Media, methods, cells and secreted factors for stem cell culture and therapy

Abstract

To provide novel culture medium compositions, stem-cell culture methods and therapy methods for the purpose of inducing, activating or priming naive multipotent stem cells, which are mesenchymal stem cells in some embodiments, into discrete, multipotent, type 1 or type 2 cell phenotypes that selectively promote or suppress inflammation and immunity.SOLUTION: Described herein are methods and culture media useful for inducing polarization in multipotent stem cells. Additionally described herein are multipotent cells produced by the methods and culture media of this disclosure that are useful therapeutic agents. Also described herein are extracellular vesicles and factors secreted by multipotent cells produced by the methods and culture media of this disclosure that are useful therapeutic agents.SELECTED DRAWING: None

Description

Technical Field

[0001] Pluripotent stem cells and mesenchymal stem cells can be induced from a drug-free or unstimulated state into an inflammatory (type 1) or anti-inflammatory (type 2) phenotype by selective stimulation of Toll-like receptors.

Summary of the Invention

Problems to be Solved by the Invention

[0002] The present disclosure is directed to drug-free pluripotent stem cells and, in some embodiments, mesenchymal stem cells, for the purpose of inducing, activating, or priming individual pluripotent type 1 or type 2 cell phenotypes that selectively promote or suppress inflammation and immunity. Accordingly, the present disclosure provides useful treatments where modulation of the inflammatory response of the immune system is desirable (e.g., cancer, optic neuritis). Compared to conventional methods, in the present invention, cells may be introduced into a homogeneous type 2 population that more efficiently migrates to the lesion site and consistently expresses anti-inflammatory cytokines at a higher level, thus expected to have higher efficacy and effectiveness of the cells. Compared to conventional methods, in the present invention, cells may be induced into a homogeneous type 1 population with unique pro-inflammatory properties, consistently expressing certain cytokines leading to tumor necrosis at a higher level and having the ability to efficiently migrate into the intratumoral microenvironment, thus expected to have higher safety, efficacy, and effectiveness of the cells.

Means for Solving the Problems

[0003] The present disclosure provides novel stem cell culture and treatment methods and culture medium compositions for introducing, polarizing, activating, or priming individual uniform cell phenotypes for selectively promoting or suppressing inflammation and immunity, providing a significant effect superior to known culture media and methods used in cell-based therapy. The methods and medium compositions are usable for cell-based therapy and are more uniform and predictable and can be used to provide a population of pluripotent stem cells that are grown in vitro, induced, polarized, activated, or primed. The effects of various embodiments of the present disclosure are that, upon introduction into a patient, they can be used to induce, polarize, activate, or prime the culture of pluripotent stem cells into a uniform and individual phenotype that behaves predictably. In some embodiments, the pluripotent stem cells are mesenchymal stem cells.

[0004] The present disclosure also provides by-products of novel stem cell culture, treatment methods, and culture medium compositions for the purpose of producing by-products. In certain embodiments, the by-product is a conditioned medium created by pluripotent stem cells using the novel stem cell culture method and composition of the present disclosure. In certain embodiments, the by-product is an extracellular vesicle created by pluripotent stem cells using the novel stem cell culture method and composition of the present disclosure. In some embodiments, the pluripotent stem cells are mesenchymal stem cells.

[0005] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the medium comprising a Toll-like receptor 3 (TLR3) ligand and a second inducer, the second inducer comprising a different Toll-like receptor ligand, neurotransmitter, neurotransmitter-like molecule, non-erythropoietin polypeptide, or lipid. In certain embodiments, the second inducer is selected from the list consisting of histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, α-melanocyte stimulating hormone (α-MSH), and leukotriene B4. In certain embodiments, the TLR3 ligand comprises poly(I:C), poly(A:U), or a combination thereof. In certain embodiments, the medium further comprises i) hypoxic conditions, ii) hypoxia mimetics, iii) erythropoietin, or iv) any combination of i), ii), or iii). In certain embodiments, the hypoxia mimetic comprises cobalt chloride, desferrioxamine, or a combination thereof. In certain embodiments, the medium further comprises a pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population comprises a mesenchymal stem cell population. In certain embodiments, the medium results in more than 20-fold induction of CXCL9 mRNA compared to a non-stimulated pluripotent stem cell population. In certain embodiments, the medium results in more than 2-fold induction of any one miRNA selected from the list consisting of miR-Let7a / d, miR-17_1, miR-222, miR-92a, and miR-1260a compared to a non-stimulated pluripotent stem cell population. In certain embodiments, a pluripotent stem cell population, obtained from the medium. In certain embodiments, the pluripotent cell population consists of a mesenchymal cell population. In certain embodiments, the pluripotent cell population consists essentially of a mesenchymal cell population. In certain embodiments, the pluripotent stem cell population is used in the treatment of inflammatory or autoimmune diseases. In certain embodiments, a method of treating an inflammatory or autoimmune disease, comprising administering to a subject a therapeutically effective amount of an immunologically polarized pluripotent stem cell population obtained from the medium described in this paragraph.In one embodiment, a method of creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising contacting the non-stimulated pluripotent stem cell population with the medium described in this paragraph. In one embodiment, the pluripotent stem cell population comprises a mesenchymal cell population. In one embodiment, the pluripotent stem cell population consists essentially of a mesenchymal cell population. In one embodiment, the method further comprises administering to a subject, in a therapeutically effective amount, the immunologically polarized pluripotent stem cell population described in this paragraph. In one embodiment described herein, a method of creating extracellular vesicles, comprising contacting a non-stimulated pluripotent stem cell population with the medium described in this paragraph. In one embodiment, the pluripotent stem cell population comprises a mesenchymal stem cell population. In one embodiment, the pluripotent stem cell population consists essentially of a mesenchymal stem cell population. In one embodiment, the extracellular vesicles are separated and / or purified from the medium.

[0006] In one embodiment, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the medium comprising a first inducer that is a Toll-like receptor 4 (TLR4) ligand and a second inducer, wherein the second inducer is a Toll-like receptor ligand, neurotransmitter, neurotransmitter-like molecule, non-erythropoietin polypeptide, or lipid different from the first inducer. In one embodiment, the second inducer is selected from the list consisting of histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, alpha-melanocyte stimulating hormone (α-MSH), and leukotriene B4. In one embodiment, the TLR4 ligand comprises lipopolysaccharide, aminoalkylglucosaminide 4-phosphate, or a combination thereof. In one embodiment, the medium further comprises i) hypoxic conditions, ii) hypoxia mimetic, iii) erythropoietin, or iv) any combination of i), ii), or iii). In one embodiment, the hypoxia mimetic comprises cobalt chloride, desferrioxamine, or a combination thereof. In one embodiment, the medium further comprises a pluripotent stem cell population. In one embodiment, the pluripotent stem cell population comprises a mesenchymal stem cell population. In one embodiment, the medium results in an induction of more than 20-fold of TNFSF10 mRNA compared to the non-stimulated pluripotent stem cell population. In one embodiment, the medium results in an induction of more than 2-fold of any one miRNA selected from miR-146, miR-155, miR-1305, miR-575, and miR-1973 compared to the non-stimulated mesenchymal stem cell population. In one embodiment herein, a pluripotent stem cell population is taken from the medium disclosed in this paragraph. In one embodiment, the pluripotent stem cell population comprises a mesenchymal stem cell population. In one embodiment, the pluripotent stem cell population consists essentially of a mesenchymal cell population. In one embodiment, the pluripotent stem cell population is used for the treatment of tumors or cancers. In one embodiment herein, a method for treating a tumor or cancer, comprising administering to a subject, in a therapeutically effective amount, a pluripotent stem cell population taken from the medium disclosed in this paragraph. In one embodiment, the pluripotent stem cell population comprises a mesenchymal cell population.In certain embodiments, the pluripotent stem cell population consists essentially of a mesenchymal cell population. In certain embodiments herein, a method of creating an immunologically polarized pluripotent stem cell population from an unstimulated mesenchymal stem cell population is described, the method comprising contacting the unstimulated pluripotent stem cell population with the medium disclosed in this paragraph. In certain embodiments, the pluripotent stem cell population comprises a mesenchymal stem cell population. In certain embodiments, the pluripotent stem cell population consists essentially of a mesenchymal stem cell population. In certain embodiments, the method further comprises administering to a subject an immunologically polarized pluripotent stem cell population in a therapeutically effective amount. In certain embodiments herein, a method of creating extracellular vesicles is described, the method comprising contacting a pluripotent stem cell population with the medium disclosed in this paragraph. In certain embodiments, the pluripotent stem cell population comprises a mesenchymal stem cell population. In certain embodiments, the pluripotent stem cell population consists essentially of a mesenchymal stem cell population. In certain embodiments, the extracellular vesicles are separated and / or purified from the medium.

[0007] In certain embodiments, there are provided isolated and purified extracellular vesicles that comprise at high levels glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSN), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2) protein, or any one or more of multiple portions thereof, where high levels are as compared to levels in untreated, unstimulated mesenchymal stem cells. In certain embodiments, the isolated and purified extracellular vesicles comprise at high levels any two or more of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSM), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2). In certain embodiments, the isolated and purified extracellular vesicles comprise at high levels all seven of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSN), vimentin (VIM), actin (ACTB), and annexin A2 (ANXA2). In certain embodiments, the isolated and purified extracellular vesicles further comprise a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, the isolated and purified extracellular vesicles are for use in treating a subject afflicted with a tumor or cancer. In certain embodiments, a method of treating a subject's cancer or tumor, comprising administering to the subject a therapeutically effective amount of the isolated and purified extracellular vesicles described in this paragraph.

[0008] In one embodiment, there is provided a separated and purified extracellular vesicle that contains, at a high level, myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2) protein, or any one or more of a plurality of portions thereof, where the high level is in comparison to the level in unstimulated mesenchymal stem cells without drug administration. In one embodiment, the separated and purified extracellular vesicle contains, at a high level, any two or more of myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2). In one embodiment, the separated and purified extracellular vesicle contains, at a high level, all four of myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), and tissue factor pathway inhibitor 2 (TFPI2). In one embodiment, the separated and purified extracellular vesicle further contains an inactive component selected from a pharmaceutically acceptable excipient, diluent, or carrier. In one embodiment, the separated and purified extracellular vesicle is used in the treatment of a subject suffering from a tumor or cancer. In one embodiment, there is provided a method for treating a cancer or tumor in a subject, the method including administering, in a therapeutically effective amount, the separated and purified extracellular vesicle described in this paragraph.

[0009] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the medium comprising a first and a second inducer, the first inducer being a Toll-like receptor 3 (TLR3) ligand, and the second inducer being a Toll-like receptor ligand different from the first inducer, a neurotransmitter, a neurotransmitter-like molecule, a polypeptide other than erythropoietin, or a lipid, is described herein. In certain embodiments, the second inducer is histamine. In certain embodiments, the second inducer is acetylcholine. In certain embodiments, the second inducer is glutamate. In certain embodiments, the second inducer is norepinephrine. In certain embodiments, the second inducer is epinephrine. In certain embodiments, the second inducer is serotonin. In certain embodiments, the second inducer is melatonin. In certain embodiments, the second inducer is lipoxin A4. In certain embodiments, the second inducer is α-melanocyte stimulating hormone. In certain embodiments, the second inducer is leukotriene B4. In certain embodiments, the TLR3 ligand is poly(I:C). In certain embodiments, the TLR3 ligand is poly(A:U). In certain embodiments, the medium further comprises a third inducer, the third inducer comprising hypoxic conditions, hypoxia mimics, or erythropoietin. In certain embodiments, the hypoxia mimic is cobalt chloride. In certain embodiments, the hypoxia mimic is desferrioxamine. In certain embodiments, the medium further comprises a fourth inducer, the fourth inducer being different from the third inducer and comprising hypoxic conditions, hypoxia mimics, or erythropoietin. In certain embodiments, the hypoxia mimic is cobalt chloride. In certain embodiments, the hypoxia mimic is desferrioxamine. In certain embodiments, the medium further comprises a pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is a non-stimulated pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is a stimulated pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is an induced pluripotent stem cell. In certain embodiments, the pluripotent stem cell population is an adult pluripotent stem cell population.In certain embodiments, the pluripotent stem cell population is a human pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is a mesenchymal stem cell population. In certain embodiments, the medium does not contain serum derived from humans or animals. In certain embodiments, the medium can cause more than 20-fold induction of CXCL9 mRNA in the stimulated stem cell population compared to unstimulated pluripotent stem cells. In certain embodiments, the medium can cause more than 30-fold induction of CXCL9 mRNA compared to unstimulated pluripotent stem cells. In certain embodiments, the medium can cause more than 50-fold induction of CXCL9 mRNA compared to unstimulated pluripotent stem cells. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of rheumatoid arthritis. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of diabetic neuropathy. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of Crohn's disease. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of acute lung injury. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of multiple sclerosis. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of acute optic neuritis. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of Krabbe disease.

[0010] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the medium comprising a first and a second inducer, the first inducer being a Toll-like receptor 4 (TLR4) ligand, and the second inducer being a Toll-like receptor ligand different from the first inducer, a neurotransmitter, a neurotransmitter-like molecule, a polypeptide other than erythropoietin, or a lipid. In certain embodiments, the second inducer is histamine. In certain embodiments, the second inducer is acetylcholine. In certain embodiments, the second inducer is glutamate. In certain embodiments, the second inducer is norepinephrine. In certain embodiments, the second inducer is epinephrine. In certain embodiments, the second inducer is serotonin. In certain embodiments, the second inducer is melatonin. In certain embodiments, the second inducer is lipoxin A4. In certain embodiments, the second inducer is α-melanocyte stimulating hormone. In certain embodiments, the second inducer is leukotriene B4. In certain embodiments, the TLR4 ligand is LPS. In certain embodiments, the TLR4 ligand is aminoalkyl glucosaminide-4-phosphate. In certain embodiments, the medium further comprises a third inducer, the third inducer being hypoxia conditions, hypoxia mimetic, or comprising erythropoietin. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the hypoxia mimetic is desferrioxamine. In certain embodiments, the medium further comprises a fourth inducer, the fourth inducer being different from the third inducer and being hypoxia conditions, hypoxia mimetic, or comprising erythropoietin. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the hypoxia mimetic is desferrioxamine. In certain embodiments, the medium further comprises a pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is a non-stimulated pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is a stimulated pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is an induced pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is an adult pluripotent stem cell population.In certain embodiments, the pluripotent stem cell population is a human pluripotent stem cell population. In certain embodiments, the pluripotent stem cell population is a mesenchymal stem cell population. In certain embodiments, the medium does not contain serum derived from humans or animals. In certain embodiments, the medium can produce more than 20-fold induction of TNFSF10 mRNA in the stimulated stem cell population compared to the stimulated pluripotent stem cells. In certain embodiments, the medium can produce more than 30-fold induction of TNFSF10 mRNA compared to the unstimulated pluripotent stem cells. The medium can produce more than 50-fold induction of TNFSF10 mRNA compared to the unstimulated pluripotent stem cells. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of ovarian cancer. In certain embodiments, the immunologically polarized pluripotent stem cell population described in this paragraph is used for the treatment of breast cancer.

