Induction medium and methods for stem cell culture and therapy

Abstract

To provide novel MSC stem cell culture and therapy, and culture medium compositions for inducing, activating, or priming discrete uniform cell phenotypes to selectively promote or suppress inflammation and immunity, thereby yielding polarized, activated, or induced cells for use in cell-based therapy.SOLUTION: Provided is an induction medium for creating an immunologically polarized mesenchymal stem cell population from an unstimulated mesenchymal stem cell population, the induction medium comprising: a. a Toll-like receptor 3 (TLR3) ligand, b. erythropoietin, and c. 0.5-2% oxygen or a hypoxia mimetic, wherein the immunologically polarized mesenchymal stem cell population possesses anti-inflammatory characteristics marked by expression of anti-inflammatory or immunosuppressive mediators.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] <Statement regarding federally funded research> This invention was made with U.S. government support under NIH grants 1R43AR061902-01 and 1P20RR20152-01; and Department of Defense grants OC073102 and OC110218. The U.S. government has certain rights in this invention. Summary of the Invention

[0002] The present invention provides novel stem cell culture and therapy methods and culture medium compositions for inducing, activating, or priming a distinct, homogeneous cell phenotype to selectively promote or suppress inflammation and immunity, offering significant advantages over known culture media and methods used in cell therapy. The present invention can be used to provide more homogeneous and predictable ex vivo expanded and induced, primed, or activated mesenchymal stem cell (MSC) populations that can be used for cell therapy. There is a long-felt need in the art for improved methods for providing the large numbers of homogeneous, effective stem cells needed for cell therapy. An advantage of various embodiments of the present invention is that they can be used to induce, activate, or prime mesenchymal stem cell cultures toward a homogeneous, discrete phenotype that behaves in a predictable manner after introduction into a patient.

[0003] In certain embodiments, disclosed herein is an induction medium for generating an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the induction medium comprising a Toll-like receptor 3 (TLR3) ligand, erythropoietin, and 0.5-2% oxygen or a hypoxia mimetic, wherein the immunologically polarized mesenchymal stem cell population has anti-inflammatory characteristics marked by the expression of anti-inflammatory or immunosuppressive mediators. In certain embodiments, the Toll-like receptor 3 (TLR3) ligand is poly(I:C). In certain embodiments, the Toll-like receptor 3 (TLR3) ligand is poly(I:C). ) ligand is poly(A:U). In certain embodiments, the erythropoietin is present at a concentration of less than 10 ng / mL. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the cobalt chloride is present at a concentration between 5 μM and 500 μM. In certain embodiments, the induction medium further comprises interleukin 4 (IL-4). In certain embodiments, the induction medium further comprises interleukin 13 (IL-13). In certain embodiments, the induction medium is free of human or animal-derived serum. In certain embodiments, the induction medium is a concentrated solution. In certain embodiments, disclosed herein are populations of mesenchymal stem cells treated with induction medium. In certain embodiments, disclosed herein are populations of human mesenchymal stem cells treated with induction medium. In certain embodiments, disclosed herein are populations of canine, feline, or equine mesenchymal stem cells treated with induction medium. In certain embodiments, disclosed herein are populations of mesenchymal stem cells treated with induction medium. In certain embodiments, disclosed herein are populations of mesenchymal stem cells treated with an induction medium, wherein the cells are characterized by increased expression of CXCL9 mRNA compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, disclosed herein are populations of mesenchymal stem cells treated with an induction medium, wherein the cells are characterized by increased expression of OAS1 mRNA compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, disclosed herein are populations of mesenchymal stem cells treated with an induction medium, wherein the cells are characterized by increased expression of ISG15 mRNA compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, disclosed herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated with an induction medium, wherein the disease is an inflammatory or autoimmune disease. In certain embodiments, the inflammatory or autoimmune disease is rheumatoid arthritis. In certain embodiments, the inflammatory or autoimmune disease is inflammatory bowel disease. In certain embodiments, the inflammatory or autoimmune disease is acute optic neuritis. In certain embodiments, the inflammatory or autoimmune disease is Krabbe disease.In certain embodiments, the inflammatory or autoimmune disease is diabetic retinopathy. In certain embodiments, the inflammatory or autoimmune disease is Crohn's disease. In certain embodiments, the inflammatory or autoimmune disease is acute lung injury.

[0004] In certain embodiments, disclosed herein is an induction medium for generating an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the induction medium comprising a Toll-like receptor 4 (TLR4) ligand, erythropoietin, and 0.5-2% oxygen or a hypoxia mimetic, wherein the immunologically polarized mesenchymal stem cell population has proinflammatory characteristics characterized by the expression of proinflammatory mediators. In certain embodiments, the Toll-like receptor 4 (TLR4) ligand is lipopolysaccharide (LPS). In certain embodiments, the Toll-like receptor 4 (TLR4) ligand is an aminoalkylaminomethylpropional (AMA)-1-hydroxybenzoate (AMA). In certain embodiments, the induction medium is arginine 4-phosphate. In certain embodiments, the erythropoietin is present at a concentration of less than 10 ng / mL. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the cobalt chloride is present at a concentration between 5 μM and 500 μM. In certain embodiments, the induction medium further comprises interferon. In certain embodiments, the induction medium further comprises tumor necrosis factor alpha (TNFα). In certain embodiments, the induction medium does not contain serum of human or animal origin. In certain embodiments, the induction medium is a concentrated solution. In certain embodiments, disclosed herein is a population of mesenchymal stem cells treated with an induction medium. In certain embodiments, disclosed herein is a population of human mesenchymal stem cells treated with an induction medium. In certain embodiments, disclosed herein is a population of canine, feline, or equine mesenchymal stem cells treated with an induction medium. In certain embodiments, disclosed herein is a population of mesenchymal stem cells treated with an induction medium, wherein the mesenchymal stem cells were derived from pluripotent stem cells. In certain embodiments, disclosed herein are populations of mesenchymal stem cells treated with an induction medium, wherein the cells are characterized by increased expression of TNFSF10 (TRAIL) mRNA compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, disclosed herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated with an induction medium, wherein the disease is cancer. In some embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is uveal melanoma. In certain embodiments, disclosed herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated with an induction medium, wherein the disease is a viral disease. In certain embodiments, disclosed herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated with an induction medium, wherein the disease is a bacterial infection.

[0005] In certain embodiments, disclosed herein is an induction medium for generating an immunologically polarized mesenchymal stem cell population from an unstimulated mesenchymal stem cell population, the induction medium comprising poly(I:C) at a concentration between 0.1 μg / mL and 100 μg / mL, erythropoietin at a concentration less than 10 ng / mL, and cobalt chloride at a concentration between 5 μM and 500 μM, wherein the immunologically polarized mesenchymal stem cell population has anti-inflammatory properties and is characterized by increased expression of CXCL9, OAS1, and ISG15 mRNA compared to the unstimulated mesenchymal stem cell population.

[0006] In certain embodiments, disclosed herein is an induction medium for generating an immunologically polarized mesenchymal stem cell population from an unstimulated mesenchymal stem cell population, the induction medium comprising LPS at a concentration between 0.1 ng / mL and 1 μg / mL, erythropoietin at a concentration less than 10 ng / mL, and cobalt chloride at a concentration between 5 μM and 500 μM, wherein the immunologically polarized mesenchymal stem cell population has pro-inflammatory characteristics and is characterized by increased expression of TNFSF10 (TRAIL) compared to the unstimulated mesenchymal stem cell population.

[0007] In certain embodiments, disclosed herein are methods for generating an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the method comprising contacting the unstimulated mesenchymal stem cell population with a composition comprising a Toll-like receptor 3 (TLR3) ligand, erythropoietin, and hypoxia or a hypoxia mimetic, wherein the immunologically polarized mesenchymal stem cell population has anti-inflammatory characteristics characterized by the expression of anti-inflammatory or immunosuppressive mediators. In certain embodiments, the TLR3 ligand is poly(I:C). In certain embodiments, the TLR3 ligand is poly(I:C). In certain embodiments, the composition is a soluble (A:U) solution. In certain embodiments, the erythropoietin is present at a concentration of less than 10 ng / mL. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the cobalt chloride is present at a concentration between 5 μM and 500 μM. In certain embodiments, the composition further comprises interleukin 4 (IL-4). In certain embodiments, the composition further comprises interleukin 13 (IL-13). In certain embodiments, the composition does not contain serum of human or animal origin. In certain embodiments, the composition is a concentrated solution. In certain embodiments, the population of unstimulated mesenchymal stem cells is simultaneously contacted with a Toll-like receptor 3 ligand, erythropoietin, and hypoxia or a hypoxia mimetic. In certain embodiments, the composition is contacted with the population of unstimulated mesenchymal stem cells for at least 30 minutes, but for less than 8 hours. In certain embodiments, the method further comprises monitoring expression of CXCL9 at either the RNA or protein level. In certain embodiments, the method further comprises monitoring expression of OAS1 at either the RNA or protein level. In certain embodiments, the method further comprises monitoring the expression of ISG15 at either the RNA or protein level. In embodiments, provided herein are populations of mesenchymal stem cells treated by the method. In certain embodiments, provided herein are populations of human mesenchymal stem cells treated by the method. In certain embodiments, provided herein are populations of canine, feline, or equine mesenchymal stem cells treated by the method. In certain embodiments, provided herein are populations of mesenchymal stem cells treated by the method, wherein the mesenchymal stem cells have been derived from pluripotent stem cells. In certain embodiments, provided herein are populations of mesenchymal stem cells treated by the method, wherein the cells have been derived from unstimulated mesenchymal stem cells. The cells are characterized by increased CXCL9 mRNA expression compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, provided herein is a population of mesenchymal stem cells treated by the method, wherein the cells are characterized by increased OAS1 mRNA expression compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, provided herein is a population of mesenchymal stem cells treated by the method, wherein the cells are characterized by increased ISG15 mRNA expression compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, provided herein is a composition for treating a disease comprising a population of mesenchymal stem cells treated by the method, wherein the disease is an inflammatory or autoimmune disease. In certain embodiments, the inflammatory or autoimmune disease is rheumatoid arthritis. In certain embodiments, the inflammatory or autoimmune disease is inflammatory bowel disease. In certain embodiments, the inflammatory or autoimmune disease is acute optic neuritis. In certain embodiments, the inflammatory or autoimmune disease is Krabbe disease. In certain embodiments, the inflammatory or autoimmune disease is diabetic retinopathy. In certain embodiments, the inflammatory or autoimmune disease is Crohn's disease. In certain embodiments, the inflammatory or autoimmune disease is acute lung injury.

[0008] In certain embodiments, disclosed herein is a method for generating an immunologically polarized mesenchymal stem cell population from an unstimulated mesenchymal stem cell population, the method comprising contacting the unstimulated mesenchymal stem cell population with a composition comprising a Toll-like receptor 4 (TLR4) ligand, erythropoietin, and hypoxia or a hypoxia mimetic, wherein the immunologically polarized mesenchymal stem cell population has proinflammatory characteristics characterized by the expression of proinflammatory or immunosuppressive mediators. In certain embodiments, the TLR4 ligand is lipopolysaccharide (LPS). In certain embodiments, the TLR4 ligand is aminoalkyl glucosaminide 4-phosphate. In certain embodiments, the erythropoietin is present at a concentration of 1 ng / mL. In certain embodiments, the hypoxia mimetic is cobalt chloride. In certain embodiments, the cobalt chloride is present at a concentration between 5 μM and 500 μM. In certain embodiments, the composition further comprises interferon. In certain embodiments, the composition further comprises tumor necrosis factor alpha (TNFα). In certain embodiments, the composition does not contain serum of human or animal origin. In certain embodiments, the composition is a concentrated solution. In certain embodiments, a population of unstimulated mesenchymal stem cells is simultaneously contacted with a Toll-like receptor 4 ligand, erythropoietin, and hypoxia or a hypoxia mimetic. In certain embodiments, the composition is contacted with the population of unstimulated mesenchymal stem cells for at least 30 minutes, but for less than 8 hours. In certain embodiments, the method further comprises monitoring TNFSF10 (TRAIL) expression at either the RNA or protein level. In certain embodiments, provided herein is a population of mesenchymal stem cells treated by the method. In certain embodiments, provided herein is a population of human mesenchymal stem cells treated by the method. In certain embodiments, provided herein is a population of canine, feline, or equine mesenchymal stem cells treated by the method. In certain embodiments, provided herein is a population of mesenchymal stem cells treated by the method, wherein the mesenchymal stem cells are derived from pluripotent stem cells.In certain embodiments, provided herein are populations of mesenchymal stem cells treated by the method, wherein the cells are characterized by increased expression of TNFSF10 (TRAIL) mRNA compared to a population of unstimulated mesenchymal stem cells. In certain embodiments, provided herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated by the method, wherein the disease is cancer. In some embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is uveal melanoma. In certain embodiments, provided herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated by the method, wherein the disease is a viral disease. In certain embodiments, provided herein are compositions for treating a disease comprising a population of mesenchymal stem cells treated by the method, wherein the disease is a bacterial infection.

