A separation step of separating exosomes from mesenchymal stem cells or their culture, A method for manufacturing a pharmaceutical composition for treating cancer, comprising the steps.

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