[0011] In one embodiment, a method of creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the method comprising contacting the non-stimulated pluripotent stem cell population with a medium containing a first and a second inducer, wherein the first inducer is a Toll-like receptor 3 (TLR3) ligand and the second inducer is a neurotransmitter, a polypeptide other than erythropoietin, or a lipid. In one embodiment, the second inducer is histamine. In one embodiment, the second inducer is acetylcholine. In one embodiment, the second inducer is glutamate. In one embodiment, the second inducer is norepinephrine. In one embodiment, the second inducer is epinephrine. In one embodiment, the second inducer is serotonin. In one embodiment, the second inducer is melatonin. In one embodiment, the second inducer is lipoxin A4. In one embodiment, the second inducer is α-melanocyte stimulating hormone. In one embodiment, the second inducer is leukotriene B4. In one embodiment, the TLR3 ligand is poly(I:C). In one embodiment, the TLR3 ligand is poly(A:U). In one embodiment, the medium further contains a third inducer, the third inducer comprising hypoxic conditions, hypoxia mimics, erythropoietin, or any combination thereof. In one embodiment, the hypoxia mimic is cobalt chloride. In one embodiment, the hypoxia mimic is desferrioxamine. In one embodiment, the medium further contains a fourth inducer, the fourth inducer being different from the third inducer and comprising hypoxic conditions, hypoxia mimics, erythropoietin, or any combination thereof. In one embodiment, the hypoxia mimic is cobalt chloride. In one embodiment, the hypoxia mimic is desferrioxamine. In one embodiment, the pluripotent stem cell population is an induced pluripotent stem cell population. In one embodiment, the pluripotent stem cell population is an adult pluripotent stem cell population. In one embodiment, the pluripotent stem cell population is a human pluripotent stem cell population. In one embodiment, the pluripotent stem cell population is a mesenchymal stem cell population. In one embodiment, the medium does not contain human or animal-derived serum.In certain embodiments, the method can result in an induction of more than 20-fold of CXCL9 mRNA in the stimulated stem cell population compared to the unstimulated pluripotent stem cell population. In certain embodiments, the method can result in an induction of more than 30-fold of CXCL9 mRNA compared to the unstimulated pluripotent stem cell population. In certain embodiments, the method can result in an induction of more than 50-fold of CXCL9 mRNA in the medium compared to the unstimulated pluripotent stem cell population. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is described herein. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of rheumatoid arthritis. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of diabetic neuropathy. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of Crohn's disease. In certain embodiments, the pluripotent stem cell population created by the aforementioned method is used for the treatment of acute lung injury. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of multiple sclerosis. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of acute optic neuritis. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of Krabbe disease.

[0012] In one embodiment, a method of creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the method comprising contacting the non-stimulated pluripotent stem cell population with a medium comprising a first and a second inducer, the first inducer being a Toll-like receptor 4 (TLR4) ligand and the second inducer being a neurotransmitter, a polypeptide other than erythropoietin, or a lipid, is described herein. In one embodiment, the second inducer is histamine. In one embodiment, the second inducer is acetylcholine. In one embodiment, the second inducer is glutamate. In one embodiment, the second inducer is norepinephrine. In one embodiment, the second inducer is epinephrine. In one embodiment, the second inducer is serotonin. In one embodiment, the second inducer is melatonin. In one embodiment, the second inducer is lipoxin A4. In one embodiment, the second inducer is α-melanocyte stimulating hormone. In one embodiment, the second inducer is leukotriene B4. In one embodiment, the TLR4 ligand is LPS. In one embodiment, the TLR4 ligand is aminoalkyl glucosaminide 4-phosphate. In one embodiment, the medium further comprises a third inducer, the third inducer comprising hypoxic conditions, reoxygenation mimics, erythropoietin, or any combination thereof. In one embodiment, the reoxygenation mimic is cobalt chloride. In one embodiment, the reoxygenation mimic is desferrioxamine. In one embodiment, the medium further comprises a fourth inducer, the fourth inducer being different from the third inducer and comprising hypoxic conditions, reoxygenation mimics, erythropoietin, or any combination thereof. In one embodiment, the reoxygenation mimic is cobalt chloride. In one embodiment, the reoxygenation mimic is desferrioxamine. In one embodiment, the pluripotent stem cell population is an induced pluripotent stem cell population. In one embodiment, the pluripotent stem cell population is an adult pluripotent stem cell population. In one embodiment, the pluripotent stem cell population is a human pluripotent stem cell population. In one embodiment, the pluripotent stem cell population is a mesenchymal stem cell population.In certain embodiments, the medium does not contain serum derived from humans or animals. In certain embodiments, the method can result in an induction of more than 20-fold of TNFSF10 mRNA in the stimulated stem cell population compared to the unstimulated pluripotent stem cell population. In certain embodiments, the method can result in an induction of more than 30-fold of TNFSF10 mRNA compared to the unstimulated pluripotent stem cell population. In certain embodiments, the method can result in an induction of more than 50-fold of TNFSF10 mRNA compared to the unstimulated pluripotent stem cell population. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is described herein. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of ovarian cancer. In certain embodiments, the polarized pluripotent stem cell population created by the aforementioned method is used for the treatment of breast cancer.

[0013] In certain embodiments, factors secreted from a population of stimulated pluripotent stem cells are described herein. In certain embodiments, the factors are isolated from a medium that has been contacted with the stimulated pluripotent stem cells. In certain embodiments, the isolation method includes any one or more of centrifugation, extraction, precipitation, filtration, and freezing. In certain embodiments, the factors are pro-inflammatory. In certain embodiments, the factors are anti-inflammatory. In certain embodiments, the factors are extracellular vesicles. In certain embodiments, the factors are cytokines. In certain embodiments, the factors are chemokines. In certain embodiments, the factors are miRNAs. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with Toll-like receptor 3 (TLR3). In certain embodiments, the TLR3 ligand is poly(I:C). In certain embodiments, the TLR3 ligand is poly(A:U). In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with Toll-like receptor 4 (TLR4). In certain embodiments, the TLR4 ligand is lipopolysaccharide. In certain embodiments, the TLR4 ligand is aminoalkyl glucosaminide 4-phosphate. In certain embodiments, the inducer is hypoxia or hypoxia mimetic. In certain embodiments, the hypoxia condition is a hypoxia mimetic. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the hypoxia mimetic is desferrioxamine. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with erythropoietin. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with histamine. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with acetylcholine. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with glutamate. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with epinephrine. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with serotonin. In certain embodiments, the population of stimulated pluripotent stem cells is stimulated with lipoxin A4. In certain embodiments, the population of stimulated pluripotent cells is stimulated with α-melanocyte stimulating hormone.In certain embodiments, the stimulated pluripotent stem cell population is stimulated with leukotriene B4. In certain embodiments, the stimulated pluripotent stem cell population is stimulated without the presence of human or animal-derived serum. In certain embodiments, the stimulated pluripotent cell population is an induced pluripotent stem cell population. In certain embodiments, the stimulated pluripotent cell population is an adult pluripotent stem cell population. In certain embodiments, the stimulated pluripotent cell population is a human pluripotent stem cell population. In certain embodiments, the stimulated pluripotent stem cell population is a mesenchymal stem cell population. In certain embodiments, the stimulated pluripotent cell population is a type 1 mesenchymal stem cell population. In certain embodiments, the type 1 mesenchymal stem cell population expresses TNFSF10 mRNA at a higher level compared to the unstimulated mesenchymal stem cell population. In certain embodiments, the stimulated pluripotent cell population is a type 2 mesenchymal stem cell population. In certain embodiments, the type 2 mesenchymal stem cell population expresses CXCL9 mRNA at a higher level compared to the unstimulated mesenchymal stem cell population. In certain embodiments, the factor is used for the treatment of rheumatoid arthritis. In certain embodiments, the factor is used for the treatment of diabetic neuropathy. In certain embodiments, the factor is used for the treatment of Crohn's disease. In certain embodiments, the factor is used for the treatment of acute lung injury. In certain embodiments, the factor is used for the treatment of multiple sclerosis. In certain embodiments, the factor is used for the treatment of acute optic neuritis. In certain embodiments, the factor is used in a treatment method for the treatment of Krabbe disease.

[0014] In certain embodiments, extracellular vesicles secreted from a mesenchymal stem cell population stimulated with a medium containing a TLR4 ligand are described herein. In certain embodiments, the medium contains erythropoietin and hypoxic conditions or hypoxia mimics. In certain embodiments, the extracellular vesicles are used for the treatment of ovarian cancer. In certain embodiments, the extracellular vesicles are used for the treatment of breast cancer.

[0015] Described herein are extracellular vesicles secreted from a mesenchymal stem cell population stimulated with a medium containing a TLR3 ligand. In certain embodiments, the medium comprises erythropoietin and hypoxic conditions or hypoxia mimics. In certain embodiments, the extracellular vesicles are those used for the treatment of rheumatoid arthritis. In certain embodiments, the extracellular vesicles are those used for the treatment of diabetic neuropathy. In certain embodiments, the extracellular vesicles are those used for the treatment of Crohn's disease. In certain embodiments, the extracellular vesicles are those used for the treatment of acute lung injury. In certain embodiments, the extracellular vesicles are those used for the treatment of multiple sclerosis. In certain embodiments, the extracellular vesicles are those used for the treatment of acute optic neuritis. In certain embodiments, the extracellular vesicles are those used for the treatment of Krabbe disease.

[0016] In one embodiment, a method of creating factors secreted from a stimulated pluripotent stem cell population, the method comprising contacting a non-stimulated pluripotent stem cell population with a medium containing an inducer, culturing the medium with the non-stimulated pluripotent stem cell population, and collecting the medium, wherein culturing the medium creates a stimulated pluripotent stem cell population from the non-stimulated pluripotent stem cell population, and as a result, results in the secretion of at least one factor from the stimulated pluripotent stem cell population, is described herein. In one embodiment, the method further comprises separating the factor from the medium. In one embodiment, separating comprises any one or more of centrifugation, extraction, precipitation, filtration, and freezing. In one embodiment, the factor is pro-inflammatory. In one embodiment, the factor is anti-inflammatory. In one embodiment, the factor is an extracellular vesicle. In one embodiment, the factor is a cytokine. In one embodiment, the factor is a chemokine. In one embodiment, the factor is a miRNA. In one embodiment, the inducer is a Toll-like receptor 3 (TLR3) ligand. In one embodiment, the TLR3 ligand is poly(I:C). In one embodiment, the TLR3 ligand is poly(A:U). In one embodiment, the inducer is a Toll-like receptor 4 (TLR4) ligand. In one embodiment, the TLR4 ligand is lipopolysaccharide. In one embodiment, the TLR4 ligand is aminoalkyl glucosaminide 4-phosphate. In one embodiment, the inducer is hypoxia or hypoxia mimetic. In one embodiment, the hypoxia mimetic is cobalt chloride. In one embodiment, the hypoxia mimetic is desferrioxamine. In one embodiment, the inducer is erythropoietin. In one embodiment, the inducer is histamine. In one embodiment, the inducer is acetylcholine. In one embodiment, the inducer is glutamate. In one embodiment, the inducer is epinephrine. In one embodiment, the inducer is serotonin. In one embodiment, the inducer is lipoxin A4. In one embodiment, the inducer is melanocyte-stimulating hormone. In one embodiment, the inducer is leukotriene B4.In certain embodiments, the medium does not contain serum derived from humans or animals. In certain embodiments, the stimulated pluripotent cell population is an induced pluripotent stem cell population. In certain embodiments, the stimulated pluripotent cell population is an adult pluripotent stem cell population. In certain embodiments, the stimulated pluripotent cell population is a human pluripotent stem cell population. In certain embodiments, the stimulated pluripotent stem cells are a mesenchymal stem cell population. In certain embodiments, the stimulated pluripotent cells are a type 1 mesenchymal stem cell population. In certain embodiments, the type 1 mesenchymal stem cell population expresses TNFSF10 mRNA at a higher level than the unstimulated mesenchymal stem cell population. In certain embodiments, the stimulated pluripotent cell population is a type 2 mesenchymal stem cell population. In certain embodiments, the type 2 mesenchymal stem cell population expresses CXCL9 at a higher level than the unstimulated mesenchymal stem cell population. In certain embodiments, the factor is one used for the treatment of ovarian cancer. In certain embodiments, the factor is one used for the treatment of breast cancer. In certain embodiments, the factor is one used for the treatment of rheumatoid arthritis. In certain embodiments, the factor is one used for the treatment of diabetic neuropathy. In certain embodiments, the factor is one used for the treatment of Crohn's disease. In certain embodiments, the factor is one used for the treatment of acute lung injury. In certain embodiments, the factor is one used for the treatment of multiple sclerosis. In certain embodiments, the factor is one used for the treatment of acute optic neuritis. In certain embodiments, the factor is one used for the treatment of Krabbe disease.

[0017] In certain embodiments, a method of creating an extracellular preparation secreted from a mesenchymal stem cell population, comprising contacting an unstimulated mesenchymal stem cell population with a medium containing a TLR4 ligand, culturing the medium with the unstimulated mesenchymal stem cell population, and collecting the medium, wherein culturing the medium creates a stimulated mesenchymal stem cell population from the unstimulated mesenchymal stem cell population, resulting in the secretion of at least one extracellular vesicle from the stimulated mesenchymal stem cell population. In certain embodiments, the medium contains erythropoietin and hypoxic conditions or hypoxia mimics. In certain embodiments, the extracellular vesicles are those used for the treatment of ovarian cancer.