[0009] <Usefulness of the present invention> There is a need for improved therapeutic methods and cell culture methods and media for inducing, activating, or priming homogenous populations of MSCs (stem cells, mesenchymal stem cells, bone marrow stromal cells, multipotent stromal cells, and pluripotent stem cells) derived from various adult tissues. Clinical applications of MSCs require reproducible cell culture and expansion methods that provide sufficient numbers of cells with appropriate quality and consistent therapeutic efficacy. Different culture media and methods have met with varying degrees of success. Further improvements to MSC culture media and methods are still needed to ensure increased yields of primed, activated, or induced cells for use in cell therapies that are safe and have consistent and reproducible therapeutic effects.

[0010] The therapeutic efficacy of induced, activated, or primed MSCs compared with uninduced conventional MSCs has been demonstrated in preclinical disease models. Anti-inflammatory induced MSC treatment alleviated pain and inflammation in models of painful diabetic peripheral neuropathy, rheumatoid arthritis, inflammatory bowel disease, and acute lung injury in a significantly improved manner compared with conventional MSC treatment. Furthermore, anti-inflammatory induced MSC treatment improved clinical scores, gait, and motor function in preclinical models of multiple sclerosis (EAE) and Krabbe disease. In an immunocompetent murine ovarian cancer model, immune-enhancing antitumor induced MSC-based immunotherapy led to attenuation of tumor growth and spread, whereas conventional MSC treatment promoted tumor growth and spread.

[0011] <Scientific Basis of the Invention> Stimulation of specific Toll-like receptors (TLRs) influences the immunoregulatory responses of MSCs. Toll-like receptors recognize "danger" signals, and their activation leads to profound cellular and systemic responses that recruit innate and adaptive host immune cells. Danger signals that trigger TLRs are released following most tissue lesions. Because danger signals recruit immune cells to sites of injury, the inventors reasoned that MSCs might be recruited in a similar manner. The inventors observed that MSCs express several TLRs (e.g., TLR3 and TLR4, known in the art) and that their migration, invasion, and secretion of immunoregulatory factors are significantly affected by specific TLR agonist engagement. In particular, the inventors observed diverse outcomes for MSCs following stimulation of TLR3 compared to TLR4 using a low-level, short-term TLR-priming protocol. Based on these findings, the inventors proposed a new paradigm for MSCs inspired by the monocyte literature. Specifically, MSCs can be polarized (induced, activated, or primed) by downstream TLR signaling into two homogeneously acting phenotypes classified as MSC1 and MSC2. TLR4-primed MSCs, i.e., MSC1, mostly express pro-inflammatory mediators, whereas TLR3-primed MSCs, i.e., MSC2, mostly express anti-inflammatory or immunosuppressive mediators. Furthermore, the inventors have shown that allogeneic (non-autologous) co-culture of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably suppresses T lymphocyte activity after MSC2 co-culture. We demonstrated that this resulted in suppression of T cell proliferation and permissive T lymphocyte activation in co-culture with MSC1. Induction of MSCs into the immunostimulatory MSC1 phenotype by TLR4 activation or the anti-inflammatory MSC2 phenotype by TLR3 activation would ensure homogeneous and defined cells, solving an industrial bottleneck by providing defined and predictable cells for use in cell therapy applications.

[0012] Erythropoietin, also known as EPO, is a glycoprotein hormone that regulates erythropoiesis, or the production of red blood cells. It is a cytokine or cell signaling molecule for erythropoiesis (red blood cell) precursors in the bone marrow. Human EPO has a molecular weight of 34 kDa and is also called hematopoietin or hemopoietin. EPO is produced by interstitial fibroblasts in the kidney, which are closely associated with peritubular capillaries and tubules of the renal tubular epithelium, and by perisinusoidal cells in the liver. While hepatic production predominates early in development (fetal and postnatal), the kidney is the predominant site of EPO production in adults. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain's response to neuronal injury by providing a pro-survival anti-apoptotic (programmed cell death) signal. EPO is also involved in wound healing processes. Synthetic erythropoietin has also been produced by recombinant DNA technology in cell culture. Additionally, several different pharmaceutical EPO-like agents are available with various glycosylation patterns, collectively referred to as erythropoiesis-stimulating agents (ESAs). EPO is used in the present invention as a means to prevent premature cell death and to prolong the survival of generated, primed, activated, or induced cells used in cell therapy.

[0013] A consistent oxygen supply is a key factor affecting all major aspects of cell biology, including survival, proliferation, differentiation, and migration. Typically, mammalian cells (but not stem cells) require a consistent supply of oxygen to maintain robust energy production and preserve normal cell function and survival. In contrast, mammalian stem cells appear to proliferate and survive in the hypoxic environment of bone marrow (with oxygen tensions ranging from 0.5% to 7%). Several studies have shown that a hypoxic environment is required to maintain stem cell proliferation and self-renewal capacity in bone marrow. In particular, the effect of reduced oxygen tension has been described as a general method for improving the engraftment potential of MSCs in cell therapy, even after short-term culture. A hypoxic environment is used in the present invention as a means to maintain the self-renewal and proliferation potential of generated, primed, activated, or induced cells used in cell therapy.

[0014] <Definitions and Preferred Values> For clarity of understanding, terms are defined and preferred values ​​are specified herein and, where appropriate, throughout the text.

[0015] The term "cancer" refers to a disease caused by the uncontrolled division of cells in a part of the body. Cancer includes, but is not limited to, leukemia, lymphoma, melanoma, carcinoma, sarcoma, adenoma, or other malignant tumors or neoplasms caused by genetic, environmental, or stochastic mechanisms.

[0016] The term "stem cell" refers to a cell that can give rise to multiple different types of cells. The term "mesenchymal stem cell" or "MSC" refers to a stem cell originally derived from mesenchyme. The term refers to a cell that can differentiate into at least two or more of osteoblasts, chondrocytes, adipocytes, or myocytes. MSCs can be isolated from any type of adult tissue. Typically, MSCs are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. In a preferred embodiment of the present invention, MSCs are obtained from bone marrow or adipose tissue, which are themselves obtained from adipose tissue.

[0017] The term "pluripotent" and the alternative term "pluripotent" refer to cells that can give rise to multiple cell types of different tissue lineages. The terms "pluripotent" or "pluripotent" also encompass induced pluripotent or pluripotent stem cells, or cells that have been induced to the pluripotent stage using any chemical or genetic means. In certain embodiments, the pluripotent or pluripotent stem cells of the present disclosure are mesenchymal stem cells.

[0018] The cells of the present disclosure are derived from any cell of any mammalian species, including human, primate, canine, feline, equine, bovine, caprine, ovine, and porcine. The cells are primary cells or immortalized cell lines.

[0019] The term "cellular therapy" or "cell-based therapy" refers to, but is not limited to, the replacement of damaged tissue or organs. Or refers to the transplantation of human or animal cells to prevent, treat, or ameliorate one or more symptoms associated with a disease or disorder, such as repair, modulation of the immune response, and reduction of inflammatory symptoms and cancer.

[0020] The term "subject" refers to an animal, preferably a mammal, including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, or mouse) or a primate (e.g., a monkey or human). In a preferred embodiment, the subject is a human.

[0021] The terms "treat," "treatment," and "treating," when used directly with reference to a patient or subject, refer to the amelioration of one or more symptoms associated with a disorder, including, but not limited to, any cancer, any tumor or neoplasm, an inflammatory disorder, an autoimmune disease, or an immune-mediated disease, including rejection of transplanted organs and tissues, wherein the amelioration results from the administration of immunomodulatory cells produced by the present invention, or a pharmaceutical composition comprising immunomodulatory cells produced by the present invention, to a subject in need of such treatment.

[0022] The term "unstimulated" refers to a cell population that has not been treated, polarized, or induced by the methods of the present disclosure. Fresh or frozen primary isolated mesenchymal stem cells are considered unstimulated. Cells previously treated with a compound or composition lacking at least one of a Toll-like receptor ligand, erythropoietin, hypoxia, or hypoxia mimetic are considered unstimulated.

[0023] The terms "repair" and "repairing" when used directly in reference to damaged tissue refer to the amelioration of such damage by direct mechanisms, such as regeneration of damaged tissue, and by indirect mechanisms, for example, by allowing tissue formation by reducing inflammation.

[0024] "Allogeneic" means from different individuals of the same species. Individuals are said to be allogeneic when they have different genes at one or more loci. In contrast, "autologous" means from the same individual.

[0025] The term "immune disease" refers to a disease in a subject characterized by cell, tissue and / or organ damage caused by the subject's immune response.

[0026] The term "autoimmune disease" refers to a disease in a subject characterized by cell, tissue, and / or organ damage caused by an immune response against the subject's own cells, tissues, and / or organs. Illustrative, non-limiting examples of autoimmune diseases that can be treated with the immunomodulatory cells produced by the present invention include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue immune deficiency syndrome (CF1DS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibrositis-fibromyositis, glomerulonephritis, Graves' disease, and gibberellosis. Lance-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjögren's syndrome, Goodpasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu's arteritis, Vasculitis such as temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, antiglomerular basement membrane disease, antiphospholipid syndrome, autoimmune diseases of the nervous system, familial Mediterranean fever, Lambert-Eaton syndrome, sympathetic ophthalmia These include rheumatoid arthritis, polyendocrinopathy, and psoriasis.

[0027] "Immune disorders" include autoimmune diseases and immune-mediated inflammatory diseases.

[0028] "Immune-mediated inflammatory disease" means any disease characterized by chronic or acute inflammation resulting from, involving, or caused by dysregulation of the normal immune response; for example, Crohn's disease, type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Hashimoto's disease, 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 disease, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, and post-organ transplant rejection.

[0029] The term "immunomodulation" refers to the alteration, amplification, inhibition or reduction of one or more biological activities of the immune system, including, but not limited to, downregulation of immune responses, enhancement of immune responses, alteration of inflammatory states mediated by changes in cytokine profiles, cytotoxic activity and antibody production, and their effects on immune and immune-related cells.

[0030] The term "inflammatory disorder" refers to a disease in a subject characterized by inflammation, e.g., chronic inflammation. Illustrative, non-limiting examples of inflammatory disorders include, but are not limited to, 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's arteritis, temporal arteritis, and lymphomatoid granulomatosis), post-traumatic vascular angioplasty (e.g., restenosis after angioplasty), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacterial infection.

[0031] The term "viral disease" refers to a disease caused by a virus. Suitable diseases include, but are not limited to, influenza, adenovirus infection, respiratory syncytial disease, rhinovirus infection, herpes simplex, chickenpox (varicella), measles (Rubeola), rubella (Rubella), mumps (epidemic), smallpox (variola), Kawasaki disease, yellow fever, dengue fever, Includes hepatitis A, hepatitis B, NANB hepatitis, viral gastroenteritis, viral fever, cytomegalovirus disease, AIDS (HIV), rabies, polio, Ebola virus, hemorrhagic fever, Epstein-Barr, and diseases including cancer caused by the viruses that cause any of the previous diseases.

[0032] The term "bacterial infection" includes, but is not limited to, any infection caused by a medically relevant bacterium, such as whooping cough, leprosy, tuberculosis, toxic shock syndrome, food poisoning, Salmonella, E. coli poisoning, Staphylococcus aureus, reus, Clostridium difficile, sepsis, Lyme disease, cholera, and dysentery.