[0018] In one embodiment, a method of creating extracellular vesicles secreted from a mesenchymal stem cell population, the method comprising contacting a non-stimulated mesenchymal stem cell population with a medium containing a TLR3 ligand, culturing the medium with the non-stimulated mesenchymal stem cell population, and collecting the medium, wherein culturing the medium creates a stimulated mesenchymal stem cell population from the non-stimulated mesenchymal stem cell population, and as a result, at least one extracellular vesicle is secreted from the stimulated mesenchymal stem cell population, is described herein. In one embodiment, the medium contains erythropoietin and hypoxic conditions or hypoxia mimics. In one embodiment, the extracellular vesicles are those used for the treatment of rheumatoid arthritis. In one embodiment, the extracellular vesicles are those used for the treatment of diabetic neuropathy. In one embodiment, the extracellular vesicles are used for the treatment of Crohn's disease. In one embodiment, the extracellular vesicles are used in the treatment of acute lung injury. In one embodiment, the extracellular vesicles are used in the treatment of multiple sclerosis. In one embodiment, the extracellular vesicles are used in the treatment of acute optic neuritis. In one embodiment, the extracellular vesicles are used in the treatment of Krabbe disease.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5A

Figure 5B

Mode for Carrying Out the Invention

[0020] The method described in this specification is useful for inducing pluripotent stem cells to have either pro-inflammatory or anti-inflammatory properties. The combination of Toll-like receptor (TLR) binding (TLR4 for pro-inflammatory properties and TLR3 for anti-inflammatory properties) and a second agent results in a stronger phenotype than when TLR binding is used alone. Such a stronger phenotype (either pro-inflammatory or anti-inflammatory) is therapeutically useful. For example, pro-inflammatory pluripotent stem cells are useful in the fight against neoplasms (such as tumors or cancers), and anti-inflammatory pluripotent stem cells are useful in the fight against inflammatory or autoimmune disorders.

[0021] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising a first and a second inducer, wherein the first inducer is a Toll-like receptor 3 (TLR3) ligand and the second inducer is a Toll-like receptor ligand different from the first inducer, a neurotransmitter, a neurotransmitter-like molecule, a polypeptide other than erythropoietin, or a lipid, is described herein. In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising a first and a second inducer, wherein the first inducer is a Toll-like receptor 4 (TLR4) ligand and the second inducer is a Toll-like receptor ligand different from the first inducer, a neurotransmitter, a neurotransmitter-like molecule, a polypeptide other than erythropoietin, or a lipid, is described herein. In certain embodiments, a method for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising contacting the non-stimulated pluripotent stem cell population with a medium containing a first and a second inducer, wherein the first inducer is a Toll-like receptor 3 (TLR3) ligand and the second inducer is a neurotransmitter, a polypeptide other than erythropoietin, or a lipid, is described herein. In certain embodiments, a method for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising contacting the non-stimulated pluripotent stem cell population with a medium containing a first and a second inducer, wherein the first inducer is a Toll-like receptor 4 (TLR4) ligand and the second inducer is a neurotransmitter, a polypeptide other than erythropoietin, or a lipid, is described herein. In certain embodiments, factors secreted from a stimulated pluripotent stem cell population are described herein. In certain embodiments, extracellular vesicles secreted from a mesenchymal stem cell population stimulated with a medium containing a TLR4 ligand are described herein. In certain embodiments, extracellular vesicles secreted from a mesenchymal stem cell population stimulated with a medium containing a TLR3 ligand are described herein. In certain embodiments, the medium comprises erythropoietin and hypoxic conditions or hypoxia mimics.In one embodiment, a method for creating a factor secreted from a stimulated pluripotent stem cell population, the method comprising contacting a non-stimulated pluripotent stem cell population with a medium containing an inducer, culturing the medium with the non-stimulated pluripotent stem cell population, and collecting the medium, wherein culturing the medium creates a stimulated pluripotent stem cell population from the non-stimulated pluripotent stem cell population, and as a result, a method for producing the secretion of at least one factor from the stimulated pluripotent stem cell population is described herein. In one embodiment, a method for creating extracellular vesicles secreted from a mesenchymal stem cell population, the method comprising contacting a non-stimulated mesenchymal stem cell population with a medium containing a TLR4 ligand, culturing the medium with the non-stimulated mesenchymal stem cell population, and collecting the medium, wherein culturing the medium creates a stimulated mesenchymal stem cell population from the non-stimulated mesenchymal stem cell population, and as a result, a method for producing the secretion of at least one extracellular vesicle from the stimulated mesenchymal stem cell population is described herein. In one embodiment, a method for creating extracellular vesicles secreted from a mesenchymal stem cell population, the method comprising contacting a non-stimulated mesenchymal stem cell population with a medium containing a TLR3 ligand, culturing the medium with the non-stimulated mesenchymal stem cell population, and collecting the medium, wherein culturing the medium creates a stimulated mesenchymal stem cell population from the non-stimulated mesenchymal stem cell population, and as a result, a method for producing the secretion of at least one extracellular vesicle from the stimulated mesenchymal stem cell population is described herein. In one embodiment, the medium comprises erythropoietin and hypoxic conditions or hypoxia mimics. <Specific Definitions>

[0022] Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein in the specification and claims, the singular forms of the indefinite and definite articles also include plural references unless the context clearly dictates otherwise. Any reference to "or" in this specification is intended to include "and / or" unless otherwise stated.

[0023] As used herein, unless otherwise indicated, the term "about" refers to an amount close to the recited amount, e.g., an amount that falls within 10%, 5%, or 1% thereof.

[0024] The term "pluripotent stem cell" means a cell capable of giving rise to a number of different types of cells. The term "mesenchymal stem cell" originally means a stem cell derived from mesenchymal tissue. This term refers to a cell capable of differentiating into at least two or more of osteoblasts, chondrocytes, adipocytes, or myocytes. Mesenchymal stem cells (MSCs) are isolated from any type of adult tissue. Generally, mesenchymal stem cells are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. In a preferred embodiment, MSCs are obtained from aspirated adipose tissue obtained from bone marrow or adipose tissue. The terms "pluripotency" or "multipotency" also include pluripotent stem cells, induced pluripotent stem cells, or cells induced to a multipotent stage using any chemical or genetic means. In certain embodiments, the pluripotent or multipotent stem cells of the present disclosure are mesenchymal stem cells.

[0025] The term "biological activity" refers to one or more of transcription, translation, post-translational protein modifications such as phosphorylation or acetylation or degradation, secretion, cell division, apoptosis, or cell motility. A substance that induces biological activity will increase or decrease any of these biological activities.

[0026] The term "population" with reference to the pluripotent and mesenchymal stem cells described herein refers to a plurality of cells that are molecularly identical or highly similar. The population is isolated from human tissue or can be expanded in culture. Any population provided herein can be "consisting essentially of" the recited cells such as pluripotent stem cells or mesenchymal stem cells. As used herein, "consisting essentially of" means that the cell population is substantially pure from the perspective of cell composition. For example, the cells can be about 80%, 90%, 95%, 97%, 98%, or more than 99% pure.

[0027] The term "cell therapy" or "cell-based therapy" refers to the transplantation of human or animal cells for the prevention, treatment, or improvement of one or more symptoms associated with a disease or disorder, including, without limitation, the replacement or repair of damaged tissue or organs, the regulation of immune responses, and the reduction of inflammatory symptoms and cancer. Cell therapy can be achieved by administering pluripotent or mesenchymal stem cells harvested from an induction medium in a therapeutically effective amount.

[0028] The term "subject" refers to a mammal, including an animal, preferably a non-primate (e.g., cow, pig, horse, cat, dog, rat, or mouse) or a primate (e.g., monkey or human). In certain embodiments, the subject is a human.

[0029] The term "hypoxic condition" or "hypoxia" refers to culturing cells or a medium under conditions of oxygen deprivation, relative to the normal physiological oxygen level of about 11%.

[0030] The terms "treating", "treatment", and "treatment thereof", when used directly with reference to a patient or subject, refer to improving one or more symptoms associated with a disorder, including, without limitation, any cancer, any tumor or neoplasm, an inflammatory disorder, an autoimmune disease, or an immune-mediated disease including rejection of a transplanted organ and tissue, where the improvement is achieved as a result of administering to a subject in need of such treatment a medicament composition comprising immunomodulatory (i.e., immunologically polarized) cells produced by the present invention or immunomodulatory cells produced by the present invention.

[0031] The term "unstimulated" or "untreated" refers to a cell population that has not been subjected to treatment, polarization, or induction by the methods of the present disclosure. Fresh or frozen primary isolated mesenchymal stem cells are considered unstimulated or untreated. Cells that have already been treated with a compound or composition lacking at least one of a Toll-like receptor ligand, erythropoietin, hypoxia, or hypoxia mimetic are considered unstimulated.

[0032] The term "autoimmune disorder" refers to a disorder in a subject characterized by damage to the subject's own cells, tissues, and / or organs caused by the subject's immune response.

[0033] The term "inflammatory disorder" refers to a disorder characterized by enhanced activation of immune cells and secretion of cytokines, chemokines, or other factors that cause activation or recruitment of immune cells.

[0034] The term "extracellular vesicle" refers to cell-derived vesicles present in eukaryotic fluids including blood, lymph, urine, saliva, and conditioned media of cell cultures. These extracellular vesicles are derived from cell endosomes and the plasma membrane and can contain DNA, RNA (including miRNA), proteins, polypeptides, lipids, and small molecules. Extracellular vesicles are small vesicles derived from cells and include exosomes, microvesicles, and apoptotic bodies. In certain embodiments, the extracellular vesicles comprise exosomes, consist of exosomes, or consist essentially of exosomes.

[0035] In certain embodiments, the methods and compositions of the subject matter described herein are used for the treatment of disorders. In certain embodiments, the methods and compositions of the subject matter described herein are used for the treatment of human disorders. In certain embodiments, the methods and compositions of the subject matter described herein are used for the treatment of disorders in livestock. In certain embodiments, the methods and compositions of the subject matter described herein are used for the treatment of disorders in animals other than humans. In certain embodiments, the animals other than humans are dogs, cats, horses, cows, pigs, sheep, or goats. <Medium>

[0036] In certain embodiments, the media described herein are for use in culturing cells, stem cells, pluripotent stem cells, and mesenchymal stem cells derived from humans or other animals. In certain embodiments, the media described herein are for use in creating individual phenotypes in cells, stem cells, pluripotent stem cells, or mesenchymal stem cells derived from humans or other animals. In certain embodiments, the media described herein include standard media components for cell culture in addition to the components specifically described herein. For example, in certain non-limiting embodiments, the media include vitamins, minerals, salts, sugars, amino acids, pyruvate, hormones, human serum, fetal bovine serum, fetal calf serum, lipids, proteins, dyes, pH buffers, and combinations thereof. In certain embodiments, the media are concentrated culture supplements. In certain embodiments, the media are at least 2-fold concentrated. In certain embodiments, the media are at least 10-fold concentrated. In certain embodiments, the media are at least 100-fold concentrated. In certain embodiments, the media are dry powder formulations that are reconstituted by the addition of any suitable aqueous solvent, such as water, as a non-limiting example. In certain embodiments, the media are sterilized. In certain embodiments, the media are packaged and supplied in any suitable container. In certain embodiments, the media are sterile filtered. In certain embodiments, the media can contain formulations of standard culture media, such as MEM, DMEM, or RPMI, as non-limiting examples. In certain embodiments, the media are serum-free and derived from humans or other animals. <TLR4 ligand>

[0037] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising a first and a second inducer, the first inducer comprising a Toll-like receptor (TLR) ligand, and the second inducer comprising a molecule having biological activity, is described herein. In certain embodiments, the TLR ligand comprises a Toll-like receptor 4 (TLR4) ligand. In certain embodiments, the TLR4 ligand comprises lipopolysaccharide. In certain embodiments, the TLR4 ligand comprises aminoalkylglucosaminide 4-phosphate. In certain embodiments, the TLR4 ligand is at a concentration of from about 10 pg / mL to about 10 μg / mL, from about 100 pg / mL to about 10 μg / mL, from about 1 ng / mL to about 1 μg / mL, from about 5 ng / mL to about 1 μg / mL, from about 10 ng / mL to about 1 μg / mL, from about 100 ng / mL to about 1 μg / mL, from about 5 ng / mL to about 50 ng / mL, or from about 5 ng / mL to about 25 ng / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 10 pg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 100 pg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 1 ng / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 10 ng / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 100 ng / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 1 μg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of at least 10 μg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of less than 1 g / mL. In certain embodiments, the TLR4 ligand is present at a concentration of less than 100 mg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of less than 10 mg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of less than 1 mg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of less than 500 μg / mL. In certain embodiments, the TLR4 ligand is present at a concentration of less than 200 μg / mL.In certain embodiments, the TLR4 ligand is present at a concentration of less than 100 μg / mL. <TLR3 ligand>

[0038] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, the medium comprising a first and a second inducer, the first inducer comprising a Toll-like receptor (TLR) ligand, and the second inducer comprising a molecule having biological activity, is described herein. In certain embodiments, the TLR ligand comprises a Toll-like receptor 3 (TLR3) ligand. In certain embodiments, the TLR3 ligand comprises poly(I:C). In certain embodiments, the TLR3 ligand comprises poly(A:U). In certain embodiments, the TLR3 ligand is at a concentration of about 10 pg / mL to about 100 μg / mL, about 100 pg / mL to about 100 μg / mL, about 1 ng / mL to about 100 μg / mL, about 5 ng / mL to about 100 μg / mL, about 10 ng / mL to about 100 μg / mL, about 100 ng / mL to about 100 μg / mL, about 0.1 μg / mL to about 50 μg / mL, about 0.1 μg / mL to about 10 μg / mL, about 0.25 μg / mL to about 7.5 μg / mL, about 0.5 μg / mL to about 5 μg / mL, about 1 μg / mL to about 2.5 μg / mL, or about 1 μg / mL to about 1.5 μg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 10 pg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 100 pg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 1 ng / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 10 ng / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 100 ng / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 1 μg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of at least 10 μg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of less than 1 g / mL. In certain embodiments, the TLR3 ligand is present at a concentration of less than 100 mg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of less than 10 mg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of less than 1 mg / mL.In certain embodiments, the TLR3 ligand is present at a concentration of less than 500 μg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of less than 200 μg / mL. In certain embodiments, the TLR3 ligand is present at a concentration of less than 100 μg / mL. <Additional agent>

[0039] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising a first and a second inducer, the first inducer comprising a Toll-like receptor (TLR) ligand, and the second inducer comprising a molecule having biological activity, is described herein. In certain embodiments, referring to FIG. 1, the second inducer comprises an inhibitor or activator of the TLR signaling pathway. In certain embodiments, the TLR ligand comprises a Toll-like receptor 3 (TLR3) ligand. In certain embodiments, the TLR ligand comprises a Toll-like receptor 4 (TLR4) ligand. In certain embodiments, the second inducer comprises a molecule having biological activity. In certain embodiments, the second inducer is a Toll-like receptor ligand, neurotransmitter, death transmitter-like molecule, small molecule, polypeptide, or lipid different from the first inducer. In certain embodiments, the second inducer is selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the second inducer is histamine. In certain embodiments, the second inducer is acetylcholine. In certain embodiments, the second inducer is α-melanocyte stimulating hormone. In certain embodiments, the second inducer is glutamate. In certain embodiments, the second inducer is norepinephrine. In certain embodiments, the second inducer is epinephrine. In certain embodiments, the second inducer is serotonin. In certain embodiments, the second inducer is melatonin. In certain embodiments, the second inducer is lipoxin A4. In certain embodiments, the second inducer is lipoxin B4. In certain embodiments, the second inducer is leukotriene B4. In certain embodiments, the second inducer is not erythropoietin. In certain embodiments, the second inducer is not hypoxia or hypoxia mimetic. In certain embodiments, the second inducer is not cobalt chloride. In certain embodiments, the second inducer is not desferrioxamine.In certain embodiments, histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, alpha melanocyte stimulating hormone (α-MSH), and leukotriene B4 are all useful for increasing or enhancing MCS2 induction. In certain embodiments, histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, alpha melanocyte stimulating hormone (α-MSH), and leukotriene B4 are all useful for increasing or enhancing MSC1 induction. In certain embodiments, acetylcholine, glutamate, and lipoxin A4 are useful for increasing or enhancing MSC2 induction. In certain embodiments, serotonin, melatonin, lipoxin A4, and leukotriene B4 are useful for increasing or enhancing MSC1 induction. <Acetylcholine (AcH)>

[0040] In certain embodiments, the second inducer comprises acetylcholine. In certain embodiments, the concentration of AcH is from about 1 nanomolar to about 100 micromolar. In certain embodiments, the concentration of AcH is at least 1 nanomolar. In certain embodiments, the concentration of AcH is at least 10 nanomolar. In certain embodiments, the concentration of AcH is at least 100 nanomolar. In certain embodiments, the concentration of AcH is at least 1 micromolar. In certain embodiments, the concentration of AcH is at least 10 micromolar. In certain embodiments, the concentration of AcH is less than 100 micromolar. In certain embodiments, the concentration of AcH is less than 10 micromolar. In certain embodiments, the concentration of AcH is less than 1 micromolar. In certain embodiments, the concentration of AcH is less than 100 nanomolar. In certain embodiments, the concentration of AcH is less than 10 nanomolar. <Alpha melanocyte stimulating hormone (MSH)>

[0041] In certain embodiments, the second inducer comprises α-melanocyte stimulating hormone (MSH). In certain embodiments, the concentration of MSH is from about 100 picomoles to about 100 nanomoles. In certain embodiments, the concentration of MSH is at least 100 picomoles. In certain embodiments, the concentration of MSH is at least 1 nanomole. In certain embodiments, the concentration of MSH is at least 10 nanomoles. In certain embodiments, the concentration of MSH is less than 100 nanomoles. In certain embodiments, the concentration of MSH is less than 1 nanomole. <Melatonin (MEL)>

[0042] In certain embodiments, the second inducer comprises melatonin (MEL). In certain embodiments, the concentration of MEL is from about 1 nanomole to about 100 micromoles. In certain embodiments, the concentration of MEL is at least 1 nanomole. In certain embodiments, the concentration of MEL is at least 10 nanomoles. In certain embodiments, the concentration of MEL is at least 100 nanomoles. In certain embodiments, the concentration of MEL is at least 1 micromole. In certain embodiments, the concentration of MEL is at least 10 micromoles. In certain embodiments, the concentration of MEL is less than 100 micromoles. In certain embodiments, the concentration of MEL is less than 100 micromoles. In certain embodiments, the concentration of MEL is less than 10 micromoles. In certain embodiments, the concentration of MEL is less than 1 micromole. In certain embodiments, the concentration of MEL is less than 100 nanomoles. In certain embodiments, the concentration of MEL is less than 10 nanomoles. <Serotonin (5-HT)>

[0043] In certain embodiments, the second inducer comprises serotonin (5-HT). In certain embodiments, the concentration of 5-HT is from about 100 picomoles to about 100 micromoles. In certain embodiments, the concentration of 5-HT is at least 100 picomoles. In certain embodiments, the concentration of 5-HT is at least 1 nanomole. In certain embodiments, the concentration of 5-HT is at least 10 nanomoles. In certain embodiments, the concentration of 5-HT is at least 100 nanomoles. In certain embodiments, the concentration of 5-HT is at least 1 micromole. In certain embodiments, the concentration of 5-HT is at least 10 micromoles. In certain embodiments, the concentration of 5-HT is less than 100 micromoles. In certain embodiments, the concentration of 5-HT is less than 10 micromoles. In certain embodiments, the concentration of 5-HT is less than 1 micromole. In certain embodiments, the concentration of 5-HT is less than 100 nanomoles. In certain embodiments, the concentration of 5-HT is less than 10 nanomoles. In certain embodiments, the concentration of 5-HT is less than 1 nanomole. <Glutamate (GLU)>

[0044] In certain embodiments, the second inducer comprises glutamate (GLU). In certain embodiments, the concentration of GLU is from about 10 nanomoles to about 1 millimole. In certain embodiments, the concentration of GLU is at least 10 nanomoles. In certain embodiments, the concentration of GLU is at least 100 nanomoles. In certain embodiments, the concentration of GLU is at least 1 micromole. In certain embodiments, the concentration of GLU is at least 10 micromoles. In certain embodiments, the concentration of GLU is at least 100 micromoles. In certain embodiments, the concentration of GLU is less than 1 millimole. In certain embodiments, the concentration of GLU is less than 100 micromoles. In certain embodiments, the concentration of GLU is less than 10 micromoles. In certain embodiments, the concentration of GLU is less than 1 micromole. In certain embodiments, the concentration of GLU is less than 100 nanomoles. <Norepinephrine (NEPI)>

[0045] In certain embodiments, the second inducer comprises norepinephrine (NEPI). In certain embodiments, the concentration of NEPI is from about 100 picomoles to about 100 nanomoles. In certain embodiments, the concentration of NEPI is at least 100 picomoles. In certain embodiments, the concentration of NEPI is at least 1 nanomole. In certain embodiments, the concentration of NEPI is at least 10 nanomoles. In certain embodiments, the concentration of NEPI is less than 100 nanomoles. In certain embodiments, the concentration of NEPI is less than 10 nanomoles. In certain embodiments, the concentration of NEPI is less than 1 nanomole. <Histamine (HIS)>

[0046] In certain embodiments, the second inducer comprises histamine (HIS). In certain embodiments, the concentration of HIS is from about 10 nanomoles to about 1 millimole. In certain embodiments, the concentration of HIS is at least 10 nanomoles. In certain embodiments, the concentration of HIS is at least 100 nanomoles. In certain embodiments, the concentration of HIS is at least 1 micromole. In certain embodiments, the concentration of HIS is at least 10 micromoles. In certain embodiments, the concentration of HIS is at least 100 micromoles. In certain embodiments, the concentration of HIS is less than 1 millimole. In certain embodiments, the concentration of HIS is less than 100 micromoles. In certain embodiments, the concentration of HIS is less than 10 micromoles. In certain embodiments, the concentration of HIS is less than 1 micromole. In certain embodiments, the concentration of HIS is less than 100 nanomoles. <Lipoxin A4 (LXA4)>

[0047] In certain embodiments, the second inducer comprises lipoxin A4 (LXA4). In certain embodiments, the concentration of LXA4 is from about 100 picomoles to about 1 millimole. In certain embodiments, the concentration of LXA4 is at least 100 picomoles. In certain embodiments, the concentration of LXA4 is at least 1 nanomole. In certain embodiments, the concentration of LXA4 is at least 10 nanomoles. In certain embodiments, the concentration of LXA4 is at least 100 nanomoles. In certain embodiments, the concentration of LXA4 is at least 1 micromole. In certain embodiments, the concentration of LXA4 is at least 10 micromoles. In certain embodiments, the concentration of LXA4 is at least 100 micromoles. In certain embodiments, the concentration of LXA4 is less than 1 millimole. In certain embodiments, the concentration of LXA4 is less than 100 micromoles. In certain embodiments, the concentration of LXA4 is less than 10 micromoles. In certain embodiments, the concentration of LXA4 is less than 1 micromole. In certain embodiments, the concentration of LXA4 is less than 100 nanomoles. In certain embodiments, the concentration of LXA4 is less than 10 nanomoles. In certain embodiments, the concentration of LXA4 is less than 1 nanomole. <Leukotriene B4 (LTB4)>

[0048] In certain embodiments, the second inducer comprises leukotriene B4 (LTB4). In certain embodiments, the concentration of LTB4 is from about 100 picomoles to about 1 millimole. In certain embodiments, the concentration of LTB4 is at least 100 picomoles. In certain embodiments, the concentration of LTB4 is at least 1 nanomole. In certain embodiments, the concentration of LTB4 is at least 10 nanomoles. In certain embodiments, the concentration of LTB4 is at least 100 nanomoles. In certain embodiments, the concentration of LTB4 is at least 1 micromole. In certain embodiments, the concentration of LTB4 is at least 10 micromoles. In certain embodiments, the concentration of LTB4 is at least 100 micromoles. In certain embodiments, the concentration of LTB4 is less than 1 millimole. In certain embodiments, the concentration of LTB4 is less than 100 micromoles. In certain embodiments, the concentration of LTB4 is less than 100 micromoles. In certain embodiments, the concentration of LTB4 is less than 1 micromole. In certain embodiments, the concentration of LTB4 is less than 100 nanomoles. In certain embodiments, the concentration of LTB4 is less than 10 nanomoles. In certain embodiments, the concentration of LTB4 is less than 1 nanomole. <Hypoxia>

[0049] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising a first and a second inducer, the first inducer comprising a Toll-like receptor (TLR) ligand, and the second inducer comprising a molecule having biological activity. In certain embodiments, the molecule having biological activity comprises a different Toll-like receptor ligand, a neurotransmitter, a neurotransmitter-like molecule, a non-erythropoietin polypeptide, or a lipid. In certain embodiments, the molecule having biological activity is selected from the list consisting of histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, α-melanocyte stimulating hormone (α-MSH), and leukotriene B4. In certain embodiments, the TLR ligand comprises a Toll-like receptor 3 (TLR3) ligand. In certain embodiments, the TLR ligand comprises a Toll-like receptor 4 (TLR4) ligand. In certain embodiments, the second inducer is selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the third inducer is a reoxygenation condition or hypoxia mimetic. In certain embodiments, the third inducer comprises erythropoietin.

[0050] In certain embodiments, the media described herein include hypoxic conditions. In certain embodiments, hypoxic conditions include culturing in a hypoxic or anoxic environment. In certain embodiments, a hypoxic environment has less than 10% oxygen. In certain embodiments, a hypoxic environment has less than 8% oxygen. In certain embodiments, a hypoxic environment has less than 6% oxygen. In certain embodiments, a hypoxic environment has less than 5% oxygen. In certain embodiments, a hypoxic environment has less than 4% oxygen. In certain embodiments, a hypoxic environment has less than 3% oxygen. In certain embodiments, a hypoxic environment has less than 2.5% oxygen. In certain embodiments, a hypoxic environment has less than 2.0% oxygen. In certain embodiments, a hypoxic environment has less than 1.5% oxygen. In certain embodiments, a hypoxic environment has less than 1.0% oxygen. In certain embodiments, a hypoxic environment has less than 0.5% oxygen. In certain embodiments, a hypoxic environment has effectively 0% oxygen. In certain embodiments, a hypoxic environment has from 0.5% to 3.0% oxygen. In certain embodiments, a hypoxic environment has from 0.5% to 2.5% oxygen. In certain embodiments, a hypoxic environment has from 0.5% to 2.0% oxygen. In certain embodiments, a hypoxic environment has from 0.5% to 1.5% oxygen. In certain embodiments, a hypoxic environment has from 0.5% to 1.0% oxygen. In certain embodiments, a hypoxic environment has from 1.0% to 2.0% oxygen. In certain embodiments, a hypoxic environment has from 1.5% to 2.0% oxygen.

[0051] In certain embodiments, the media described herein includes hypoxia mimicking. In certain embodiments, the hypoxia mimicking includes cobalt chloride. In certain embodiments, cobalt chloride is present at a concentration of about 50 μM. In certain embodiments, cobalt chloride is present at a concentration of about 100 μM. In certain embodiments, cobalt chloride is present at a concentration of about 200 μM. In certain embodiments, cobalt chloride is present at a concentration of about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of about 400 μM. In certain embodiments, cobalt chloride is present at a concentration of about 500 μM. In certain embodiments, cobalt chloride is present at a concentration of about 600 μM. In certain embodiments, cobalt chloride is present at a concentration of about 700 μM. In certain embodiments, cobalt chloride is present at a concentration of about 800 μM. In certain embodiments, cobalt chloride is present at a concentration of about 900 μM. In certain embodiments, cobalt chloride is present at a concentration of about 1 mM. In certain embodiments, cobalt chloride is present at a concentration of from about 10 μM to about 1 mM. In certain embodiments, cobalt chloride is present at a concentration of from about 10 μM to about 800 μM. In certain embodiments, cobalt chloride is present at a concentration of from about 10 μM to about 500 μM. In certain embodiments, cobalt chloride is present at a concentration of from about 10 μM to about 400 μM. In certain embodiments, cobalt chloride is present at a concentration of from about 10 μM to about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of from about 50 μM to about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of from about 100 μM to about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of from about 150 μM to about 300 μM.

[0052] In certain embodiments, the media described herein include hypoxia mimics. In certain embodiments, the hypoxia mimics include deferoxamine. In certain embodiments, deferoxamine is present at a concentration of about 50 μM. In certain embodiments, deferoxamine is present at a concentration of about 200 μM. In certain embodiments, deferoxamine is present at a concentration of about 300 μM. In certain embodiments, deferoxamine is present at a concentration of about 400 μM. In certain embodiments, deferoxamine is present at a concentration of about 500 μM. In certain embodiments, deferoxamine is present at a concentration of about 600 μM. In certain embodiments, deferoxamine is present at a concentration of about 700 μM. In certain embodiments, deferoxamine is present at a concentration of about 800 μM. In certain embodiments, deferoxamine is present at a concentration of about 900 μM. In certain embodiments, deferoxamine is present at a concentration of about 1 mM. In certain embodiments, deferoxamine is present at a concentration of from about 10 μM to 1 mM. In certain embodiments, deferoxamine is present at a concentration of from about 10 μM to 800 μM. In certain embodiments, deferoxamine is present at a concentration of from about 10 μM to 500 μM. In certain embodiments, deferoxamine is present at a concentration of from about 10 μM to 400 μM. In certain embodiments, deferoxamine is present at a concentration of from about 10 μM to 300 μM. In certain embodiments, deferoxamine is present at a concentration of from about 50 μM to 300 μM. In certain embodiments, deferoxamine is present at a concentration of from about 100 μM to 300 μM. In certain embodiments, deferoxamine is present at a concentration of from about 150 μM to 300 μM.