[0033] By "isolated cell population" is meant a cell population, isolated from the human or animal body, that is substantially free of one or more other cell populations normally associated with the cell population in vivo or in vitro.

[0034] The term "ligand inducer" refers to an agent that results in increased production of such ligand. A ligand inducer for a Toll-like receptor (TLR) ligand produces increased TLR ligand and is therefore essentially equivalent to the TLR ligand itself.

[0035] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encode proteins that present cell surface antigens. In humans, these genes are called human leukocyte antigen (HLA) genes. The abbreviations MHC and HLA are used interchangeably.

[0036] The term "population of cells" refers to any number of cells greater than 1, but includes at least 1 x 10 cells, at least 1 x 10 4 of cells, at least 1 x 10 5 of cells, at least 1 x 10 6 of cells, at least 1 x 10 7 of cells, at least 1 x 10 8 A population of cells refers to at least 1 x 10 cells, at least 1 x 10 cells, or more cells. A population of cells also refers to cells in batch formation grown in a bioreactor or other industrial process intended to culture large amounts of cells.

[0037] In preferred embodiments of the invention, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% (by cell number) of the stem cells in the population of progenitor cells are undifferentiated MSCs.

[0038] The term "significant expression" or its synonyms "positive" and "+" when used in reference to a cell surface marker means that more than 20%, preferably more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% of the cells in a cell population express the cell surface marker. Expression of cell surface markers can be determined, for example, by flow cytometry for specific cell surface markers using conventional methods and equipment (e.g., a BECKMAN COULTER EPICS XL FACS system used with commercially available antibodies and standard protocols known in the art), which demonstrates a signal for a specific cell surface marker in flow cytometry that exceeds the background signal using conventional methods and equipment. The background signal is defined as the signal intensity given by a nonspecific antibody of the same isotype as the specific antibody used to detect each surface marker in conventional FACS analysis. For a marker to be considered positive, the observed specific signal must be 20% stronger, preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, 500%, 1000%, 5000%, 10000%, or more than the intensity of the background signal using conventional methods and equipment. Furthermore, commercially available and known monoclonal antibodies against the cell surface markers (e.g., cell receptors and transmembrane proteins) can be used to identify relevant cells.

[0039] mRNA expression can be determined by any suitable technique, including, but not limited to, gene expression arrays (gene chips), mRNA-SEQ, Northern blots, or polymerase chain reaction (PCR), including quantitative PCR (qPCR) methods involving the use of reverse transcriptase. qPCR methods include, but are not limited to, probe-based quantification, such as TaqMan®; dye-based quantification, such as SYBR Green; and digital PCR. qPCR methods can be absolute quantification methods or methods that use relative quantification methods that require normalization to housekeeping genes such as actin, GAPDH, or ribosomal subunits.

[0040] <Incorporated by reference> All applications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual application, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 shows gene expression data generated using PCR arrays from MSCs polarized to have anti-inflammatory characteristics using the TLR3 ligand poly(I:C). Gray indicates at least a two-fold induction of gene expression; black indicates a two-fold decrease in gene expression; and double (thick) boxes indicate genes that were upregulated more than two-fold and selected for further validation in Figure 2 (except for PIAS2, which was decreased by at least two-fold). [Figure 2] Figure 2 shows validation of selected genes from the experiment in Figure 1 using qPCR (A and B). Error bars indicate SEM. [Figure 3] FIG. 3 shows the induction of IL-6 and IL-8 secretion in MSC cells after treatment with a TLR4 ligand (MSC1) or a TLR4 ligand plus EPO and cobalt chloride (MSC1*). [Figure 4] FIG. 4 shows the induction of CCL5 and CXCL10 secretion in MSC cells after treatment with a TLR3 ligand (MSC2) or a TLR3 ligand plus EPO and cobalt chloride (MSC2*). [Figure 5] FIG. 5 shows the induction of the MSC1 phenotype by expression of TNFSF10 (TRAIL) in MSC cells from different human donors after treatment with TLR4 ligand (no*) or TLR4 ligand plus EPO and cobalt chloride (with*). [Figure 6] FIG. 6 shows the induction of the MSC2 phenotype by expression of CXCL9 in MSC cells from different human donors after treatment with TLR3 ligand (no*) or TLR3 ligand plus EPO and cobalt chloride (with*). [Figure 7]FIG. 7 shows the time course of induction of TNFSF10 (TRAIL) expression in MSC cells from MSCs after treatment with a TLR4 ligand (MSC1) or a TLR4 ligand plus EPO and cobalt chloride (MSC1*). [Figure 8] FIG. 8 shows the time course of induction of CXCL9 expression in MSC cells from MSCs after treatment with a TLR3 ligand (MSC2) or a TLR3 ligand plus EPO and cobalt chloride (MSC2*). [Figure 9] Figure 9 shows a transwell migration assay of unstimulated (negative control) MSCs compared with MSCs stimulated with TLR4 ligand (MSC1), TLR4 ligand plus EPO and cobalt chloride (MSC1*), TLR3 ligand (MSC2), and TLR3 ligand plus EPO and cobalt chloride (MSC2*). Error bars indicate SEM. [Figure 10] Figure 10 shows cell proliferation / viability assays of unstimulated (negative control) MSCs compared with MSCs stimulated with TLR4 ligand (MSC1), TLR4 ligand plus EPO and cobalt chloride (MSC1*), TLR3 ligand (MSC2), and TLR3 ligand plus EPO and cobalt chloride (MSC2*). Error bars indicate SEM. [Figure 11] Figure 11 shows validation of the qPCR assay for measuring TNFSF10 (TRAIL) expression: an agarose gel of primer PCR amplification products of TNFSF10 (TRAIL) is shown. [Figure 12] FIG. 12 (A) shows the time course of CXCL9 expression in MSCs polarized to MSC2; (B) shows an agarose gel of primer PCR amplification products of CXCL9. DETAILED DESCRIPTION OF THE INVENTION

[0042] The practice of the present invention (other than the invention itself) employs conventional techniques of cell culture, molecular biology, and microbiology, which are within the skill of those in the art.

[0043] The present invention provides an inducing, activating, polarizing, or priming culture induction medium for a population of mesenchymal stem cells, comprising a Toll-like receptor (TLR) ligand or a TLR ligand inducer in combination with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic, as well as additional standard components of cell culture media known in the art and described herein.

[0044] The present invention also provides a culture-medium induction supplement comprising a Toll-like receptor (TLR) ligand or TLR ligand inducer in combination with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic, which can be added to an otherwise existing culture medium. Such a supplement may be appropriate when an abnormal component or abnormal concentration of another component is appropriate for a particular situation.

[0045] The present invention also provides a sealed culture vessel containing the culture induction medium or medium induction supplement of the present invention.

[0046] The present invention also provides a method for preparing a culture induction medium as disclosed herein, the method comprising the steps of: (a) obtaining a culture medium; and (b) adding to the culture medium a Toll-like receptor (TLR) ligand or a TLR ligand inducer in combination with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic.

[0047] The present invention also provides a composition, the composition comprising: (a) a culture medium according to the present invention; and (b) stem cells.

[0048] The present invention also provides a composition, the composition comprising (a) a culture medium according to the present invention; and (b) a solid surface. In certain embodiments, the solid surface is a tissue culture-compatible surface, including tissue culture plates, flasks, and bottles of any size used for 2D cell culture. In certain embodiments, the solid surface is a microcarrier or any other support matrix for cells used in 3D culture.

[0049] The present invention also provides the use of the culture medium of the present invention to induce, activate or prime a population of mesenchymal stem cells.

[0050] The present invention also provides an ex vivo method for inducing, activating, or priming a population of mesenchymal stem cells, the method comprising the steps of: (a) providing a population of mesenchymal stem cells; (b) providing a culture medium of the present invention; (c) contacting the stem cells with the culture medium; and (d) culturing the cells under appropriate conditions.

[0051] In one aspect, the present invention provides the use of a Toll-like receptor (TLR) ligand or TLR ligand inducer in combination with erythropoietin and exposed to hypoxia or a hypoxia mimetic in the manufacture of a cell therapy agent. Accordingly, in one embodiment, the present invention also provides a method for the manufacture of a cell therapy agent, the method comprising the steps of: (a) providing a population of mesenchymal stem cells; (b) providing a culture medium of the present invention; (c) contacting the stem cells with the culture medium; and (d) culturing the cells under appropriate conditions. The present invention also provides the use of a composition, the composition comprising: (a) a culture medium of the present invention; and (b) stem cells for the manufacture of a cell therapy agent. The present invention also provides the use of a composition, the composition comprising: (a) a culture medium of the present invention; and (b) a solid surface for the manufacture of a cell therapy agent.

[0052] The cell therapy agents are intended for use in the treatment, repair, prevention, and / or amelioration of one or more conditions associated with damaged tissue or inflammatory and / or immune disorders, such as immune-mediated diseases, including, but not limited to, autoimmune diseases, inflammatory disorders, and rejection of transplanted organs and tissues, and cancer. The cell therapy agents of the present invention comprise a prophylactically or therapeutically effective amount of stem cells and a pharmaceutical carrier. Particularly preferred are mesenchymal stem cells. Examples of dosages and administration regimens for each of these cell types are known in the art. Suitable pharmaceutical carriers are known in the art and preferably are those approved by U.S. federal or state regulatory authorities or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. The composition may also contain a small amount of a pH buffer, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Science" by E.W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to a subject. The formulation should suit the mode of administration. In preferred embodiments, the agent is sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[0053] In certain embodiments, the disclosed methods, cells, and induction media are for the treatment of acute or chronic pain. In certain embodiments, the pain is not associated with a specific diagnosis. In certain embodiments, the pain is associated with trauma. In certain embodiments, the pain is back pain. In certain embodiments, the pain is associated with a herniated disc or degenerative disc disease. In certain embodiments, the pain is neuropathic. In certain embodiments, the pain is caused by sciatica.

[0054] In certain embodiments, the disclosed methods, cells, and induction media are for the treatment of cancer. In certain embodiments, the disclosed methods, cells, and induction media are for the treatment of tumors. In certain embodiments, the disclosed methods, cells, and induction media are for enhancing the treatment of cancer. In certain embodiments, the cancer is: adult acute lymphoblastic leukemia; childhood acute lymphoblastic leukemia; adult acute myeloid leukemia; childhood acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdomyosarcoma-like tumor; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; osteosarcoma, osteosarcoma, and malignant fibrous histiocytoma; brain stem glioma; brain tumor; central nervous system embryonal tumor; astrocytoma; craniopharyngioma; ependymoblastoma; brain tumor, ependymoma; medulloblastoma; medulloepithelioma ;Pineal parenchymal tumors showing intermediate differentiation;Supratentorial primitive neuroectodermal tumors and pineoblastomas;Brain and spinal cord tumors;Breast cancer;Male breast cancer;Bronchial tumors;Burkitt's lymphoma;Carcinoid tumors;Central nervous system atypical teratoid / rhabdomyosarcomatoid tumors;Central nervous system embryonal tumors;Central nervous system (CNS) lymphomas, cervical cancer;Primary;Cervical cancer;Chordoma;Chronic lymphocytic leukemia;Chronic myelogenous leukemia;Chronic myeloproliferative syndromes;Colon cancer;Colorectal cancer;Craniopharyngioma;Malignant cutaneous T-cell lymphoma;Central nervous system embryonal tumors;Endometrial cancer ;Ependymoblastoma;Ependymoma;Esophageal cancer;Esthesioneuroblastoma;Ewing's sarcoma family tumors;Extracranial germ cell tumors;Extragonadal germ cell tumors;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinoblastoma;Gallbladder cancer;Gastric (stomach) cancer;Gastrointestinal carcinoid tumors;Gastrointestinal stromal tumors (GIST);Extracranial germ cell tumors;Extragonadal germ cell tumors;Ovarian germ cell tumors;Gestational trophoblastic tumors;Glioma;Hairy cell leukemia;Head and neck cancer;Cardiac cancer;Hepatocellular (liver) carcinoma, adult (primary);Hepatocellular (liver) carcinoma;Combined Histiocytosis, Langerhans cell;Adult Hodgkin's lymphoma;Childhood Hodgkin's lymphoma;Hypopharyngeal carcinoma;Intraocular melanoma;Insulinoma (endocrine pancreas);Kaposi's sarcoma;Kidney (renal cell) carcinoma;Kidney carcinoma;Langerhans cell histiocytosis;Laryngeal carcinoma;Childhood laryngeal carcinoma;Leukemia, acute lymphoblastic, adult;Leukemia, acute lymphoblastic, childhood;Leukemia, acute myeloid, adult;Leukemia, acute myeloid, childhood;Leukemia, chronic lymphocytic;Leukemia, chronic myeloid;Leukemia, hairy cell;Lip and oral cavity cancer;Liver cancer, adult (primary);Liver cancer; Lung cancer, non-small cell; Lung cancer, small cell; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-cell; Lymphoma, Hodgkin, adult; Lymphoma, Hodgkin, childhood; Lymphoma, non-Hodgkin, adult; Lymphoma, non-Hodgkin, childhood; Lymphoma, primary central nervous system (CNS); Macroglobulinemia, Waldenstrom; Malignant fibrous histiocytoma of bone and osteosarcoma; Medulloblastoma; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma, adult malignant tumor; Mesothelioma; Metastatic squamous cell carcinoma of the neck of unknown primary; Oral cancer; Multiple endocrine neoplasia; Multiple myeloma / plasma cell neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplastic / myeloproliferative neoplasm; Myeloid leukemia, chronic; Myeloid leukemia, adult acute; Myeloid leukemia, Acute childhood; multiple myeloma; myeloproliferative syndromes, chronic; cancer of the nasal cavity and paranasal sinuses; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin's lymphoma, adult; non-Hodgkin's lymphoma, childhood; non-small cell lung cancer; oral cavity cancer; oral cavity, lip, and oropharynx cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial carcinoma; ovarian germ cell tumor; ovarian low-malignant potential tumor; pancreatic cancer; pancreatic cancer, islet cell tumor; papillomatosis; cancer of the paranasal sinuses and nasal cavity; parathyroid carcinoma; penile cancer; pharyngeal cancer; pineal parenchymal tumor with intermediate differentiation; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; primary central nervous system (CNS) lymphoma; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell carcinoma; respiratory tract cancer with chromosome 15 alterations Cancer); Retinoblastoma; Rhabdomyosarcoma; Salivary gland carcinoma; Salivary gland carcinoma; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi's; Sarcoma, soft tissue, adult; Sarcoma, soft tissue, childhood; Sarcoma, uterine; Sezary syndrome; Skin cancer (non-melanoma); Skin cancer; Skin cancer (melanoma); Skin cancer, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma, adult; Soft tissue sarcoma, childhood; Squamous cell carcinoma; Cervical carcinoma of unknown primary Squamous cell carcinoma, metastatic; gastric cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, skin; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, gestational; carcinoma of unknown primary site, renal pelvis and ureter, transitional cell carcinoma; urethral cancer; uterine cancer, endometrium; uterine sarcoma; uveal melanoma; vaginal cancer; vulvar cancer; Waldenstrom's macroglobulinemia or Wilms' tumor.

[0055] In certain embodiments, the disclosed methods, cells, and induction media are intended for administration to a subject in need of treatment for cancer, autoimmune disease, or inflammatory disorder. In certain embodiments, the disclosed methods, cells, and induction media encompass different administration routes. In certain embodiments, the administration route is subcutaneous, intraparietal, intramuscular, intravenous, intratumoral, intraocular, intraretinal, intravitreal, or intracranial.

[0056] In certain embodiments, the disclosed methods, cells, and induction media are for administration to a subject in need of treatment for cancer, autoimmune disease, or immune-mediated inflammatory disease. In certain embodiments, the disclosed methods, cells, and induction media encompass different dosing frequencies. In certain embodiments, the disclosed cells and agents are administered once daily, once weekly, once monthly, or once yearly. In certain embodiments, the disclosed cells and methods are for administration twice daily, twice weekly, twice monthly, or twice yearly. In certain embodiments, the disclosed cells and methods are for administration three times daily, three times weekly, three times monthly, or three times yearly. In certain embodiments, the disclosed cells and methods are for administration four times daily, four times weekly, four times monthly, or four times yearly. In certain embodiments, after primary treatment, a maintenance dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. In certain embodiments, the maintenance dose is continued for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. In certain embodiments, at least 1 x 10 cells are administered per administration. In certain embodiments, at least 2 x 10 cells are administered per administration. In certain embodiments, at least 3 x 10 cells are administered per administration. In certain embodiments, at least 4 x 10 cells are administered per administration. In certain embodiments, at least 5 x 10 cells are administered per administration. In certain embodiments, at least 6 x 10 cells are administered per administration. In certain embodiments, cells are administered. In certain embodiments, at least 7 x 10 cells are administered per administration. In certain embodiments, at least 8x10 cells are administered per dose. In certain embodiments, at least 9x10 cells are administered per dose. In certain embodiments, at least 1x10 cells are administered per dose. In certain embodiments, at least 2x10 cells are administered per dose. In certain embodiments, at least 3x10 cells are administered per dose.In certain embodiments, at least 4x10 cells are administered per dose. In certain embodiments, at least 5x10 cells are administered per dose. In certain embodiments, at least 6x10 cells are administered per dose. In certain embodiments, at least 7x10 cells are administered per dose. In certain embodiments, at least 8x10 cells are administered per dose. In certain embodiments, at least 9x10 cells are administered per dose. In certain embodiments, at least 1x10 cells are administered per dose. In certain embodiments, at least 2x10 cells are administered per dose. In certain embodiments, at least 3x10 cells are administered per dose. In certain embodiments, at least 4x10 cells are administered per dose. In certain embodiments, at least 5x10 cells are administered per dose. In certain embodiments, at least 6x10 cells are administered per dose. In certain embodiments, at least 7x10 cells are administered per dose. In certain embodiments, at least 8x10 cells are administered per dose. In certain embodiments, at least 9x10 cells are administered per dose. In certain embodiments, at least 1x10 cells are administered per dose. In certain embodiments, at least 2x10 cells are administered per dose. In certain embodiments, at least 3x10 cells are administered per dose.

[0057] The medicaments of the present invention may be in a variety of forms, including semi-solid and liquid dosage forms, such as lyophilized preparations, liquid solutions or suspensions, injectable and infusible solutions, with the medicaments preferably being injectable.

[0058] In certain embodiments, the medicament is for the treatment or repair of damaged tissue (preferably mesenchymal tissue) and / or for the treatment, modulation, prevention, and / or amelioration of one or more symptoms associated with inflammatory and / or immune disorders. Accordingly, the methods and cells of the present invention are for use in treating any disorder characterized by any or all of the above symptoms. A representative, non-exhaustive list of such disorders is provided in the definitions section. Particularly preferred are medicaments for the treatment of immune-mediated inflammatory diseases. Even more preferred are medicaments for the treatment of diabetes, rheumatoid arthritis (RA), inflammatory bowel disease (IBD, including Crohn's disease and / or ulcerative colitis), and multiple sclerosis (MS). The present invention also provides the use of a Toll-like receptor (TLR) ligand or TLR ligand inducer in combination with erythropoietin and exposed to hypoxia or a hypoxia mimetic for the culture of mesenchymal stem cells.

[0059] The specific components and component ratios of the culture medium, supplements, and compositions of the present invention can vary according to specific needs and applications.Similarly, the exact steps of the methods of the present invention can vary according to specific needs and applications.The culture medium, supplements, methods, compositions, and uses of the present invention can be optimized by routine experimentation.For example, if the desired outcome is an anti-inflammatory therapeutic effect, the culture medium, supplement, or composition specifically contains a TLR3 ligand or TLR ligand inducer combined with erythropoietin and exposed to hypoxia or a hypoxia mimic (cobalt chloride or desferrioxamine); in contrast, if the desired outcome is an immunostimulatory therapeutic effect, the culture medium, supplement, or composition specifically contains a TLR4 ligand or TLR ligand inducer combined with erythropoietin and exposed to hypoxia or a hypoxia mimic.The amount of each component described herein can be optimized independently of other components by routine optimization, or one or more components can be added or removed. Culture media can be tested for their ability to support mesenchymal stem cells by testing their induction, activation, or priming, along with or in place of known culture media or methods. The culture media, supplements, methods, compositions, and uses of the present invention are described in more detail below.

[0060] The induction medium of the present invention comprises a Toll-like receptor (TLR) ligand or a TLR ligand inducer in combination with erythropoietin and exposed to hypoxia or a hypoxic mimic. In one aspect, the induction medium of the present invention comprises a Toll-like receptor (TLR) ligand or a TLR ligand inducer. In an alternative aspect, the induction medium of the present invention comprises erythropoietin and is exposed to hypoxia or a hypoxic mimic. In a further aspect, the induction medium of the present invention comprises a Toll-like receptor (TLR) ligand or a TLR ligand inducer in combination with erythropoietin and is exposed to hypoxia or a hypoxic mimic. In certain embodiments, the TLR ligand is a TLR4 ligand. In certain embodiments, the TLR ligand is a TLR3 ligand.

[0061] The induction medium of the present invention may include a combination of two or more, three or more, four, five, six, seven, eight, nine, ten or more Toll-like receptor (TLR) ligands or TLR ligand inducers in combination with erythropoietin (EPO) and exposure to hypoxia or a hypoxia mimetic.

[0062] The induction medium of the present invention may comprise between about 0.10 picomolar (pM) and about 100 millimolar (mM) TLR ligand or TLR ligand inducer, at a concentration of about 10 micromolar to about 1 mM, in combination with between about 0.5 mU / mL and about 100 mU / mL erythropoietin (EPO), and exposed to about 0.5 to about 2% oxygen conditions (hypoxia) or a hypoxia mimetic, such as cobalt chloride or desferrioxamine, or other combinations of the TLR ligand or TLR ligand inducer, erythropoietin, and hypoxia described above.

[0063] The TLR3 ligand used in the induction medium may be IL4, IL13, poly(A:U), poly(I:C), or a combination thereof, and may be delivered by incubation, transfection, transduction, by a carrier molecule, or a combination thereof. Preferably, the TLR3 ligand or agonist is poly(I:C).

[0064] The TLR4 ligand used in the induction medium may be aminoalkyl glucosaminide 4-phosphate, interferon, TNF-alpha, GM-CSF, lipopolysaccharide (LPS), or a combination thereof, and may be delivered by incubation, transfection, transduction, a carrier molecule, or a combination thereof. Preferably, the TLR4 ligand or agonist is LPS.

[0065] The TLR3 ligand or agonist may be provided in a culture medium or supplement as noted above in an amount 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, and preferably about 1 μg / mL to about 1.5 μg / mL.

[0066] In certain embodiments, the TLR3 ligand is poly(I:C) and is provided in an amount 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 5 μg / mL, and about 1 μg / mL to about 2.5 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 1 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 2 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 3 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 4 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 5 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 6 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 7 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 8 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 9 μg / mL. In certain embodiments, poly(I:C) is provided in an amount of about 10 μg / mL. In certain embodiments, poly(I:C) is provided in an amount less than about 100 ng / mL. In certain embodiments, poly(I:C) is provided in an amount of less than about 50 ng / mL. In certain embodiments, poly(I:C) is provided in an amount of less than about 20 ng / mL. In certain embodiments, poly(I:C) is provided in an amount of less than about 10 ng / mL. In certain embodiments, poly(I:C) is provided in an amount of less than about 50 ng / mL.

[0067] The TLR4 ligand or agonist may be provided in a culture medium or supplement as noted above in an amount of about 10 pg / mL to about 10 μg / mL, about 100 pg / mL to about 10 μg / mL, about 1 ng / mL to about 1 μg / mL, about 5 ng / mL to about 1 μg / mL, about 10 ng / mL to about 1 μg / mL, about 100 ng / mL to about 1 μg / mL, preferably about 5 ng / mL to about 50 ng / mL, and also preferably about 5 ng / mL to about 25 ng / mL.