[0053] In certain embodiments, the medium described herein contains erythropoietin. In certain embodiments, the medium of the present invention contains recombinant erythropoietin. In certain embodiments, the medium of the present invention contains human recombinant erythropoietin. In certain embodiments, the amount of erythropoietin is from about 0.1 ng / mL to about 1.0 mg / mL. In certain embodiments, the amount of erythropoietin is from about 0.1 ng / mL to about 100 ng / mL. In certain embodiments, the amount of erythropoietin is from about 0.1 ng / mL to about 50 ng / mL. In certain embodiments, the amount of erythropoietin is from about 0.1 ng / mL to about 10 ng / mL. In certain embodiments, the amount of erythropoietin is from about 0.1 ng / mL to about 1.0 ng / mL. In certain embodiments, the amount of erythropoietin is from about 0.2 ng / mL to about 0.8 ng / mL. In certain embodiments, the amount of erythropoietin is from about 0.3 ng / mL to about 0.6 ng / mL. In certain embodiments, the amount of erythropoietin is less than 10 mg / mL. In certain embodiments, the amount of erythropoietin is less than 5 mg / mL. In certain embodiments, the amount of erythropoietin is less than 1 mg / mL. In certain embodiments, the amount of erythropoietin is less than 100 ng / mL. In certain embodiments, the amount of erythropoietin is less than 30 ng / mL. In certain embodiments, the amount of erythropoietin is less than 10 ng / mL. In certain embodiments, the amount of erythropoietin is less than 5 ng / mL. In certain embodiments, the amount of erythropoietin is less than 4 ng / mL. In certain embodiments, the amount of erythropoietin is less than 1 ng / mL. In certain embodiments, the amount of erythropoietin is less than 0.8 ng / mL. In certain embodiments, the amount of erythropoietin is less than 1 ng / mL. In certain embodiments, the amount of erythropoietin is less than 5 U / mL. In certain embodiments, the amount of erythropoietin is less than 1 U / mL. In certain embodiments, the amount of erythropoietin is less than 0.5 U / mL. In certain embodiments, the amount of erythropoietin is less than 0.1 U / mL. In certain embodiments, the amount of erythropoietin is less than 0.05 U / mL.

[0054] In certain embodiments, a medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising a first and a second inducer, the first inducer comprising a Toll-like receptor (TLR) ligand, the second inducer comprising a molecule having biological activity, is described herein. In certain embodiments, the TLR ligand comprises a Toll-like receptor 3 (TLR3) ligand. In certain embodiments, the TLR ligand comprises a Toll-like receptor 4 (TLR4). In certain embodiments, the second inducer is selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the medium comprises a third inducer. In certain embodiments, the third inducer comprises hypoxic conditions or hypoxia mimics. In certain embodiments, the third inducer comprises erythropoietin. In certain embodiments, the medium comprises a fourth inducer. In certain embodiments, the fourth inducer comprises erythropoietin. In certain embodiments, the fourth inducer comprises hypoxic conditions or hypoxia mimics. <Pluripotent stem cells>

[0055] In certain embodiments, methods and culture media for use with pluripotent stem cells are described herein. In certain embodiments, methods and culture media for use with a population of pluripotent stem cells are described herein. In certain embodiments, the medium comprises one or more pluripotent stem cells. In certain embodiments, the medium comprises one or more drug-free pluripotent stem cells. In certain embodiments, the medium comprises one or more unstimulated pluripotent stem cells. In certain embodiments, the medium comprises one or more stimulated pluripotent stem cells. In certain embodiments, the pluripotent stem cells are of human origin. In certain embodiments, the pluripotent stem cells are primary human cells. In certain embodiments, the pluripotent stem cells are isolated from blood. In certain embodiments, the pluripotent stem cells are isolated from cord blood. In certain embodiments, the pluripotent stem cells are isolated from the placenta. In certain embodiments, the pluripotent stem cells are isolated from dental pulp. In certain embodiments, the pluripotent stem cells are isolated from menstrual blood. In certain embodiments, the pluripotent stem cells are isolated from a fetus. In certain embodiments, the pluripotent stem cells are isolated from aspirated adipose tissue. In certain embodiments, the pluripotent stem cells are isolated from a biopsy. <Mesenchymal stem cells>

[0056] In certain embodiments, methods and culture media for use with mesenchymal stem cells are described herein. In certain embodiments, methods and culture media for use with mesenchymal stem cell populations are described herein. In certain embodiments, the medium comprises one or more mesenchymal stem cells. In certain embodiments, the medium comprises one or more unprimed mesenchymal stem cells. In certain embodiments, the medium comprises one or more unstimulated mesenchymal stem cells. In certain embodiments, the medium comprises one or more stimulated mesenchymal stem cells. In certain embodiments, the mesenchymal stem cells are of human origin. In certain embodiments, the mesenchymal stem cells are primary human cells. In certain embodiments, the mesenchymal stem cells are isolated from blood. In certain embodiments, the mesenchymal stem cells are isolated from umbilical cord blood. In certain embodiments, the mesenchymal stem cells are isolated from the placenta. In certain embodiments, the mesenchymal stem cells are isolated from dental pulp. In certain embodiments, the mesenchymal stem cells are isolated from menstrual blood. In certain embodiments, the mesenchymal stem cells are isolated from a fetus. In certain embodiments, the mesenchymal stem cells are isolated from liposuction tissue. In certain embodiments, the mesenchymal stem cells are isolated from a biopsy. <Type 1 polarized pluripotent stem cells>

[0057] In certain embodiments, methods for inducing immunological polarization in pluripotent or mesenchymal stem cells are described herein. In certain embodiments, media for inducing immunological polarization in pluripotent stem cells are described herein. In certain embodiments, polarized pluripotent stem cells are described herein. In certain embodiments, the cells exhibit type 1 polarization. Type 1 polarization is characterized by the expression or release of pro-inflammatory mediators. Cells treated with a TLR4 ligand will exhibit type 1 polarization. Type 1 polarization is characterized by the expression of TNFSF10 (TRAIL). In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 2-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 5-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 10-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 50-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 100-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 200-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 500-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 1000-fold that of unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce the expression of TNFSF10 at 10000-fold that of unstimulated cells.

[0058] In certain embodiments, type 1 polarized pluripotency or mesenchymal stem cells are described herein. In certain embodiments, type 1 polarized pluripotent stem cells express TNFSF10. In certain embodiments, type 1 polarized pluripotent stem cells express more than 2-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 5-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 10-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 50-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 100-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 500-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 1000-fold TNFSF10 compared to non-stimulated cells. In certain embodiments, type 1 polarized pluripotent stem cells express more than 10000-fold TNFSF10 compared to non-stimulated cells.

[0059] In certain embodiments, type 1 polarized pluripotency or mesenchymal stem cells are described herein. In certain embodiments, type 1 polarized pluripotent stem cells express certain miRNAs at high levels compared to non-stimulated pluripotent stem cells. In certain embodiments, the supplemented miRNAs include any one or more of miR-146, miR-155, miR-1305, miR-575, and miR-1973. In certain embodiments, the supplemented miRNAs include any one, two, three, four, or all five of miR-146, miR-155, miR-1305, miR-575, and miR-1973. The level of any one of these miRNAs can be supplemented by at least about 2-fold, 3-fold, 4-fold, or 5-fold or more compared to a non-stimulated pluripotent stem cell population. In certain embodiments, type 1 polarized pluripotent stem cells express certain miRNAs at reduced levels compared to non-stimulated pluripotent stem cells. In certain embodiments, the reduced miRNA includes miRNA-24-3p. The level of this miRNA can be reduced by at least about 2-fold, 3-fold, 4-fold, or 5-fold or more compared to a non-stimulated pluripotent cell population. <Type 2 polarized pluripotent stem cells>

[0060] In certain embodiments, methods are described herein for inducing immunological type 2 polarization in pluripotent or mesenchymal stem cells. In certain embodiments, media for inducing type 2 polarization in pluripotent stem cells are described herein. Type 2 polarization is characterized by the expression or release of anti-inflammatory mediators. Cells treated with a TLR3 ligand will exhibit type 2 polarization. Type 2 polarization is characterized by the expression of CXCL9. In certain embodiments, the methods and culture media of the present disclosure induce the expression of CXCL9. In certain embodiments, the methods and culture media of the present disclosure induce a two-fold increase in CXCL9 expression compared to unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce a five-fold increase in CXCL9 expression compared to unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce a ten-fold increase in CXCL9 expression compared to unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce a fifty-fold increase in CXCL9 expression compared to unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce a hundred-fold increase in CXCL9 expression compared to unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce a two-hundred-fold increase in CXCL9 expression compared to unstimulated cells. In certain embodiments, the methods and culture media of the present disclosure induce a five-hundred-fold increase in CXCL9 expression compared to unstimulated cells.

[0061] In certain embodiments, type 2 polarized pluripotency or mesenchymal stem cells are described herein. In certain embodiments, type 2 polarized pluripotent stem cells express CXCL9. In certain embodiments, type 2 polarized pluripotent stem cells express more than 2-fold CXCL9 compared to unstimulated cells. In certain embodiments, type 2 polarized pluripotent stem cells express more than 5-fold CXCL9 compared to unstimulated cells. In certain embodiments, type 2 polarized pluripotent stem cells express more than 10-fold CXCL9 compared to unstimulated cells. In certain embodiments, type 2 polarized pluripotent stem cells express more than 50-fold CXCL9 compared to unstimulated cells. In certain embodiments, type 2 polarized pluripotent stem cells express more than 100-fold CXCL9 compared to unstimulated cells. In certain embodiments, type 2 polarized pluripotent stem cells express more than 200-fold CXCL9 compared to unstimulated cells. In certain embodiments, type 2 polarized pluripotent stem cells express more than 500-fold CXCL9 compared to unstimulated cells.

[0062] In certain embodiments, type 2 polarized pluripotent or mesenchymal stem cells that express any of the following genes at least 2-fold when compared to a non-stimulated cell population are described herein. Namely, CXCL9, EGFR, IRF1, A2M, FAS, IL2RG, MMP3, GBP1, ISG15, FCGR1, NFKB1, NOS2A, USF1, YY1, JAK2, STA2, STAT4, STAT5, SOCS1, or IRF1. In certain embodiments, type 2 polarized pluripotent stem cells express any of the following genes at least 2-fold when compared to a non-stimulated cell population. Namely, EPOR, F2R, STAM, PDGFRA, PIAS2, MYC, SH2B1, or CSF2RB. In certain embodiments, type 2 polarized pluripotent stem cells express any of the following genes at least 10-fold when compared to a non-stimulated cell population. Namely, CXCL9, GBP1, ISG15, SOCS1, MMP3, JAK2, or IRF1. In certain embodiments, type 2 polarized pluripotent stem cells express any of the following genes at least 20-fold when compared to a non-stimulated cell population. Namely, CXCL9, GBP1, ISG15, or SOCS1.

[0063] In certain embodiments, type 2 polarized pluripotent or mesenchymal stem cells are described herein. In certain embodiments, type 2 polarized pluripotent stem cells express certain miRNAs at high levels that are not harmful to non-stimulated pluripotent stem cells. In certain embodiments, the supplemented miRNAs include any one or more of miR-Let7a / d, miR-17_1, miR-222, miR-92a, and miR-1260a. In certain embodiments, the supplemented miRNAs include any one, two, three, four, or all five of miR-Let7a / d, miR-17_1, miR-222, miR-92a, or miR-1260a. In certain embodiments, type 2 polarized pluripotent stem cells express certain miRNAs at reduced levels compared to non-stimulated pluripotent stem cells. In certain embodiments, the reduced miRNA includes miRNA-222. The level of this miRNA can be reduced by at least about 2-fold, 3-fold, 4-fold, or 5-fold or more compared to the non-stimulated pluripotent cell population. <Cell homogeneity>

[0064] In certain embodiments, the methods and culture media of the present disclosure can increase the homogeneity of polarized pluripotent cells after culture. In some embodiments, more than 60% of the cells express a type 1 marker. In some embodiments, more than 70% of the cells express a type 1 marker. In some embodiments, more than 80% of the cells express a type 1 marker. In some embodiments, more than 90% of the cells express a type 1 marker. In some embodiments, more than 95% of the cells express a type 1 marker. In some embodiments, more than 98% of the cells express a type 1 marker. In one embodiment, the type 1 marker is TNFSF10. The marker can be measured by any suitable method, such as immunohistochemistry, ELISA, flow cytometry, Western blot, qRT-PCR, Northern blot, or digital PCR.