[0068] In certain embodiments, the TLR4 ligand is LPS. In certain embodiments, LPS is present in an amount of about 10 pg / mL to about 10 μg / mL, about 100 pg / mL to about 10 μg / mL, about 1 ng / mL to about 1 μg / mL, about 5 ng / mL to about 1 μg / mL, about 10 ng / mL to about 1 μg / mL, about 100 ng / mL to about 1 μg / mL, preferably about 5 ng / mL to about 50 ng / mL, and also preferably about 5 ng / mL to about 25 ng / mL. In certain embodiments, LPS is present at a concentration of about 5 ng / mL. In certain embodiments, LPS is present at a concentration of about 10 ng / mL. In certain embodiments, LPS is present at a concentration of about 15 ng / mL. In certain embodiments, LPS is present at a concentration of about 20 ng / mL. In certain embodiments, LPS is present at a concentration of about 25 ng / mL. In certain embodiments, LPS is present at a concentration of about 30 ng / mL. In certain embodiments, LPS is present at a concentration of about 35 ng / mL. In certain embodiments, LPS is present at a concentration of about 40 ng / mL. In certain embodiments, LPS is present at a concentration of about 45 ng / mL. In certain embodiments, LPS is present at a concentration of about 50 ng / mL. In certain embodiments, LPS is present at a concentration of less than about 100 ng / mL. In certain embodiments, LPS is present at a concentration of less than about 50 ng / mL. In certain embodiments, LPS is present at a concentration of less than about 20 ng / mL. In certain embodiments, LPS is present at a concentration of less than about 10 ng / mL.

[0069] In certain embodiments, the induction medium of the present invention includes incubation in a hypoxic or oxygen-deficient environment. In certain embodiments, the hypoxic environment has less than 2% oxygen. In certain embodiments, the hypoxic environment has less than 1.5% oxygen. In certain embodiments, the hypoxic environment has less than 1.0% oxygen. In certain embodiments, the hypoxic environment has less than 0.5% oxygen. In certain embodiments, the hypoxic environment effectively has 0% oxygen. In certain embodiments, the hypoxic environment has between 0.5% and 2.0% oxygen. In certain embodiments, the hypoxic environment has between 0.5% and 1.5% oxygen. In certain embodiments, the hypoxic environment has between 0.5% and 1.0% oxygen. In certain embodiments, the hypoxic environment has between 1.0% and 2.0% oxygen. In certain embodiments, the hypoxic environment has between 1.5% and 2.0% oxygen.

[0070] In certain embodiments, the induction medium of the present invention comprises 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 about 10 μM to about 1 mM. In certain embodiments, cobalt chloride is present at a concentration of about 10 μM to about 800 μM. In certain embodiments, cobalt chloride is present at a concentration of about 10 μM to about 500 μM. In certain embodiments, cobalt chloride is present at a concentration of about 10 μM to about 400 μM. In certain embodiments, cobalt chloride is present at a concentration of about 10 μM to about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of about 50 μM to about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of about 100 μM to about 300 μM. In certain embodiments, cobalt chloride is present at a concentration of about 150 μM to about 300 μM.

[0071] In certain embodiments, the induction medium of the present invention comprises desferrioxamine. In certain embodiments, desferrioxamine is present at a concentration of about 50 μM. In certain embodiments, desferrioxamine is present at a concentration of about 200 μM. In certain embodiments, desferrioxamine is present at a concentration of about 300 μM. In certain embodiments, desferrioxamine is present at a concentration of about 400 μM. In certain embodiments, desferrioxamine is present at a concentration of about 500 μM. In certain embodiments, desferrioxamine is present at a concentration of about 600 μM. In certain embodiments, desferrioxamine is present at a concentration of about 700 μM. In certain embodiments, desferrioxamine is present at a concentration of about 800 μM. In certain embodiments, desferrioxamine is present at a concentration of about 900 μM. In certain embodiments, desferrioxamine is present at a concentration of about 1 mM. In certain embodiments, desferrioxamine is present at a concentration of about 10 μM to about 1 mM. In certain embodiments, desferrioxamine is present at a concentration of about 10 μM to about 800 μM. In certain embodiments, desferrioxamine is present at a concentration of about 10 μM to about 500 μM. In certain embodiments, desferrioxamine is present at a concentration of about 10 μM to about 400 μM. In certain embodiments, desferrioxamine is present at a concentration of about 10 μM to about 300 μM. In certain embodiments, desferrioxamine is present at a concentration of about 50 μM to about 300 μM. In certain embodiments, desferrioxamine is present at a concentration of about 100 μM to about 300 μM. In certain embodiments, desferrioxamine is present at a concentration of about 150 μM to about 300 μM.

[0072] In certain embodiments, the induction medium of the present invention comprises erythropoietin. In certain embodiments, the induction medium of the present invention comprises recombinant erythropoietin. In certain embodiments, the induction medium of the present invention comprises human recombinant erythropoietin. In certain embodiments, the amount of erythropoietin is between about 0.1 ng / mL and about 1.0 mg / mL. In certain embodiments, the amount of erythropoietin is between about 0.1 ng / mL and about 100 ng / mL. In certain embodiments, the amount of erythropoietin is between about 0.1 ng / mL and about 50 ng / mL. In certain embodiments, the amount of erythropoietin is between about 0.1 ng / mL and about 10 ng / mL. In certain embodiments, the amount of erythropoietin is between about 0.1 ng / mL and about 1.0 ng / mL. In certain embodiments, the amount of erythropoietin is between about 0.2 ng / mL and about 0.8 ng / mL. In certain embodiments, the amount of erythropoietin is between about 0.3 ng / mL and 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.

[0073] Cell induction media typically contain numerous components required to support the maintenance of cultured cells. Therefore, the induction media of the present invention usually contain many other components in addition to a Toll-like receptor (TLR) ligand or TLR ligand inducer in combination with erythropoietin and exposure to hypoxia or a hypoxia mimetic (cobalt chloride or desferrioxamine). Appropriate component combinations can be readily formulated by those skilled in the art in light of the following disclosure. Induction media according to the present invention will generally be a nutrient solution containing standard cell culture components, such as amino acids, vitamins, trace metals, inorganic salts, a carbon-energy source, and buffers, as described in more detail below.

[0074] The induction medium of the present invention may contain serum. Serum contains cellular and non-cellular factors and components that may be necessary for viability and proliferation. Serum obtained from any suitable source may be used, including fetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), porcine serum, sheep serum, rabbit serum, rat serum (RS), etc. If the MSCs are of human origin, it is also within the scope of the present invention for the cell induction medium to be supplemented with preferably autologous human serum. It is understood that serum may be heat-inactivated at 55-65°C if it is deemed necessary to inactivate components of the complement cascade. If used, serum substitutes may be used at between about 2% and about 40% by volume of the medium according to conventional techniques.

[0075] In other embodiments, the induction medium of the present invention may contain a serum replacement. A variety of different serum replacement formulations, such as, but not limited to, serum albumin, serum transferrin, selenium, and recombinant proteins, including, but not limited to, insulin, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), are commercially available and known to those skilled in the art. When used, serum replacement may be used at between about 2% and about 40% by volume of the medium according to conventional techniques. In other embodiments, the induction medium of the present invention may be serum-free and / or serum replacement-free. A serum-free medium is a medium that does not contain any type of animal serum. A serum-free medium is preferred to avoid the possibility of xeno-contamination of stem cells. A serum replacement-free medium is a medium that is not supplemented with a commercial serum replacement formulation.

[0076] The induction medium of the present invention is usually formulated in deionized distilled water. The induction medium of the present invention is typically sterilized before use to prevent contamination, for example, by ultraviolet light, heat, irradiation, or filtration. The induction medium can also be frozen (e.g., at -20°C or -80°C) for storage or transportation. Antimicrobial agents are also typically used in the medium to reduce contamination by bacteria, mycoplasma, and fungi. The medium may contain one or more antimicrobial or antibiotic agents to prevent contamination. Typically, the antibiotic or antifungal compound used is a penicillin / streptomycin mixture, but can also include, but is not limited to, amphotericin (Fungizone®), ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, and the like.

[0077] In one embodiment of the present invention, the induction medium is a medium conditioned by the addition of cells induced by a Toll-like receptor (TLR) ligand or a TLR ligand inducer in combination with erythropoietin and exposed to hypoxia or a hypoxia mimic (cobalt chloride or desferrioxamine). The conditioned medium is produced by culturing a population of cells in the induction medium for a time sufficient to condition the medium and then harvesting the conditioned medium. When used, the conditioned medium can be conditioned on mammalian cells, such as mouse cells or human cells. A variety of different types of mammalian cells can be used to produce a conditioned medium suitable for inducing mesenchymal stem cells.

[0078] The induction medium can be a 1x formulation or a concentrated formulation, for example, a 2x to 250x concentrated medium formulation. In a 1x formulation, each component in the medium is at a concentration intended for cell induction. In a concentrated formulation, one or more components are present at a higher concentration than the concentration intended for cell induction. The induction medium can be concentrated using known methods, such as salt precipitation or selective filtration. The concentrated medium can be diluted for use with water (preferably deionized and distilled) or any suitable solution, such as aqueous saline, aqueous buffer, or culture medium.

[0079] Induction media as disclosed herein may, under appropriate conditions, be capable of inducing, activating, or priming a population of stem cells in a pluripotent, undifferentiated, and proliferative state for a single passage or population doubling. Stem cells are considered to be in a pluripotent, undifferentiated, and proliferative state if they exhibit the characteristics as described elsewhere in more detail herein. Suitable conditions may be selected by one skilled in the art from conditions commonly used for culturing mesenchymal stem cells.

[0080] As noted elsewhere herein, the present invention also provides a sealed container containing the induction medium of the present invention. A sealed container is preferred for transporting or storing the induction medium to prevent contamination. The container can be any suitable container, such as a bioreactor, flask, plate, bottle, jar, vial, or bag. As noted elsewhere herein, the present invention also provides a method for preparing an induction medium, the method comprising: (a) obtaining a culture medium; and (b) adding a Toll-like receptor (TLR) ligand or a TLR ligand inducer combined with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic (cobalt chloride or desferrioxamine) to the culture medium. Depending on the specific components to be included in the induction medium, various different methods for preparing the induction medium are envisioned. For example, a method for preparing an induction medium may include (a) obtaining a culture medium; and (b) adding to the culture medium a TLR ligand or a TLR ligand inducer in combination with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic (cobalt chloride or desferrioxamine). In one embodiment, a method for preparing an induction medium may include (a) obtaining a culture medium; and (b) adding to the culture medium a TLR ligand, EPO, and cobalt chloride.

[0081] The induction medium of the present invention can be used to induce, activate, or prime a population of mesenchymal stem cells. Thus, the present invention provides the use of an induction medium as disclosed herein to induce, activate, or prime a population of mesenchymal stem cells toward a distinct and uniform phenotype for cell therapy.

[0082] In certain embodiments, the induction medium disclosed herein induces or decreases the expression of specific genes, which can be measured by methods known to those skilled in the art, including, but not limited to, PCR; qPCR; qRT-PCR; semi-quantitative RT-PCR; digital PCR; Northern blot; mRNA-SEQ; microarray, etc. In certain embodiments, the induction medium disclosed herein increases or decreases protein levels, which can be measured by methods known to those skilled in the art, including, but not limited to, antibody-based assays; enzyme-linked immunosorbent assay (ELISA); immunoblot or Western blot; flow cytometry, mass spectrometry, etc. In certain embodiments, the induction medium disclosed herein induces the activation or relaxation of cell signaling pathways, which can be measured by methods known to those skilled in the art, including, but not limited to, kinase assays; protein phosphorylation / dephosphorylation measurements; protein ubiquitination / deubiquitination measurements; protein acetylation / deacetylation measurements; protein degradation / stability measurements; measurement of second messengers such as calcium or diacylglycerol; or monitoring the cleavage of inactive to active forms.

[0083] In certain embodiments, the induction media disclosed herein result in a measurable change in gene expression, protein levels, or cell signaling pathways in a cell population. In certain embodiments, the change is an increase in gene expression, protein levels, or cell signaling. In certain embodiments, the change is a statistically significant change measured between an unstimulated or control sample and a stimulated or test sample. In certain embodiments, the change between an unstimulated or control sample and a stimulated or test sample is at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more increase. In certain embodiments, the change between an unstimulated or control sample and a stimulated or test sample is at least a 100-fold or more increase. In certain embodiments, the change between an unstimulated or control sample and a stimulated or test sample is at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more decrease. In certain embodiments, the change between the unstimulated or control sample and the stimulated or test sample is at least a 100-fold or greater decrease.

[0084] In certain embodiments, induction medium containing a TLR3 ligand induces mRNA expression of any of the following genes by at least two-fold when compared to a population of unstimulated cells: CXCL9; EGFR; IRF1; A2M; FAS; IL2RG; MMP3; GBP1; ISG15; FCGR1; NFKB1; NOS2A; USF1; YY1; JAK2; STA2, STAT4; STAT5; SOCS1; or IRF1. In certain embodiments, induction medium containing a TLR3 ligand reduces mRNA expression of any of the following genes by at least two-fold when compared to a population of unstimulated cells: EPOR; F2R; STAM; PDGFRA; PIAS2; MYC; SH2B1; or CSF2RB. In certain embodiments, induction medium containing a TLR3 ligand induces mRNA expression of any of the following genes by at least ten-fold when compared to a population of unstimulated cells: CXCL9; GBP1; ISG15; SOCS1; MMP3; JAK2 or IRF1. In certain embodiments, induction medium containing a TLR3 ligand induces mRNA expression of any of the following genes by at least 20-fold when compared to a population of unstimulated cells: CXCL9; GBP1; ISG15; or SOCS1. In certain embodiments, induction medium containing a TLR3 ligand induces mRNA expression of CXCL9 by at least 100-fold when compared to a population of unstimulated cells.

[0085] In certain embodiments, induction medium containing a TLR4 ligand induces TNFSF10 (TRAIL) mRNA expression by at least 2-fold, 10-fold, 100-fold, or 1000-fold when compared to a population of unstimulated cells.

[0086] The present invention also provides an ex vivo method for inducing, activating, or priming a population of mesenchymal stem cells, the method comprising the steps of: (a) providing a population of mesenchymal stem cells; (b) providing an induction medium as disclosed herein; (c) contacting the stem cells with the induction medium; and (d) culturing the stem cells under appropriate conditions.

[0087] In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein induce or decrease the expression of specific genes, which can be measured by methods known to those of skill in the art, including, but not limited to, PCR; qPCR; qRT-PCR; semi-quantitative RT-PCR; digital PCR; Northern blot; mRNA-SEQ; microarray; etc. In certain embodiments, the induction media disclosed herein increase or decrease protein levels, which can be measured by methods known to those of skill in the art, including, but not limited to, antibody-based assays; enzyme-linked immunosorbent assay (ELISA); immunoblot or Western blot; flow cytometry, mass spectrometry, etc. In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein induce the activation or relaxation of cell signaling pathways that can be measured by methods known to those skilled in the art, including, but not limited to, kinase assays; protein phosphorylation / dephosphorylation measurements; protein ubiquitination / deubiquitination measurements; protein acetylation / deacetylation measurements; protein degradation / stability measurements; measurement of second messengers such as calcium or diacylglycerol; or monitoring the cleavage of inactive to active forms.

[0088] In certain embodiments, the ex vivo methods of inducing, activating, or priming a mesenchymal stem cell population disclosed herein result in a measurable change in gene expression, protein levels, or cell signaling pathways in the stem cell population. In certain embodiments, the change is an increase in gene expression, protein levels, or cell signaling. In certain embodiments, the change is a statistically significant change measured between an unstimulated or control sample and a stimulated or test sample. In certain embodiments, the change between an unstimulated or control sample and a stimulated or test sample is at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more increase. In certain embodiments, the change between an unstimulated or control sample and a stimulated or test sample is at least a 100-fold or more increase. In certain embodiments, the change between the unstimulated or control sample and the stimulated or test sample is at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more decrease, hi certain embodiments, the change between the unstimulated or control sample and the stimulated or test sample is at least a 100-fold or more decrease.

[0089] In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein with a TLR3 ligand induce at least two-fold the mRNA expression of any of the following genes compared to an unstimulated cell population: CXCL9; EGFR; IRF1; A2M; FAS; IL2RG; MMP3; GBP1; ISG15; FCGR1; NFKB1; NOS2A; USF1; YY1; JAK2; STA2, STAT4; STAT5; SOCS1; or IRF1. In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein with a TLR3 ligand decrease at least two-fold the mRNA expression of any of the following genes compared to an unstimulated cell population: EPOR; F2R; STAM; PDGFRA; PIAS2; MYC; SH2B1; or CSF2RB. In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein with a TLR3 ligand induce at least 10-fold the mRNA expression of any of the following genes compared to an unstimulated cell population: CXCL9; GBP1; ISG15; SOCS1; MMP3; JAK2; or IRF1. In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein with a TLR3 ligand induce at least 20-fold the mRNA expression of any of the following genes compared to an unstimulated cell population: CXCL9, GBP1, ISG15, or SOCS1. In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein with a TLR3 ligand induce at least 100-fold the mRNA expression of CXCL9 compared to an unstimulated cell population.

[0090] In certain embodiments, the ex vivo methods of inducing, activating, or priming a population of mesenchymal stem cells disclosed herein with a TLR4 ligand induce TNFSF10 (TRAIL) mRNA expression by at least 2-fold, 10-fold, 100-fold, or 1000-fold compared to an unstimulated cell population.

[0091] The present invention also provides a method of cell therapy, comprising the steps of: (a) providing a population of mesenchymal stem cells; (b) providing an induction medium of the present invention; (c) contacting the stem cell population with the induction medium; and (d) culturing the cells under appropriate conditions.

[0092] The methods of the present invention may include culturing cells attached to a solid surface as described elsewhere herein. For example, a method may be provided that includes the steps of: (a) providing a population of mesenchymal stem cells; (b) providing an induction medium as disclosed herein; (c) contacting the stem cells with the induction medium; and (d) culturing the cells attached to the solid surface under appropriate conditions. The present invention also provides the use of an induction medium and a solid surface as disclosed herein to expand a population of mesenchymal stem cells. Mesenchymal stem cells may adhere, attach, or be seeded on a support. Typically, cells are plated at a desired density, such as between about 100 cells / cm and about 100,000 cells / cm (e.g., between about 500 cells / cm and about 50,000 cells / cm, or more specifically, between about 1,000 cells / cm and about 20,000 cells / cm), before inducing, activating, or priming the stem cells. In certain embodiments, the cell density is between 200-10,000 cells / cm 2 .

[0093] It will be appreciated that the steps of the methods disclosed herein may be performed in any suitable order or simultaneously, and need not be performed in the order listed. For example, in the above methods, the step of providing a population of mesenchymal stem cells may be performed before, after, or simultaneously with the step of providing an induction medium.

[0094] The methods and uses of the present invention may include induction media or supplements as disclosed herein. Thus, in some embodiments, the methods of the present invention may be methods without serum and / or blood substitutes. In some embodiments, the methods of the present invention may be used to induce cells without contact with a layer of feeder cells.

[0095] A preferred method and use of the present invention is for inducing, activating or priming a population of mesenchymal stem cells, resulting in cells being expanded and then cryopreserved and used in cell therapy.

[0096] Preferably, the stem cell population is of adult origin, such as bone marrow-derived or adipose tissue-derived cells, and more preferably, the stem cells are a population of mesenchymal stem cells.

[0097] Conditions for stem cell culture are known to those skilled in the art. Preferably, the culture is carried out in the presence of a solid support suitable for the attachment of mesenchymal stem cells.

[0098] The method of production may optionally further comprise steps (a), (b), and (c): (a) passaging the cells into a culture medium as disclosed herein; (b) further culturing the cells under appropriate conditions; and (c) inducing, activating, or priming the cells.

[0099] It has been shown that ex vivo expansion of MSCs without inducing differentiation can be achieved for extended periods of time using, for example, a number of suitable, specially screened sera, such as fetal bovine serum or human serum. Methods for measuring viability and yield are known in the art (e.g., trypan blue exclusion).

[0100] If desired, any of the steps and procedures for separating cells of the cell populations of the present invention can be performed manually. Alternatively, such cell separation processes can be facilitated and / or automated by one or more suitable devices, examples of which are known in the art.

[0101] The practice of the present invention may be carried out using a suitable cell culture vessel as a support. Cell culture vessels of various shapes and sizes (e.g., flasks, single- or multi-well plates, single- or multi-well dishes, bottles, jars, vials, bags, bioreactors) and made from a variety of different materials (e.g., plastic or glass) are known in the art. An appropriate cell culture vessel can be readily selected by one of ordinary skill in the art.

[0102] The present invention also provides a culture medium induction supplement that can be used to generate a culture induction medium as disclosed herein. A "culture medium induction supplement" is a mixture of ingredients that, by themselves, cannot support mesenchymal stem cells, but that, when combined with other cell culture medium components, enable or enhance the culture of mesenchymal stem cells. Thus, the supplement can be used to generate a functional cell culture medium of the present invention by combining the supplement with other cell culture components to generate an appropriate medium formulation. The use of culture medium supplements is well known in the art. The present invention provides a culture medium induction supplement that includes adding a TLR ligand or TLR ligand inducer in combination with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic (cobalt chloride or desferrioxamine). The supplement may contain the ligand disclosed herein. The supplement may also include one or more additional cell culture components, such as one or more cell culture components selected from the group consisting of amino acids, vitamins, inorganic salts, trace elements, a carbon-energy source, and a buffer.

[0103] The culture medium-derived supplement may be a concentrated liquid supplement (e.g., a 2- to 250-fold concentrated liquid supplement) or a dry supplement. Both liquid and dry forms of supplements are well known in the art. The supplement may be freeze-dried.

[0104] The culture medium derived supplements of the present invention will typically be sterilized before use to prevent contamination, for example, by ultraviolet light, heat, irradiation, or filtration. The culture medium derived supplements may be frozen (e.g., at -20°C or -80°C) for storage or transport.

[0105] The present invention also provides a sealed container containing the culture medium supplement of the present invention. A sealed container is preferred for transporting or storing the culture medium supplement disclosed herein to prevent contamination. The container may be any suitable container, such as a bioreactor, flask, plate, bottle, jar, vial, or bag.

[0106] A variety of materials have been used as surfaces for culturing adherent stem cells, and suitable materials can be readily selected by one of skill in the art. Preferably, the solid surface is composed of plastic, but may alternatively be composed of glass or extracellular matrix. The surface may be planar, tubular, scaffold-like, beaded, or fibrous.

[0107] The compositions of the present invention may comprise serum, or may be serum-free and / or free of serum replacement, as described elsewhere herein.

[0108] Mesenchymal stem cells for use in the present invention can be obtained using well-known methods (see below). Various types of mesenchymal stem cells are contemplated for use with the present invention, whether obtained from embryonic, fetal, or biological tissue sources, although they are preferably obtained from biological tissue sources.

[0109] The induction medium disclosed herein may be used to culture mammalian stem cells, particularly human adult stem cells. Human adult stem cells that can be used with the present invention are preferably mesenchymal stem cells. Murine or primate stem cells can also be used. In a preferred embodiment, the stem cells are human bone marrow-derived stem cells (MSCs).

[0110] Mesenchymal stem cells can be identified using their ability to differentiate into cells of all three germ layers, for example, by determining the ability of the cells to differentiate into cells that exhibit detectable expression of markers specific for all three germ layers. Unless the context requires otherwise, references to the singular (e.g., "a cell" and equivalent references) include the plural (e.g., "cells").

[0111] The induction medium of the present invention can be used to induce, activate, or prime a population of mesenchymal stem cells. Thus, the present invention provides the use of an induction medium as disclosed herein to induce, activate, or prime a population of mesenchymal stem cells toward a distinct, homogeneous phenotype for cell therapy. The distinct, homogeneous phenotype can be an anti-inflammatory MSC phenotype (MSC2) and a homogeneous, distinct, immunostimulatory, anti-tumor MSC phenotype (MSC1).