[0065] In certain embodiments, the methods and culture media of the present disclosure can increase the homogeneity of polarized pluripotent cells after culture. In some embodiments, more than 60% of the cells express a type 2 marker. In some embodiments, more than 70% of the cells express a type 2 marker. In some embodiments, more than 80% of the cells express a type 2 marker. In some embodiments, more than 90% of the cells express a type 2 marker. In some embodiments, more than 95% of the cells express a type 2 marker. In some embodiments, more than 98% of the cells express a type 2 marker. In one embodiment, the type 2 marker is CXCL9. The marker can be measured by any suitable method, such as immunohistochemistry, ELISA, flow cytometry, Western blot, qRT-PCR, Northern blot, or digital PCR. <Method>

[0066] In certain embodiments, the methods described herein enable an amount of contact between a culture medium and a pluripotent stem cell. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for less than 24 hours. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for less than 8 hours. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for less than 4 hours. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for less than 2 hours. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for less than 1 hour. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for more than 1 minute. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for more than 1 hour.. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for more than 2 hours.. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for more than 4 hours.. In certain embodiments, the pluripotent cells are contacted with the disclosed medium or inducer for more than 8 hours. <Secreted inflammation-inducing factor>

[0067] In certain embodiments, the methods and culture media described herein cause unstimulated pluripotent stem cells to secrete a therapeutically effective factor. In certain embodiments, the factor is secreted by mesenchymal stem cells. In certain embodiments, the factor is pro-inflammatory. In certain embodiments, the factor is secreted in response to stimulation with a TLR4 ligand. In certain embodiments, the TLR4 ligand comprises LPS or aminoalkyl glucosaminide. In certain embodiments, the factor is secreted in response to stimulation with a TLR4 ligand, erythropoietin, and hypoxia. In certain embodiments, the factor is secreted in response to stimulation with a TLR4 ligand, erythropoietin, hypoxia, and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and combinations thereof. In certain embodiments, the secreted factor is a cytokine. In certain embodiments, the factor is secreted in response to a TLR4 ligand and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the cytokine comprises IL-3. In certain embodiments, the cytokine comprises IL-6. In certain embodiments, the cytokine comprises IL-8. In certain embodiments, the cytokine comprises IL-17. In certain embodiments, the cytokine comprises interferon β. In certain embodiments, the secreted factor comprises a chemokine. In certain embodiments, the chemokine comprises LIF. In certain embodiments, the chemokine comprises GM-CSF. In certain embodiments, the chemokine comprises MIG. In certain embodiments, the chemokine comprises MCP-1. In certain embodiments, the chemokine comprises TRIL. In certain embodiments, the secreted factor comprises an miRNA. In certain embodiments, the miRNA comprises miR-146. In certain embodiments, the miRNA comprises miR-155.In certain embodiments, the secreted factors include a combination of cytokines, chemokines, or miRNAs. In certain embodiments, the secreted factors are not separated from the stimulated cell culture medium and are present with all of the remaining conditioned medium that can be used for treating a subject or contact a cell population administered to the subject. <Secreted anti-inflammatory factors>

[0068] In certain embodiments, the methods and culture media described herein cause stimulated pluripotent stem cells to secrete a factor having a therapeutic effect. In certain embodiments, the factor is secreted by mesenchymal stem cells. In certain embodiments, the factor is anti-inflammatory. In certain embodiments, the factor is secreted in response to stimulation with a TLR3 ligand. In certain embodiments, the TLR3 ligand comprises poly(I:C) or poly(A:U). In certain embodiments, the factor is secreted in response to stimulation with a TLR3 ligand, erythropoietin, and hypoxic conditions. In certain embodiments, the factor is secreted in response to stimulation with a TLR3 ligand, erythropoietin, hypoxic conditions, and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the secreted factor is a cytokine. In certain embodiments, the factor is secreted in response to stimulation with a TLR3 ligand and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the secreted factor is a cytokine. In certain embodiments, the cytokine comprises IL-4. In certain embodiments, the cytokine is IL-10. In certain embodiments, the cytokine comprises IL-13. In certain embodiments, the cytokine comprises TGFβ. In certain embodiments, the secreted factor comprises a chemokine. In certain embodiments, the chemokine comprises CXCL9. In certain embodiments, the chemokine comprises CCL5. In certain embodiments, the chemokine comprises CXCL10. In certain embodiments, the secreted factor comprises miRNA. In certain embodiments, the miRNA comprises miR LeT7a / d. In certain embodiments, the miRNA comprises miR-17 / 92a. In certain embodiments, the secreted factor comprises a combination of cytokines, chemokines, or miRNAs. In certain embodiments, the secreted factor comprises prostaglandin E2.In certain embodiments, the secreted factor comprises indoleamine 2,3-dioxygenase. In certain embodiments, the secreted factor is not isolated from the stimulated cell culture medium, but is present with all of the remaining conditioned medium that can be used for treating a subject or contacts a cell population to be administered to a subject. <Isolation of Secreted Factor>

[0069] In certain embodiments, the secreted factors described herein are isolated from a culture medium that has contacted the disclosed stimulated pluripotent stem cell population. In certain embodiments, the secreted factors described herein are isolated from a culture medium that has contacted the disclosed stimulated mesenchymal stem cell population. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 1 hour. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 2 hours. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 4 hours. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 8 hours. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 24 hours. In certain embodiments, the secreted factor is isolated from the culture medium by any suitable method including, but not limited to, filtration, centrifugation, precipitation, chromatography, and freezing. In certain embodiments, the secreted factor is not isolated from the culture medium. <Secreted Inflammatory-Inducing Extracellular Vesicles>

[0070] Extracellular vesicles are cell-derived vesicles present in many, and perhaps all, eukaryotic fluids, including blood, urine, and the conditioned media of cell cultures. These extracellular vesicles are derived from cell endosomes and the plasma membrane and can contain DNA, RNA (including miRNAs), proteins, polypeptides, lipids, and small molecules. Extracellular vesicles are small vesicles derived from cells and include exosomes, microvesicles, and apoptotic bodies. In certain embodiments, the extracellular vesicles include exosomes. In certain embodiments, the methods and culture media described herein cause stimulated pluripotent stem cells to secrete therapeutically effective extracellular vesicles. In certain embodiments, the extracellular vesicles are secreted by mesenchymal stem cells. In certain embodiments, the extracellular vesicles are inflammatory. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR4 ligand. In certain embodiments, the TLR4 ligand includes LPS or aminoalkyl glucosamine. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR4 ligand, erythropoietin, and hypoxic conditions. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR4 ligand, erythropoietin, hypoxic conditions, and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR4 ligand and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and any combination thereof. In certain embodiments, the extracellular vesicles contain cytokines. In certain embodiments, the extracellular vesicles contain IL-3. In certain embodiments, the extracellular vesicles contain IL-6. In certain embodiments, the extracellular vesicles contain IL-8. In certain embodiments, the extracellular vesicles contain IL-17. In certain embodiments, the extracellular vesicles contain interferon β. In certain embodiments, the extracellular vesicles contain chemokines. In certain embodiments, the extracellular vesicles contain LIF.In certain embodiments, the extracellular vesicles contain GM-CSF. In certain embodiments, the extracellular vesicles contain MIG. In certain embodiments, the extracellular vesicles contain MCP-1. In certain embodiments, the chemokine contains TRAIL. In certain embodiments, the extracellular vesicles contain miRNA. In certain embodiments, the extracellular vesicles contain miR-146. In certain embodiments, the extracellular vesicles contain miR-155. In certain embodiments, the extracellular vesicles contain a combination of cytokines, chemokines, or miRNAs. In certain embodiments, the inflammation-induced extracellular vesicles contain one or more proteins that are enhanced compared to extracellular vesicles secreted from an unstimulated, untreated pluripotent cell population. In certain embodiments, the one or more proteins contain glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSN), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2) at a higher level compared to extracellular vesicles from untreated, unstimulated pluripotent stem cells. In certain embodiments, the extracellular vesicles contain any two, three, four, five, six, or all seven of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSN), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2) at a higher level compared to extracellular vesicles from untreated, unstimulated pluripotent stem cells. In certain embodiments, any of these proteins can be enhanced 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, or 20-fold or more compared to unstimulated pluripotent cells. In certain embodiments, the inflammation-induced extracellular vesicles contain one or more proteins that are reduced compared to extracellular vesicles secreted from an unstimulated, untreated pluripotent cell population.In certain embodiments, the one or more proteins are present at reduced levels, including any one or more of thrombospondin-1 (THBS1), collagen alpha-2(VI) chain (COL6A2), decorin (DCN), insulin-like growth factor binding protein 7 (IGFBP7), lysyl oxidase homolog 2 (LOXL2), biglycan (BGN), or collagen alpha-3(VI) chain (COL6A3). In certain embodiments, the extracellular vesicles are present at reduced levels, including any two, three, four, five, six, or all seven of thrombospondin-1 (THBS1), collagen alpha-2(VI) chain (COL6A2), decorin (DCN), insulin-like growth factor binding protein 7 (IGFBP7), lysyl oxidase homolog 2 (LOXL2), biglycan (BGN), or collagen alpha-3(VI) chain (COL6A3). In certain embodiments, any of these proteins can be reduced by 2-fold, 3-fold, 5-fold, or more than 10-fold compared to unstimulated pluripotent cells. <Secreted anti-inflammatory extracellular vesicles>

[0071] In certain embodiments, the methods and culture media described herein cause stimulated pluripotent stem cells to secrete therapeutically effective extracellular vesicles. In certain embodiments, the extracellular vesicles are secreted by mesenchymal stem cells. In certain embodiments, the extracellular vesicles are anti-inflammatory. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR3 ligand. In certain embodiments, the TLR3 ligand comprises poly(I:C) or poly(A:U). In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR3 ligand, erythropoietin, and hypoxic conditions. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR3 ligand, erythropoietin, hypoxic conditions, and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and combinations thereof. In certain embodiments, the extracellular vesicles comprise cytokines. In certain embodiments, the extracellular vesicles are secreted in response to stimulation with a TLR3 ligand and an additional inducer selected from acetylcholine, α-melanocyte stimulating hormone, melatonin, serotonin, glutamate, norepinephrine, histamine, lipoxin A4, leukotriene B4, and combinations thereof. In certain embodiments, the extracellular vesicles comprise cytokines. In certain embodiments, the extracellular vesicles comprise IL-4. In certain embodiments, the extracellular vesicles comprise IL-10. In certain embodiments, the extracellular vesicles comprise IL-13. In certain embodiments, the extracellular vesicles comprise TGFβ. In certain embodiments, the extracellular vesicles comprise chemokines. In certain embodiments, the extracellular vesicles comprise CXCL9. In certain embodiments, the extracellular vesicles comprise CCL5. In certain embodiments, the extracellular vesicles comprise CXCL10. In certain embodiments, the extracellular vesicles comprise miRNAs. In certain embodiments, the extracellular vesicles comprise miR Let7a / d. In certain embodiments, the extracellular vesicles comprise miR-17 / 92a. In certain embodiments, the extracellular vesicles comprise a combination of cytokines, chemokines, or miRNAs. In certain embodiments, the extracellular vesicles comprise prostaglandin E2.In certain embodiments, the extracellular vesicles contain indoleamine 2,3-dioxygenase. In certain embodiments, the anti-inflammatory extracellular vesicles contain one or more proteins that are enhanced compared to extracellular vesicles secreted from unstimulated pluripotent cell populations. In certain embodiments, the one or more proteins contain myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2) at a higher level compared to extracellular vesicles from untreated unstimulated pluripotent stem cells. In certain embodiments, the extracellular vesicles contain any two, three, or all four of myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2) at a higher level compared to extracellular vesicles from untreated unstimulated pluripotent stem cells. In certain embodiments, any of these proteins can be enhanced 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, or 20-fold compared to unstimulated pluripotent cells. In certain embodiments, the anti-inflammatory extracellular vesicles contain one or more proteins that are reduced compared to extracellular vesicles secreted from untreated unstimulated pluripotent cell populations. In certain embodiments, the one or more proteins contain collagen alpha-1(I) chain (COL6A3), sulfhydryl oxidase-1 (QSOX1), decorin-7 (DCN), collagen alpha-2(VI) chain (COL6A2), thrombospondin (THBS1), or collagen alpha-1(I) chain (COL1A1) at a reduced level compared to extracellular vesicles from untreated unstimulated pluripotent stem cells. In certain embodiments, the extracellular vesicles contain any two, three, four, five, or all six of collagen alpha-1(I) chain (COL6A3), sulfhydryl oxidase-1 (QSOX1), decorin-7 (DCN), collagen alpha-2(VI) chain (COL6A2), thrombospondin (THBS1), or collagen alpha-1(I) chain (COL1A1) at a reduced level compared to extracellular vesicles from untreated unstimulated pluripotent stem cells. In certain embodiments, any of these proteins can be reduced 2-fold, 3-fold, 5-fold, or 10-fold or more compared to unstimulated pluripotent cells. <Isolation of Extracellular Vesicles>

[0072] In certain embodiments, the extracellular vesicles described herein are isolated from a culture medium that has been contacted with a population of stimulated pluripotent stem cells of the present disclosure. In certain embodiments, the extracellular vesicles are isolated from a culture medium that has been contacted with a population of stimulated mesenchymal stem cells of the present disclosure. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 1 hour. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 2 hours. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 4 hours. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 8 hours. In certain embodiments, the culture medium has been contacted with the stimulated cell population for more than 24 hours. The extracellular vesicles can be isolated and / or purified from the culture medium. Public methods for separating extracellular vesicles from culture media are known in the art. In certain embodiments, the extracellular vesicles are separated from the culture medium by any suitable method including, but not limited to, filtration, centrifugation, ultracentrifugation, density gradient separation, precipitation, immunoaffinity separation, chromatography, and freezing. In certain embodiments, the extracellular vesicles are not separated from the culture medium. <Treatment method>

[0073] In certain embodiments, methods, culture media, cells, secreted factors, and extracellular vesicles having a therapeutic effect are described herein. For example, FIG. 2 shows a flowchart of a non-limiting treatment protocol for the methods, culture media, cells, secreted factors, and extracellular vesicles described herein. In certain embodiments, the methods, culture media, cells, secreted factors, and extracellular vesicles described herein are useful for the treatment of cancer. In certain embodiments, the methods, culture media, cells, secreted factors, and extracellular vesicles described herein are useful for the treatment of autoimmune diseases. In certain embodiments, the methods, culture media, cells, secreted factors, and extracellular vesicles described herein are useful for the treatment of anti-inflammatory diseases. In certain embodiments, pluripotent stem cells treated with a medium containing a TLR4 ligand are useful for the treatment of cancer. In certain embodiments, other quiescent stem cells treated with a medium containing a TLR3 ligand are useful for the treatment of autoimmune or inflammatory diseases.

[0074] In certain embodiments, mesenchymal stem cells treated with a medium containing a TLR4 ligand are useful for treating cancer. In certain embodiments, mesenchymal stem cells treated with a medium containing a TLR3 ligand are useful for treating autoimmune or inflammatory diseases. In certain embodiments, pluripotent stem cells treated by a method comprising contacting the pluripotent stem cells with a TLR4 ligand are useful for treating cancer. In certain embodiments, pluripotent stem cells treated by a method comprising contacting the pluripotent stem cells with a TLR3 ligand are useful for treating autoimmune or inflammatory diseases. In certain embodiments, mesenchymal stem cells treated by a method comprising contacting the mesenchymal stem cells with a TLR4 ligand are useful for treating cancer. In certain embodiments, mesenchymal stem cells treated by a method comprising contacting the mesenchymal stem cells with a TLR3 ligand are useful for treating autoimmune or inflammatory diseases.

[0075] In certain embodiments, pluripotent stem cells treated with a medium containing a TLR4 ligand are useful for treating cancer. In certain embodiments, factors or extracellular vesicles secreted from pluripotent stem cells treated with a medium containing a TLR4 ligand are useful for treating cancer. In certain embodiments, mesenchymal stem cells treated with a medium containing a TLR3 ligand are useful for treating autoimmune or inflammatory diseases. In certain embodiments, factors or extracellular vesicles secreted from mesenchymal stem cells treated with a medium containing a TLR3 ligand are useful for treating autoimmune or inflammatory diseases.