[0112] The preferred method for induction of a uniform, isolated, anti-inflammatory MSC phenotype (MSC2) is incubation of 70-90% confluent MSCs in culture medium containing a Toll-like receptor 3 (TLR3) ligand, such as polyinosinic:polycytidylic acid (or poly(I:C); 1 μg / mL), in combination with erythropoietin (1 mU / mL or 5 ng / mL) for 1 hour, followed by exposure to hypoxia (1% oxygen) or a hypoxia mimetic (cobalt chloride or desferrioxamine, either 200 μM).

[0113] The preferred method of induction for a homogenous, isolated, immunostimulatory, anti-tumor MSC phenotype (MSC1) is incubation of 70-90% confluent MSCs with culture medium containing a Toll-like receptor 4 (TLR3) ligand, such as lipopolysaccharide (LPS, endotoxin 10 ng / mL), in combination with erythropoietin (1 mU / mL or 5 ng / mL) and exposed to hypoxia (1% oxygen) or a hypoxia mimetic (cobalt chloride or desferrioxamine, either 200 μM) for 1 hour.

[0114] TLR ligands combined with erythropoietin and exposed to hypoxia or hypoxia mimics (cobalt chloride or desferrioxamine) are added to fresh culture medium or as a culture supplement and incubated with cells for 1 hour. Following this induction step, MSCs are washed twice in culture medium or appropriate buffered saline without TLR ligand to remove cellular and culture debris. Without being bound by theory, the short incubation time (<1 hour) and minimal TLR ligand exposure at the concentrations listed above (or less) are critical to achieving the desired phenotype and further ensure the protocol mimics the gradient of danger signals that endogenous MSCs encounter and respond to at distances from the site of injury. Once washed, the induced, activated, or primed MSCs can be harvested using conventional methods, such as trypsin and EDTA for 5 seconds to 15 minutes at 37° C., or trypsin substitutes (e.g., TrypLE from Invitrogen), collagenase, dispase, accutase, or other reagents known to those skilled in the art. After cell harvest, the primed, activated, or induced MSCs can be cryopreserved using standard methods.

[0115] The TLR3 or TLR4 agonist can be delivered by incubation, transfection, transduction with a carrier molecule, or by other techniques known to those skilled in the art.

[0116] Cells may be incubated with a TLR ligand or agonist ligand in combination with erythropoietin (EPO) and exposed to hypoxia or a hypoxia mimetic (cobalt chloride or desferrioxamine) for about 1 minute to about 480 minutes, about 5 minutes to about 475 minutes, about 10 minutes to about 470 minutes, about 15 minutes to about 400 minutes, about 20 minutes to about 120 minutes, about 25 minutes to about 90 minutes, about 30 minutes to about 80 minutes, about 35 minutes to about 70 minutes, about 40 minutes to about 65 minutes, about 45 minutes to about 60 minutes, about 55 minutes to about 60 minutes, and preferably about 60 minutes. [Example]

[0117] Example 1 Induction of the MSC2 gene expression signature from human primary MSCs. For this experiment, human primary MSCs were incubated in serum-free culture medium containing 2 μg / mL poly(I:C) at 37°C and 5% CO2 for 6 hours. Subsequently, cells were washed twice, and RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA) and then processed using a TURBO DNA-Free Kit (Ambion, Austin, TX). RNA was reverse transcribed, and the resulting cDNA was used in a JAK / STAT Signaling Pasthway RT2 Profiler™ PCR Array (SuperArrayBioscience, Frederick, MD) according to the manufacturer's instructions for the iCycler iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Raw data from untreated and treated groups were analyzed using GEArray Analyzer software (SuperArray, Bethesda, MD). This array measures the RNA expression levels of 84 different genes in the JAK / STAT signaling pathway. The results are shown in Figure 1, where genes showing a 2-fold or greater induction are boxed in gray, genes showing a 2-fold or greater decrease are boxed in black, and genes that were induced 2-fold or greater and selected for further validation are boxed in double (except for PIAS2, which was decreased 2-fold or greater). Figure 2 shows further qPCR validation of the genes selected in Figure 1. Validation was performed by qPCR using SYBR green Master Mix with gene-specific primers on the same samples analyzed in Figure 1.

[0118] Example 2 MSC1 and MSC2 polarization from primary human MSCs. Primary human MSCs from donors were polarized to MSC1 or MSC2. Human MSCs were polarized using culture medium containing 10 ng / mL LPS in the absence (MSC1) or presence (MSC1*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. MSC2 cells were polarized using culture medium containing 2 μg / mL Poly(I:C) in the absence (MSC2) or presence (MSC2*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Culture supernatants were collected and then analyzed for chemokine / cytokine expression by Bio-Plex as previously described. Briefly, MSCs were plated at a density of 50,000 cells in 24-well plates, allowed to adhere overnight, and then primed with TLR agonists for 1 hour, as indicated. Conditioned culture medium was collected 48 hours later and analyzed using Bio-Plex Cytokine Assays (Human Group I & II; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. These experiments were performed at least three times on three separate MSC donor pools. MSC1 induction, regardless of formulation, resulted in significant secretion of cytokines including IL6 and IL8 (Figure 3), while MSC2 induction, regardless of formulation, resulted in significant secretion of IP10 (CXCL10) and RANTES (CCL5) (Figure 4). Error bars indicate + / - standard error of the mean (SEM).

[0119] Example 3 MSC1 and MSC2 polarization is uniform across multiple MSC sources. Human MSC donors from three different commercial sources and up to six different donors were polarized to MSC1. Human MSCs were polarized using culture medium containing 10 ng / mL LPS in either the absence (MSC1) or presence (MSC1*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Total RNA was isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix. Data were analyzed using the comparative quantitative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and presented as fold increase relative to untreated controls. TRAIL gene expression significantly increased in all donors, including combined donors, after MSC1 induction (Figure 5). Error bars represent + / - SEM. 1: 95℃ for 0:30, 2: 95℃ for 0:10, 3: 68℃ for 0:30, Plate Read, 4: GOTO2, 39 further runs, using the protocol. Human cDNA primers used: Cxcl9 forward - CTT TCCTGG CTA CTC CAT GTT reverse - GTT GGT CACTGG CTG ATC TAT AA; Trail forward - CTT CAC AGT GCT CCT GCA GT reverse - TTA GCC AACTAA AAA GGC CCC; 18Sr RNA forward - GAGGGAGCCTGAGAAACGG, reverse - GTCGGGAGTGGGTAATTTGC.

[0120] Human MSC donors from three different commercial sources and up to six different donors were polarized into MSC2. Human MSCs were polarized using culture medium containing 2 μg / mL poly(I:C) in either the absence (MSC2) or presence (MSC2*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Total RNA was isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix. Data were analyzed using the comparative quantitative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and presented as fold increase relative to untreated controls. CXCL9 gene expression significantly increased in all donors, including combined donors, after MSC2 induction (Figure 6). Error bars represent + / - SEM.

[0121] Example 4: Time course of MSC1 and MSC2 polarization. Human MSC donors were polarized to MSC1. Human MSCs were polarized using culture medium containing 10 ng / mL LPS in either the absence (MSC1) or presence (MSC1*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Cells were harvested at different times as indicated. Total RNA was isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix. Data were analyzed using the comparative quantitative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and presented as fold increase relative to the untreated control. TRAIL gene expression significantly increased 4 hours after MSC1 induction (FIG. 7). Error bars represent + / - SEM.

[0122] Human MSC donors were polarized to MSC2. Human MSCs were polarized using culture medium containing 2 μg / mL poly(I:C) in either the absence (MSC2) or presence (MSC2*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Cells were harvested at different times as indicated. Total RNA was isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix. Data were analyzed using the comparative quantitative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and presented as fold increase relative to the untreated control. CXCL9 gene expression significantly increased 4 hours after MSC2 induction (Figure 8). Error bars represent ± SEM.

[0123] Example 5 Cellular biodistribution of MSC1 and MSC2. Compared with naive MSC, experiments were conducted to determine the in vivo effects of polarized MSC. For this experiment, human naive MSC, MSC1 and MSC2 (1 million cells) were administered to wild-type mice by intraperitoneal (IP) injection, and all organs were harvested 4 hours later. Subsequently, the extracted RNA was analyzed for human GAPDH and compared with mouse GAPDH DNA to determine tissue homing. The results are shown in Table 1 below.

[0124] [Table 1]

[0125] Example 6 Incubation with erythropoietin and cobalt chloride during MSC polarization increases the migration and proliferation / survival capacity of MSC1 and MSC2 cells. To determine the migration capacity of MSCs cultured with or without erythropoietin and hypoxia, human naive MSC cells and either MSC1- or MSC2-polarized cells were added to individual transwell inserts (8 μM pores, 50,000 cells / insert). MSC1 cells were polarized using culture medium containing 10 ng / mL LPS in either the absence (MSC1) or presence (MSC1*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. MSC2 cells were polarized using culture medium containing 2 μg / mL Poly(I:C) in either the absence (MSC2) or presence (MSC2*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Membranes were placed in 24-well companion plates containing either negative control serum-free culture medium (SFM), positive control serum-containing growth culture medium (CCM), or the corresponding induction culture medium as indicated. Photomicrographs were taken after 16 hours of incubation using a Nikon Eclipse TE300 inverted fluorescence microscope. Figure 9 shows representative data for migrated MSCs counted from four representative quadrants of more than three independently performed experiments, each repeated three times (n=3). Error bars indicate SEM.

[0126] Proliferation assays were used to determine the proliferation potential and viability of MSCs cultured with or without erythropoietin and hypoxia. Human MSCs were polarized using culture medium containing 10 ng / mL LPS in the absence (MSC1) or presence (MSC1*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. MSC2 cells were polarized using culture medium containing 2 μg / mL Poly(I:C) in the absence (MSC2) or presence (MSC2*) of 0.5 ng / mL human recombinant erythropoietin and 200 μM cobalt chloride. Cells were incubated with the specified culture medium for 48 hours. Cell proliferation and viability from each sample were analyzed using a CyQUANT assay (Life Technologies, CA) and trypan blue analysis, respectively. For cell proliferation, cells were seeded in triplicate at 1 x 103 cells in 50 μl per well in 96-well plates and cultured at 37°C, 5% CO2. Samples were taken at 0, 24, 48, 72, and 96 hours after treatment and processed as described by the manufacturer (CyQUANT assay, Life Technologies, CA). Data shown are expressed relative to untreated controls. Cell proliferation assays were performed with each sample replicated along eight wells of a 96-well plate. At least three separate experiments were performed (n=3) using the same method. Error bars indicate SEM. The results are shown in (Figure 10).

[0127] Example 7 Validation of qPCR assays for CXCL9 and TNFSF10. Human MSCs were induced into MSC1. Total RNA was isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix. Data were analyzed using the comparative quantitative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and presented as fold increase over untreated controls. TRAIL gene expression was significantly increased after MSC1 induction. Error bars represent + / - SEM. Primer efficiency and product specificity (Figure 11) were established.

[0128] Human MSCs were induced to become MSC2. Total RNA was isolated, purified, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR Green Master Mix. Data were analyzed using the comparative quantitative CT method, normalizing target gene expression to the 18S rRNA housekeeping gene, and presented as fold increase over untreated controls. CXCL9 gene expression significantly increased after MSC2 induction. Error bars represent + / - SEM. The time course of gene expression (Figure 12A) and product specificity (Figure 12B) were established.

[0129] Although the present invention has been described in detail with particular reference to preferred embodiments, the principles and modes of practicing the invention are also described herein. The present invention should not be construed as being limited to the particular forms disclosed, which are illustrative rather than restrictive. Modifications, variations, and changes may be made by those skilled in the art without departing from the spirit and scope of the invention, as set forth in the following claims.

[0130] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. An induction medium for producing an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the induction medium comprising: a. Toll-like receptor 3 (TLR3) ligands, b. erythropoietin, and c. 0.5-2% oxygen or hypoxia mimic; Here, a population of immunologically polarized mesenchymal stem cells has anti-inflammatory properties characterized by the expression of anti-inflammatory or immunosuppressive mediators, in an induction medium.

2. The induction medium of claim 1 , wherein the Toll-like receptor 3 (TLR3) ligand is poly(I:C).

3. The induction medium of claim 1 , wherein the Toll-like receptor 3 (TLR3) ligand is poly(A:U).

4. 2. The induction medium of claim 1, wherein erythropoietin is present at a concentration of less than 10 ng / mL.

5. 2. The induction medium of claim 1, wherein the hypoxia mimetic is cobalt chloride.

6. 6. The induction medium of claim 5, wherein cobalt chloride is present at a concentration between 5 μM and 500 μM.

7. The induction medium of claim 1, further comprising interleukin 4 (IL-4).

8. The induction medium of claim 1, further comprising interleukin 13 (IL-13).

9. The induction medium of claim 1 , which is free of serum of human or animal origin.

10. The induction medium of claim 1 , which is a concentrated solution.

11. A population of mesenchymal stem cells treated with the induction medium of claim 1.

12. A population of human mesenchymal stem cells treated with the induction medium of claim 1.

13. A population of canine, feline, or equine mesenchymal stem cells treated with the induction medium of claim 1.

14. A population of mesenchymal stem cells treated with the induction medium of claim 1, wherein the mesenchymal stem cells are pluripotent. A population of mesenchymal stem cells derived from stem cells.

15. A population of mesenchymal stem cells treated with the induction medium of claim 1, wherein the mesenchymal stem cells are characterized by increased expression of CXCL9 mRNA compared to a population of unstimulated mesenchymal stem cells.

16. A population of mesenchymal stem cells treated with the induction medium of claim 1, wherein the mesenchymal stem cells are characterized by increased expression of OAS1 mRNA compared to a population of unstimulated mesenchymal stem cells.

17. A population of mesenchymal stem cells treated with the induction medium of claim 1, wherein the mesenchymal stem cells are characterized by increased expression of ISG15 mRNA compared to a population of unstimulated mesenchymal stem cells.

18. A composition for treating a disease comprising a population of mesenchymal stem cells treated with the induction medium of claim 1, wherein the disease is an inflammatory or autoimmune disease.

19. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is rheumatoid arthritis.

20. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is inflammatory bowel disease.

21. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is acute optic neuritis.

22. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is Krabbe disease.

23. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is diabetic retinopathy.

24. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is Crohn's disease.

25. 19. The composition of claim 18, wherein the inflammatory or autoimmune disease is acute lung injury.

26. 1. An induction medium for producing an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the induction medium comprising: a. Toll-like receptor 4 (TLR4) ligands, b. erythropoietin, and c. 0.5-2% oxygen or hypoxia mimic; wherein the immunologically polarized mesenchymal stem cell population has pro-inflammatory characteristics characterized by the expression of pro-inflammatory mediators.

27. 27. The induction medium of claim 26, wherein the Toll-like receptor 4 (TLR4) ligand is lipopolysaccharide (LPS).

28. The induction medium of claim 26, wherein the Toll-like receptor 4 (TLR4) ligand is an aminoalkyl glucosaminide 4-phosphate.

29. 27. The induction medium of claim 26, wherein erythropoietin is present at a concentration of less than 10 ng / mL.

30. 27. The induction medium of claim 26, wherein the hypoxia mimetic is cobalt chloride.

31. 31. The induction medium of claim 30, wherein cobalt chloride is present at a concentration between 5 μM and 500 μM.

32. 27. The induction medium of claim 26, further comprising an interferon.

33. 27. The induction medium of claim 26, further comprising tumor necrosis factor alpha (TNFα).

34. 27. The induction medium of claim 26, which is free of serum of human or animal origin.

35. 27. The induction medium of claim 26, which is a concentrated solution.

36. 27. A population of mesenchymal stem cells treated with the induction medium of claim 26.

37. A population of human mesenchymal stem cells treated with the induction medium of claim 26.

38. 27. A population of canine, feline, or equine mesenchymal stem cells treated with the induction medium of claim 26.

39. 27. A population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the mesenchymal stem cells are derived from pluripotent stem cells.

40. A population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the mesenchymal stem cells are characterized by increased expression of TNFSF10 (TRAIL) mRNA compared to a population of unstimulated mesenchymal stem cells.

41. 27. A composition for treating a disease comprising a population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the disease is cancer.

42. 27. A composition for treating a disease comprising a population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the disease is ovarian cancer.

43. 27. A composition for treating a disease comprising a population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the disease is uveal melanoma.

44. 27. A composition for treating a disease comprising a population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the disease is a viral disease.

45. 27. A composition for treating a disease comprising a population of mesenchymal stem cells treated with the induction medium of claim 26, wherein the disease is a bacterial infection.

46. 1. An induction medium for producing an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the induction medium comprising: a. Poly(I:C) at a concentration between 0.1 μg / mL and 100 μg / mL; b. erythropoietin at a concentration of less than 10 ng / mL, and c. Cobalt chloride at a concentration between 5 μM and 500 μM; Here, an induction medium in which a population of immunologically polarized mesenchymal stem cells has anti-inflammatory properties and is characterized by increased expression of CXCL9, OAS1 and ISG15 mRNA compared to a population of unstimulated mesenchymal stem cells.

47. 1. An induction medium for producing an immunologically polarized mesenchymal stem cell population from a population of unstimulated mesenchymal stem cells, the induction medium comprising: a. LPS at a concentration between 0.1 ng / mL and 1 μg / mL; b. erythropoietin at a concentration of less than 10 ng / mL, and c. Cobalt chloride at a concentration between 5 μM and 500 μM; wherein the immunologically polarized mesenchymal stem cell population has pro-inflammatory characteristics and is characterized by increased expression of TNFSF10 (TRAIL) compared to an unstimulated mesenchymal stem cell population.

48. 1. A method for producing a population of immunologically polarized mesenchymal stem cells from a population of unstimulated mesenchymal stem cells, the method comprising: A method comprising contacting a population of unstimulated mesenchymal stem cells with a composition comprising a Toll-like receptor 3 (TLR3) ligand, erythropoietin, and hypoxia or a hypoxia mimetic, wherein the immunologically polarized mesenchymal stem cell population has anti-inflammatory characteristics characterized by the expression of anti-inflammatory or immunosuppressive mediators.

49. 49. The method of claim 48, wherein the Toll-like receptor 3 (TLR3) ligand is poly(I:C).

50. 49. The method of claim 48, wherein the Toll-like receptor 3 (TLR3) ligand is poly(A:U).

51. 49. The method of claim 48, wherein the erythropoietin is present at a concentration of less than 10 ng / mL.

52. 49. The method of claim 48, wherein the hypoxia mimetic is cobalt chloride.

53. 53. The method of claim 52, wherein the cobalt chloride is present at a concentration between 5 μM and 500 μM.

54. 49. The method of claim 48, wherein the composition further comprises interleukin 4 (IL-4).

55. 49. The method of claim 48, wherein the composition further comprises interleukin 13 (IL-13).

56. 49. The method of claim 48, wherein the composition does not contain serum of human or animal origin.

57. 49. The method of claim 48, wherein the composition is a concentrated solution.

58. 49. The method of claim 48, wherein the population of unstimulated mesenchymal stem cells is simultaneously contacted with a Toll-like receptor 3 (TLR3) ligand, erythropoietin, and hypoxia or a hypoxia mimetic.

59. 49. The method of claim 48, wherein the composition is in contact with the population of unstimulated mesenchymal stem cells for at least 30 minutes but not for more than 8 hours.

60. 49. The method of claim 48, further comprising monitoring expression of CXCL9 at either the RNA or protein level.

61. 49. The method of claim 48, further comprising monitoring expression of OAS1 at either the RNA or protein level.

62. 49. The method of claim 48, further comprising monitoring expression of ISG15 at either the RNA or protein level.

63. 49. A population of mesenchymal stem cells treated by the method of claim 48.

64. 49. A population of human mesenchymal stem cells treated by the method of claim 48.

65. 49. A population of canine, feline, or equine mesenchymal stem cells treated by the method of claim 48.

66. 49. A population of mesenchymal stem cells treated by the method of claim 48, wherein the mesenchymal stem cells are derived from pluripotent stem cells.

67. 49. A population of mesenchymal stem cells treated by the method of claim 48, wherein the mesenchymal stem cells are characterized by increased expression of CXCL9 mRNA compared to a population of unstimulated mesenchymal stem cells.

68. A population of mesenchymal stem cells treated by the method of claim 48, wherein the mesenchymal stem cells are characterized by increased expression of OAS1 mRNA compared to a population of unstimulated mesenchymal stem cells.

69. 49. A population of mesenchymal stem cells treated by the method of claim 48, wherein the mesenchymal stem cells are characterized by increased expression of ISG15 mRNA compared to a population of unstimulated mesenchymal stem cells.

70. 49. A method for the treatment of a disease comprising a population of mesenchymal stem cells treated by the method of claim 48, wherein the disease is an inflammatory or autoimmune disease.

71. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is rheumatoid arthritis.

72. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is inflammatory bowel disease.

73. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is rheumatoid arthritis.

74. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is acute optic neuritis.

75. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is Krabbe disease.

76. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is diabetic retinopathy.

77. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is Crohn's disease.

78. 71. The method of claim 70, wherein the inflammatory or autoimmune disease is acute lung injury.

79. 1. A method for producing an immunologically polarized population of mesenchymal stem cells from a population of unstimulated mesenchymal stem cells, the method comprising contacting the population of unstimulated mesenchymal stem cells with a composition comprising a Toll-like receptor 4 (TLR4) ligand, erythropoietin, and oxygen or a hypoxia mimetic, wherein the immunologically polarized population of mesenchymal stem cells has pro-inflammatory characteristics characterized by the expression of pro-inflammatory mediators.

80. 80. The method of claim 79, wherein the Toll-like receptor 4 (TLR4) ligand is lipopolysaccharide (LPS).

81. 80. The method of claim 79, wherein the Toll-like receptor 4 (TLR4) ligand is an aminoalkyl glucosaminide 4-phosphate.

82. 80. The method of claim 79, wherein the erythropoietin is present at a concentration of less than 10 ng / mL.

83. 80. The method of claim 79, wherein the hypoxia mimetic is cobalt chloride.

84. 84. The method of claim 83, wherein the cobalt chloride is present at a concentration between 5 μM and 500 μM.

85. 80. The method of claim 79, wherein the composition further comprises an interferon.

86. 80. The method of claim 79, wherein the composition further comprises tumor necrosis factor alpha (TNFα).

87. 80. The method of claim 79, wherein the composition does not contain serum of human or animal origin.

88. 80. The method of claim 79, wherein the composition is a concentrated solution.

89. 80. The method of claim 79, wherein the population of unstimulated mesenchymal stem cells is simultaneously contacted with a Toll-like receptor 4 (TLR4) ligand, erythropoietin, and hypoxia or a hypoxia mimetic.

90. 80. The method of claim 79, wherein the composition is in contact with the population of unstimulated mesenchymal stem cells for at least 30 minutes but not for more than 8 hours.

91. 80. The method of claim 79, further comprising monitoring expression of TNFSF10 (TRAIL) at either the RNA or protein level.

92. 80. A population of mesenchymal stem cells treated by the method of claim 79.

93. 80. A population of human mesenchymal stem cells treated by the method of claim 79.

94. 80. A population of canine, feline, or equine mesenchymal stem cells treated by the method of claim 79.

95. 80. A population of mesenchymal stem cells treated by the method of claim 79, wherein the mesenchymal stem cells are derived from pluripotent stem cells.

96. A population of mesenchymal stem cells treated by the method of claim 79, wherein the mesenchymal stem cells are characterized by increased expression of TNFSF10 (TRAIL) mRNA compared to a population of unstimulated mesenchymal stem cells.

97. 80. A method for the treatment of a disease comprising a population of mesenchymal stem cells treated by the method of claim 79, wherein the disease is cancer.

98. 98. A method for the treatment of a disease comprising a population of mesenchymal stem cells treated by the method of claim 97, wherein the cancer is ovarian cancer.

99. 98. A method for the treatment of a disease comprising a population of mesenchymal stem cells treated by the method of claim 97, wherein the cancer is uveal melanoma.

100. 80. A method for the treatment of a disease comprising a population of mesenchymal stem cells treated by the method of claim 79, wherein the disease is a viral disease.

101. 80. A method for the treatment of a disease comprising a population of mesenchymal stem cells treated by the method of claim 79, wherein the disease is a bacterial infection.

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