[0076] In certain embodiments, extracellular vesicles or factors secreted from pluripotent stem cells treated by a method comprising contacting the pluripotent stem cells with a TLR4 ligand are useful for treating cancer. In certain embodiments, extracellular vesicles or factors secreted from pluripotent stem cells treated by a method comprising contacting the pluripotent stem cells with a TLR3 ligand are useful for treating autoimmune or inflammatory diseases. In certain embodiments, extracellular vesicles or factors secreted from mesenchymal stem cells treated by a method comprising contacting the mesenchymal stem cells with a TLR4 ligand are useful for treating cancer. In certain embodiments, extracellular vesicles or factors secreted from mesenchymal stem cells treated by a method comprising contacting the mesenchymal stem cells with a TLR3 ligand are useful for treating autoimmune or inflammatory diseases.

[0077] The present disclosure provides a cell therapy method comprising: (a) providing a population of pluripotent stem cells; (b) contacting the stem cell population with any of the media described herein; (c) culturing the cells under an appropriate disease; and (d) administering the stimulated cells to a subject. In certain embodiments, the present disclosure provides a cell therapy method comprising: (a) providing a population of mesenchymal stem cells; (b) contacting the stem cell population with any of the media described herein; (c) culturing the cells under an appropriate disease; and (d) administering the stimulated cells to a subject. In certain embodiments, the cell population is collected from a container containing both the cells and the medium by steps including removal from the container, centrifugation, ultracentrifugation, filtration, dialysis, washing, and suspension in a suitable diluent such as sterile neutral buffered saline. <Autoimmune and Inflammatory Diseases>

[0078] In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for the treatment of autoimmune diseases. In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for the treatment of acute optic neuritis, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, bronchiolitis obliterans, cardiomyopathy, celiac disease, chronic fatigue syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, Ménière's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular autoimmune syndromes, polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis vulgaris, psoriatic arthritis, Raynaud's disease, Reiter's syndrome, sarcoidosis, scleroderma, systemic progressive sclerosis, Sjögren's syndrome, Goodpasture's syndrome, Tietz syndrome, systemic lupus erythematosus, systemic erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as herpes zoster dermatitis, vitiligo, Wegener's granulomatosis, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, autoimmune diseases of the nervous system, familial Mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, polyglandular endocrine disorders, or psoriasis vulgaris.

[0079] In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for the treatment of inflammatory disorders. In certain embodiments, the inflammatory disorder is Crohn's disease, type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, psoriasis vulgaris, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Hashimoto's thyroiditis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, ulcerative colitis, autoimmune hemolytic anemia, infertility, myasthenia gravis, multiple sclerosis, Graves' disease, thrombocytopenic purpura, Guillain-Barré syndrome, allergy, asthma, atopic diseases, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, rejection after organ transplantation. In certain embodiments, the inflammatory disorder is acute optic neuritis, diabetic neuropathy, Krabbe disease, acute lung injury, Crohn's disease celiac disease, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), inflammatory vasculitis (e.g., polyarteritis nodosa, Wegener's granulomatosis, Takayasu arteritis, temporal arteritis, lymphomatoid granulomatosis), post-traumatic angiogenesis (e.g., restenosis after formation), undifferentiated spondyloarthritis, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, or chronic inflammation resulting from chronic viral or bacterial infection. <Cancer>

[0080] In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for the treatment of cancer. In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for the treatment of tumors. In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for the augmentation of cancer treatment. In certain embodiments, the cancer is adult acute lymphoblastic leukemia, pediatric acute lymphoblastic leukemia, adult acute myeloid leukemia, pediatric acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, bladder cancer, osteosarcoma, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, central nervous system fetal tumor, astrocytoma, craniopharyngioma, ependymoblastoma, brain tumor, ependymoma, medulloblastoma medulloblastoma, medulloepithelioma, intermediate pineal parenchymal tumor, moderately differentiated, supratentorial primitive neuroectodermal tumor and pineoblastoma, brain and spinal cord tumors, breast cancer, male breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system fetal tumor, central nervous system (CNS) lymphoma, primary cervical cancer, cervical cancer, pediatric cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, fetal tumor, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, nasal neuroblastoma, tumor of the Ewing sarcoma family, extracranial cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma eye cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, extracranial cell tumor, ovarian extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, adult (primary) hepatocellular (liver) cancer, hepatocellular (liver) cancer, histiocytosis, Langerhans cell, adult Hodgkin lymphoma, pediatric Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreatic endocrine gland), Kaposi sarcoma, kidney (renal cell) cancer, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, pediatric laryngeal cancer, leukemia, adult acute lymphoblastic leukemia, leukemia, pediatric acute lymphoblastic leukemia, leukemia, adult acute myeloid leukemia, leukemia, pediatric acute myeloid leukemia, leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell,Lip and oral cavity cancer, adult (primary) liver cancer, liver cancer, lung cancer, non-small cell, lung cancer, small cell, lymphoma, AIDS-related, lymphoma, Burkitt, lymphoma, cutaneous T-cell, adult Hodgkin lymphoma, pediatric Hodgkin lymphoma, adult non-Hodgkin lymphoma, pediatric non-Hodgkin lymphoma, primary central nervous system (CNS) lymphoma, macroglobulinemia, Waldenström, bone malignant fibrous histiocytoma and osteosarcoma, medulloblastoma medulloblastoma, medulloepithelioma, melanoma, melanoma, intraocular (eye), Merkel cell carcinoma, adult malignant mesothelioma, mesothelioma, cervical squamous cell carcinoma with occult primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasms fungating polyps, myelodysplastic syndrome, myelodysplasia / myeloproliferative neoplasm, chronic myelogenous leukemia, adult myelogenous leukemia, pediatric acute myelogenous leukemia, multiple myeloma, chronic myeloproliferative disorder, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, adult non-Hodgkin lymphoma, pediatric non-Hodgkin lymphoma, non-small cell lung cancer, mouth cancer, oral cancer, lip and oropharyngeal cancer, osteosarcoma and bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell tumor pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, moderately differentiated pineal parenchymal tumor, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, breast cancer during pregnancy, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis ureter, transitional cell carcinoma, airway cancer with chromosome 15 alteration, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, salivary gland cancer, tumor of the Ewing family of sarcomas, Kaposi sarcoma, adult soft tissue, pediatric soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (non-melanoma), skin cancer, skin cancer (melanoma), Merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, adult soft tissue sarcoma, pediatric soft tissue sarcoma, squamous cell carcinoma, metastatic occult primary squamous cervical cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumor, cutaneous T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, renal pelvis ureter transitional cell carcinoma, gestational trophoblastic tumor, unknown primary site, carcinoma of the ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine cancer, endometrial, uterine sarcoma, choroidal melanoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, or Wilms tumor., <Administration Schedule>

[0081] In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for administration to a subject in need of treatment of cancer, an autoimmune disorder, or an inflammatory disorder. In certain embodiments, the methods, cells, and induction media of the present disclosure include different routes of administration. In certain embodiments, the route of administration is subcutaneous, intracranial, intramuscular, intravenous, intratumoral, intraocular, intravitreal, or intracranial. In certain embodiments, the cells are directly administrable to the tumor site or the site of inflammation.

[0082] In certain embodiments, the methods, cells, culture media, extracellular vesicles, and secreted factors of the present disclosure are for administration to a subject in need of treatment for cancer, an autoimmune disorder, or an immune-mediated inflammatory disease. In certain embodiments, the methods, cells, and induction media of the present disclosure encompass different dosing frequencies. In certain embodiments, the cells and methods of the present disclosure are administered once a day, once a week, once a month, or once a year. In certain embodiments, the cells and methods of the present disclosure are administered twice a day, twice a week, twice a month, or twice a year. In certain embodiments, the cells and methods of the present disclosure are administered three times a day, three times a week, three times a month, or three times a year. In certain embodiments, the cells and methods of the present disclosure are administered four times a day, four times a week, four times a month, or four times a year. In certain embodiments, following an initial treatment, maintenance dosing is performed once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, or twelve times a year. In certain embodiments, the maintenance dosing is continued for at least one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, or ten years or more. In certain embodiments, at least 1×106 cells are administered per dose. In certain embodiments, at least 2×106 cells are administered per dose. In certain embodiments, at least 3×106 cells are administered per dose. In certain embodiments, at least 4×106 cells are administered per dose. In certain embodiments, at least 5×106 cells are administered per dose. In certain embodiments, at least 6×106 cells are administered per dose. In certain embodiments, at least 7×106 cells are administered per dose. In certain embodiments, at least 8×106 cells are administered per dose. In certain embodiments, at least 9×106 cells are administered per dose. In certain embodiments, at least 1×107 cells are administered per dose. In certain embodiments, at least 2×107 cells are administered per dose. In certain embodiments, at least 3×107 cells are administered per dose.In certain embodiments, at least 4×10^7 cells are administered per single dose. In certain embodiments, at least 5×10^7 cells are administered per single dose. In certain embodiments, at least 6×10^7 cells are administered per single dose. In certain embodiments, at least 7×10^7 cells are administered per single dose. In certain embodiments, at least 8×10^7 cells are administered per single dose. In certain embodiments, at least 9×10^7 cells are administered per single dose. In certain embodiments, at least 1×10^8 cells are administered per single dose. In certain embodiments, at least 2×10^8 cells are administered per single dose. In certain embodiments, at least 3×10^8 cells are administered per single dose. In certain embodiments, at least 4×10^8 cells are administered per single dose. In certain embodiments, at least 5×10^8 cells are administered per single dose. In certain embodiments, at least 6×10^8 cells are administered per single dose. In certain embodiments, at least 7×10^8 cells are administered per single dose. In certain embodiments, at least 8×10^8 cells are administered per single dose. In certain embodiments, at least 9×10^8 cells are administered per single dose. In certain embodiments, at least 1×10^9 cells are administered per single dose. In certain embodiments, at least 2×10^9 cells are administered per single dose. In certain embodiments, at least 3×10^9 cells are administered per single dose. <Pharmaceutically acceptable excipients, diluents, and carriers>

[0083] The cells, media, and extracellular vesicles of the present disclosure can be combined with pharmaceutically acceptable excipients, diluents, or carriers to improve and enhance administration, stability, uniformity, bioavailability, or any combination thereof. In certain embodiments, the extracellular vesicles or cells of the present disclosure are administered by suspension in a sterile solution. In certain embodiments, the solution contains 0.9% NaCl. In certain embodiments, the solution further contains one or more of a buffering agent such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, hydroxymethylaminomethane (Tris), a surfactant such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), or poloxamer 188, a polyol / disaccharide / polysaccharide such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, or dextran 40, an antioxidant such as ascorbic acid or methionine, and a chelating agent such as EDTA or EGTA. In certain embodiments, the extracellular vesicles of the present disclosure are transported / stored by lyophilization and reconstitution prior to administration. In certain embodiments, the lyophilized extracellular vesicle formulation contains a bulking agent such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. <Example>

[0084] The following examples are intended to be illustrative and not to limit the invention described herein. <Example 1 - Enhancement of Mesenchymal Stem Cell Priming by the Combination of Toll - like Receptors and Specific Derivatives>

[0085] Referring to FIGS. 3A and 3B, type 2 mesenchymal stem cell priming by TLR3 is further enhanced by acetylcholine (ACH), glutamate (GLU), and lipoxin A4 (LaX4). Human bone marrow-derived mesenchymal stem cells are induced into type 2 mesenchymal cells using poly(I:C) and 0.5 ng / mL of human recombinant erythropoietin. All RNA is isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR is performed using SYBR Green master mix. The data are analyzed using the quantitative comparative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and showing several-fold increases over untreated controls. CXCL9 gene expression increased significantly following induction. Error bars represent + / − standard error of the mean (SEM). As shown as two independent experiments in FIGS. 3A and 3B, stimulation of mesenchymal stem cells by TLR3 alone, or with any one of ACH (about 24-fold in FIG. 3A and about 37-fold in FIG. 3B), GLU (about 317.4-fold in FIG. 3A and about 59-fold in FIG. 3B), LAX4 (about 32-fold in FIG. 3A and about 47-fold in FIG. 3B), or all three of these combinations results in increased expression of CXCL9 compared to using TLR3 (about 8.8-fold in FIG. 3A and about 22-fold in FIG. 3B), ACH (about 3.7-fold in FIG. 3A and about 20-fold in FIG. 3B), GLU (about 1.4-fold in FIG. 3A and about 16-fold in FIG. 3B), or LAX4 (about -A-fold in FIG. 3A and about 13-fold in FIG. 3B) alone.

[0086] Referring to FIGS. 4A and 4B, the priming of type 1 mesenchymal stem cells by TLR4 is further enhanced by leukotriene B4 (LTB4), serotonin (5-HT), and melatonin (MEL). Human bone marrow-derived mesenchymal stem cells are induced into type 1 mesenchymal stem cells using lipopolysaccharide (LPS) and 0.5 ng / mL of human recombinant erythropoietin. All RNA is isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR is performed using SYBR Green Master Mix. The data are analyzed using the quantitative comparative CT method, standardizing the target gene expression to the 18S rRNA housekeeping gene, and showing several-fold increases over the untreated control. The gene expression of TNFSF10 (also known as TRAIL) increased significantly following induction. Error bars represent + / - standard error of the mean (SEM). As shown as two independent experiments in FIGS. 4A and 4B, stimulating mesenchymal stem cells with TLR4 in combination with either LTB4 (about 875,457-fold in FIG. 4A and about 15,437-fold in FIG. 4B), 5-HT (about 12,323-fold in FIG. 4B), MEL (about 5,555-fold in FIG. 4B), or all three combinations (about 22,238-fold in FIG. 4B) results in more expression of TNFSF10 than when using any of these alone. TLR4 (about 425,854-fold in FIG. 4A and about 7,567-fold in FIG. 4B), LTB4 (about 49-fold in FIG. 4A and about 49-fold in FIG. 4B), 5-HT (about 333-fold in FIG. 4B), or MEL (about 489-fold in FIG. 4B). <Example 2 - Analysis of Factors Secreted by Primed Mesenchymal Stem Cells>

[0087] The secretion of a wide range of bioactive molecules (i.e., the secretome) is an important mechanism by which MSCs achieve their therapeutic effects and may have the most impact on the target diseased tissue. We include exosomes [also referred to as extracellular vesicles, EVs or multivesicular bodies, MVBs] as known contributors to such bioactive factors. Exosomes are secreted vesicles that can affect neighboring cells. We include mitogens, extracellular matrix (ECM) proteins, angiogenesis, exosomes, and inflammatory / immunomodulatory bioactive factors as molecules potentially contributed by MSCs.

[0088] The non-drug MSC, MSC1, and MSC2 secretomes / exosomes were tested by Bio-Plex bioactive factors according to the manufacturer's instructions (Human Group I&II; Bio-Rad, Hercules, CA), miRNA analysis by Sistemic Inc (UK), Phalanx Biotech group (CA), and next-generation protein sequencing by C-MS / MS System Biosciences (SBI, CA), and then assayed by Western blot analysis, ELISA, flow cytometry, and RT-qPCR.

[0089] Lesion media from non-drug MSC, MSC1, and MSC2 were collected and purified by centrifugation prior to bioactive factor analysis. MSC1 was stimulated by treating human bone marrow-derived mesenchymal stem cells with 10 ng / mL of LPS for 48 hours. MSC2 was stimulated by treating human bone marrow-derived mesenchymal stem cells with 1 μg / mL of poly(I:C) for 48 hours. All levels were normalized by the total protein concentration in the sample and further normalized by the untreated drug MSC level. Levels greater than 4-fold were considered statistically significant. The data represent more than 3 experiments and were further assayed by ELISA or Western blot analysis. The results are shown in Table 1 below.

Table 1

[0090] The results show a distinct set of non-redundant secreted factors secreted by MSC1 and MSC2. Generally, pro-inflammatory factors are secreted by MSC1 and anti-inflammatory factors are secreted by MSC2. <Analysis of the protein content of extracellular vesicles secreted by primed marine stem cells in Example 3>

[0091] Extracellular vesicles were isolated from non-treated MSCs, MSC1, and MSC2 and analyzed by mass spectrometry [MS] using liquid chromatography [LC]-MS / ExoQuick-TC kit (System Biosciences, Inc). Briefly, the conditioned media from non-treated, MSC1, and MSC2 were collected 48 hours after culturing in serum / animal-free media and purified by centrifugation. MSC1 was stimulated by treatment of human bone marrow-derived mesenchymal stem cells for 48 hours prior to the assay using 10 ng / mL of LPS and 5 ng / mL of human recombinant erythropoietin. MSC2 was stimulated by treatment of human bone marrow-derived mesenchymal stem cells for 48 hours prior to the assay using 1 μg / mL of poly(I:C) and 5 ng / mL of human recombinant erythropoietin. The extracellular vesicle pellet was lysed in 200 μL of modified RIPA buffer (2.0% SDS, 150 mM NaCl, 50 mM Tris, pH 8.5, 1X complete protease inhibitor (Roche)) at 100 °C for 15 minutes. The lysate was purified by centrifugation and the protein concentration was determined using a Qubit fluorometer (Invitrogen). 10 μg of the extracted protein was processed by SDS-PAGE using a 10% Bis Tris NuPage mini-gel (Invitrogen) in a MES buffer system. A transfer window (2 cm lane) was performed and in-gel digestion was carried out using a ProGest robot (DigiLab). Half of each digested sample was analyzed by nano LC-MS / MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded onto a trap column and eluted at 350 nL / min on a 75 μM analytical column using a 2-hour reversed-phase gradient. Both columns were packed with Luna C18 resin (Phenomenex). For MS / MS, 15 of the most abundant ions were selected. The results are shown in Table 2 below.

Table 2

[0092] MicroRNA [miRNA] was first described in nematodes approximately 20 years ago and was found to be important in cell fate determination. The existence of miRNA in humans was first described more than a decade ago. miRNA profiling studies have demonstrated that miRNAs are selectively expressed at different developmental stages in different organs. The miRNA signatures for different tissue sources of MSCs define the patterns of miRNAs involved in maintaining stem cell properties such as proliferation, self-renewal, and differentiation potential. The MSC1 and MSC2 miRNA profiles were analyzed from stimulated cells and revealed unique expression patterns compared to untreated and unmedicated MSCs. The data represent at least three independent experiments from more than three different donors. The data are presented as relative expression compared to untreated MSC = 1. For example, the enhanced expression of the inflammation-inducing miRNA constructed in MSC1 is consistent with its predicted inflammatory phenotype. MSC1 was stimulated by treating human bone marrow-derived mesenchymal stem cells with 10 ng / mL of LPS and 5 ng / mL of human recombinant erythropoietin for 4 hours prior to the assay. MSC2 was stimulated by treating human bone marrow-derived mesenchymal stem cells with 1 μg / mL of poly(I:C) and 5 ng / mL of human recombinant erythropoietin for 4 hours prior to the assay.

[0093] Figures 5A (MSC1) and B (MSC2) show the results of these experiments. For MSC1-stimulated extracellular vesicles, the levels of miR-146 (approximately 2.7-fold), miR-155 (approximately 3.3-fold), miR-1305 (approximately 2.3-fold), miR-575 (approximately 2.1-fold), and miR-1973 (approximately 2.3-fold) were all induced more than 2-fold compared to non-stimulated MSCs, and the level of miR-24-3p was reduced by approximately 2-fold. For MSC2-stimulated extracellular vesicles, the levels of miR-Let7a / d (approximately 3.4-fold), miR-17_1 (approximately 6.5-fold), miR-92a (approximately 6.5-fold), and miR-1260a (approximately 2.4-fold) were all induced more than 2-fold compared to non-stimulated MSCs, and the level of miR-222 was reduced by approximately 2-fold.

[0094] In this specification, preferred embodiments of the present invention have been illustrated and described. However, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Those skilled in the art will come up with numerous modifications, changes, and substitutions without departing from the present invention. It should be understood that various alternatives of the embodiments of the present invention described in this specification may be adopted in the implementation of the present invention.

Claims

**Claim 1** A medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising: a. a Toll-like receptor 3 (TLR3) ligand; and b. a second inducer, wherein the second inducer comprises a different Toll-like receptor ligand, neurotransmitter, neurotransmitter-like molecule, non-erythropoietin polypeptide, or lipid. **Claim 2** The medium according to claim 1, wherein the second inducer is selected from the list consisting of histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, α-melanocyte-stimulating hormone (α-MSH), and leukotriene B4. **Claim 3** The medium according to claim 1 or 2, wherein the TLR3 ligand comprises poly(I:C), poly(A:U), or a combination thereof. **Claim 4** The medium according to any one of claims 1 to 3, further comprising i) hypoxic conditions, ii) hypoxia mimics, iii) erythropoietin, or iv) any combination of i), ii), or iii). **Claim 5** The medium according to claim 4, wherein the hypoxia mimics comprise cobalt chloride, desferrioxamine, or a combination thereof. **Claim 6** The medium according to any one of claims 1 to 5, further comprising a pluripotent stem cell population. **Claim 7** The medium according to claim 6, wherein the pluripotent stem cell population comprises a mesenchymal stem cell population. **Claim 8** The medium according to any one of claims 1 to 7, which causes more than 20-fold induction of CXCL9 mRNA compared to a non-stimulated pluripotent stem cell population. **Claim 9** The medium according to any one of claims 1 to 7, which causes more than 2-fold induction of any one miRNA selected from the list consisting of miR-Let7a / d, miR-17_1, miR-222, miR-92a, and miR-1260a in a non-stimulated pluripotent stem cell population. **Claim 10** A pluripotent stem cell population collected from the medium according to any one of claims 1 to 9. **Claim 11** The pluripotent stem cell population according to claim 10, wherein the pluripotent cell population comprises a mesenchymal cell population. **Claim 12** The pluripotent stem cell population according to claim 10, wherein the pluripotent cell population consists essentially of a mesenchymal cell population. **Claim 13** The pluripotent stem cell population according to any one of claims 10 to 12, which is used in the treatment of inflammatory or autoimmune diseases. **Claim 14** A method for treating an inflammatory or autoimmune disease, comprising administering to a subject, in a therapeutically effective amount, an immunologically polarized pluripotent stem cell population harvested from the medium of any one of claims 1 to 9.

15. A method for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, comprising contacting the non-stimulated pluripotent stem cell population with the medium of any one of claims 1 to 9.

16. The method according to claim 15, wherein the pluripotent stem cell population comprises a mesenchymal cell population.

17. The method according to claim 15, wherein the pluripotent stem cell population consists essentially of a mesenchymal cell population.

18. The method according to any one of claims 15 to 17, further comprising administering to a subject, in a therapeutically effective amount, the immunologically polarized pluripotent stem cell population.

19. A method for creating extracellular vesicles, comprising contacting a non-stimulated pluripotent stem cell population with the medium of any one of claims 1 to 9.

20. The method according to claim 19, wherein the non-stimulated pluripotent stem cells comprise a mesenchymal stem cell population.

21. The method according to claim 19, wherein the non-stimulated pluripotent stem cells consist essentially of a mesenchymal stem cell population.

22. The method according to any one of claims 19 to 21, wherein the extracellular vesicles are separated and / or purified from the medium.

23. A medium for creating an immunologically polarized pluripotent stem cell population from a non-stimulated pluripotent stem cell population, a. a Toll-like receptor 4 (TLR4) ligand; and b. a second inducer, wherein the second inducer is a different Toll-like receptor ligand, neurotransmitter, neurotransmitter-like molecule, non-erythropoietin polypeptide, or lipid.

24. The medium according to claim 23, wherein the second inducer is selected from the list consisting of histamine, acetylcholine, glutamate, norepinephrine, epinephrine, serotonin, melatonin, lipoxin A4, alpha-melanocyte stimulating hormone (α-MSH), and leukotriene B4.

25. The medium according to claim 23 or 24, wherein the TLR4 ligand comprises lipopolysaccharide or aminoalkylglucosaminide 4-phosphate.

26. The medium according to any one of claims 23 to 25, further comprising (i) hypoxic conditions, (ii) hypoxia mimics, (iii) erythropoietin, or (iv) any combination of (i), (ii), or (iii). **Claim 27** The medium according to claim 26, wherein the hypoxia mimic comprises cobalt chloride, desferrioxamine, or a combination thereof. **Claim 28** The medium according to any one of claims 23 to 27, further comprising a pluripotent stem cell population. **Claim 29** The medium according to claim 28, wherein the pluripotent stem cell population comprises a mesenchymal stem cell population. **Claim 30** The medium according to any one of claims 23 to 29, which causes more than 20-fold induction of TNFRSF10 mRNA compared to an unstimulated pluripotent stem cell population. **Claim 31** The medium according to any one of claims 23 to 29, which causes more than 2-fold induction of any one miRNA selected from the list consisting of miR-146, miR-155, miR-1305, miR-575, and miR-1973 in an unstimulated pluripotent stem cell population. **Claim 32** A pluripotent or mesenchymal stem cell population, A pluripotent stem cell population obtained from the medium according to any one of claims 23 to 31. **Claim 33** The pluripotent stem cell population according to claim 32, wherein the pluripotent stem cell population comprises a mesenchymal cell population. **Claim 34** The pluripotent stem cell population according to claim 32, wherein the pluripotent stem cell population consists essentially of a mesenchymal cell population. **Claim 35** The pluripotent stem cell population according to any one of claims 32 to 34, which is used for the treatment of tumors or cancers. **Claim 36** A method for treating tumors or cancers, comprising administering to a subject, in a therapeutically effective amount, a pluripotent stem cell population obtained from the medium according to any one of claims 23 to 31. **Claim 37** The method according to claim 36, wherein the pluripotent stem cell population comprises a mesenchymal cell population. **Claim 38** The method according to claim 36, wherein the pluripotent stem cell population consists essentially of a mesenchymal cell population. **Claim 39** A method for creating an immunologically polarized pluripotent stem cell population from an unstimulated pluripotent stem cell population, comprising contacting the unstimulated pluripotent stem cell population with the medium according to any one of claims 23 to 31. **Claim 40** The method according to claim 39, wherein the pluripotent stem cell population comprises a mesenchymal cell population. **Claim 41** The method according to claim 39, wherein the pluripotent stem cell population consists essentially of a mesenchymal cell population. **Claim 42** The method according to any one of claims 39 to 41, further comprising administering the immunologically polarized pluripotent stem cell population to a subject in a therapeutically effective amount.

43. A method for producing extracellular vesicles, the method comprising contacting a pluripotent stem cell population with the medium according to any one of claims 23 to 31.

44. The method according to claim 43, wherein the pluripotent stem cells comprise a mesenchymal stem cell population.

45. The method according to claim 43, wherein the pluripotent stem cells consist essentially of a mesenchymal stem cell population.

46. The method according to any one of claims 43 to 45, wherein the extracellular vesicles are separated and / or purified from the medium.

47. A separated and purified extracellular vesicle, which contains glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSN), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2) protein, or one or more of a part thereof, at a high level, and the high level is a comparison with non-drugged and non-stimulated mesenchymal stem cells.

48. The separated and purified extracellular vesicle according to claim 47, which contains any two or more of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSM), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2) at a high level.

49. The separated and purified extracellular vesicle according to claim 47 or 48, which contains all seven of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), junction plakoglobin (JUP), desmoplakin (DSP), moesin (MSN), vimentin (VIM), actin (ACTB), or annexin A2 (ANXA2) at a high level.

50. The separated and purified extracellular vesicle according to any one of claims 47 to 49, further comprising a pharmaceutically acceptable excipient, diluent, or carrier.

51. The separated and purified extracellular vesicle according to any one of claims 47 to 50, which is used for treating a subject suffering from a tumor or cancer.

52. A method for treating a cancer or tumor of a subject, the method comprising administering the separated and purified extracellular vesicle according to any one of claims 47 to 50 in a therapeutically effective amount.

53. A separated and purified extracellular vesicle, comprising It contains myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2) protein, or any one or more of a part thereof, at a high level, The high level is a separated and purified extracellular vesicle in comparison with non-drugged and non-stimulated mesenchymal stem cells.

54. The separated and purified extracellular vesicle according to claim 53, which contains any two or more of myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2) at a high level.

55. The separated and purified extracellular vesicle according to claim 53 or 54, which contains all four of myosin-1 (MYH1), myosin-2 (MTH2), myosin-7 (MYH-7), or tissue factor pathway inhibitor 2 (TFPI2) at a high level.

56. The separated and purified extracellular vesicle according to any one of claims 53 to 55, further comprising a pharmaceutically acceptable excipient, diluent, or carrier.

57. The separated and purified extracellular vesicle according to any one of claims 53 to 56, which is used for the treatment of a subject suffering from a tumor or cancer.

58. A method for treating a cancer or tumor of a subject, the method comprising administering the separated and purified extracellular vesicle according to any one of claims 53 to 56 in a therapeutically effective amount.

Images