BRPI0717150B1 - Uses of mesenchymal stem cells - Google Patents

Abstract

USES OF MESENCHYMAL STEM CELLS. The present invention relates to methods of treating autoimmune diseases, allergic responses, cancer, or inflammatory diseases in an animal, promoting wound healing, restoring epithelial damage, and promoting angiogenesis in an organ or tissue of an animal, by administering to the animal of mesenchymal stem cells in an effective amount.

Description

[001] The present invention was made with the support of the Government under Contract No. N66001-02-C-8068 granted by the Department of the Navy. The Government has certain rights in this invention.

[002] This application is a continuation in part of application Serial No. 11/080,298, filed March 15, 2005, which claims priority based on provisional application Serial No. 60/555,118, filed March 22, 2004, the contents of which are incorporated by reference in each aspect.

[003] This invention relates to mesenchymal stem cells. More particularly, this invention relates to novel uses for mesenchymal stem cells, including the promotion of angiogenesis in various tissues and organs, the treatment of autoimmune diseases, the treatment of allergic responses, the treatment of cancer, the treatment of diseases and inflammatory disorders, the promotion of wound healing, the treatment of inflammation, and the restoration of epithelial damage.

[004] Mesenchymal stem cells (MSCs) are multipotent stem cells that can readily differentiate into lineages that include osteoblasts, myocytes, chondrocytes, and adipocytes (Pittenger et al., Science, Vol. 284, p. 143 (1999); Haynesworth, et al., Bone, Vol. 13, p. 69 (1992); Prockop, Science, Vol. 276, p. 71 (1997)). In vitro studies have demonstrated the ability of MSCs to differentiate into muscle (Wakitani, et al., Muscle Nerve, Vol. 18, p. 1417 (1995), neuronal-like precursors (Woodbury, et al., J. Neurosci. Res ., Vol. 69, p. 908 (2002); Sanchez-Ramos, et al., Exp. Neurol., Vol. 171, p. 109 (2001)), cardiomyocytes (Toma, et al., Circulation, Vol. 105, 93 (2002); Fakuta, Artif. Organs, Vol. 25, p. 185 (2001)) and possibly other cell types. In addition, MSCs have been shown to provide effective feeder layers for hematopoietic stem cell expansion. and embryonic (Eaves, et al., Ann. N.Y. Acad. Sci., Vol. 938, p. 63 (2001); Wagers, et al., Gene Therapy, Vol. 9, p. 606 (2002)). Recent studies with a variety of animal models have shown that MSCs can be useful in restoring or regenerating injured bone, cartilage, meniscus, or myocardial tissues (DeKok, et al., Clin. Oral Implants Res., Vol. 14, p. 481 (DeKok, et al., Clin. Oral Implants Res., Vol. 14, p. 481 ( 2003)); Wu, et al. Transplantation, Vol. 75, p. 679 (2003); Noel, and others, Curr. opinion Investigation Drugs, Vol. 3, p. 1000 (2002); Ballas, et al., J. Cell. Biochem. Suppl., Vol. 38, p. 20 (2002); Mackenzie, et al., Blood Cells MoI. Dis., Vol. 27 (2002)). Several investigators have used MSCs with encouraging results for transplantation in animal models of disease, including osteogenesis imperfecta (Pereira, et al., Proc. Nat. Acad. Sci., Vol. 95, p. 1142 (1998)), parkinsonism (Schwartz, et al., Hum. Gene Ther., Vol. 10, p. 2539 (1999)), spinal cord injury (Chopp, et al., Neuroreport, Vol. 11, p. 3001 (2000); Wu, et al., J. Neurosci. Res., Vol. 72, p. 393 (2003)) and cardiac disorders (Tomita, et al., Circulation, Vol. 100, p. 247 (1999). Shake, et al. , Ann. Thorac. Surg., Vol. 73, p. 1919 (2002)). Importantly, promising results have also been reported in clinical trials for osteogenesis imperfecta (Horwitz, et al., Blood, Vol. 97, p. 1227 (2001); Horowitz, et al. Proc. Nat. Acad. Sci., Vol. 99, p.8932 (2002)) and improved grafting of heterologous bone marrow transplants (Frassoni, et al., Int. Society for Cell Therapy, SA006 (abstract) (2002); Koc, et al., J. Clin. Oncol. , Vol. 18, p. 307 (2000)).

[005] MSCs express class I major histocompatibility complex (MHC) antigen on their surface, but do not express class II MCH (Le Blanc, et al., Exp. Hematol., Vol. 31, p. 890 ( 2003); Potian, et al., J. Immunol., Vol. 171, p. 3426 (2003)) and no costimulatory B7 or CD40 molecules (Majumdar, et al., J. Biomed. Sci., Vol. 10, p. 228 (2003)), suggesting that these cells have a low immunogenic phenotype (Tse, et al., Transplantation, Vol. 75, p. 389 (2003)). MSCs also inhibit T cell proliferative responses in an MHC-independent manner (Bartholomew, et al., Exp. Hematol., Vol. 30, p. 42 (2002); Devine, et al., Cancer J., Vol. 7 , p.576 (2001); DiNicola, et al., Blood, Vol.99, p.3838 (2002)). These immunological properties of MSCs can improve your transplant engraftment and limit the ability of the recipient immune system to recognize and reject allogeneic cells after transplantation. The production of factors by MSCs, which modulate the immune response and support hematopoiesis with their ability to differentiate into appropriate cell types under local stimuli, makes them desirable stem cells for cell transplantation studies (Majumdar, et al. , Hematother, Stem Cell Res., Vol. 9, p.841 (2000); Haynesworth, et al., J. Cell. Physiol., Vol. 166, p. 585 (1996).

[006] Applicants have now examined the interactions of mesenchymal stem cells with isolated populations of immune cells, including dendritic cells (DC1 and DC2), effector T cells (Th1 and Th2), and NK cells. Based on such interactions, the applicants found that mesenchymal stem cells can regulate the production of several factors that can regulate various steps in the immune response process. Thus, mesenchymal stem cells may be employed in the treatment of disease conditions and disorders that involve the immune system, or diseases, conditions, or disorders that involve inflammation, epithelial damage, or allergic responses. Such diseases, conditions, and disorders include, but are not limited to, autoimmune diseases, allergies, arthritis, inflamed wounds, alopecia in areas (baldness), periodontal diseases, including gingivitis and periodontitis, and other diseases, conditions, or disorders that involve a immune.

[007] In addition, mesenchymal stem cells are thought to express and secrete vascular endothelial growth factor, or VEGF, which promotes angiogenesis by stimulating the formation of new blood vessels. Mesenchymal stem cells also stimulate peripheral blood mononuclear cells (PBMCs) to produce VEGF.

[008] Furthermore, mesenchymal stem cells are believed to stimulate dendritic cells (DCs) to produce Interferon-Beta (IFN-β), which promotes tumor suppression and immunity against viral infection.

[009] In accordance with one aspect of the present invention, there is provided a method of treating a disease selected from the group consisting of autoimmune diseases and graft versus host disease in an animal. The method comprises administering to the animal the mesenchymal stem cells in an amount effective to treat the disease in the animal.

[010] While the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that at least one mechanism by which mesenchymal stem cells suppress autoimmune disease and graft versus host disease is by causing the release of Interleukin-10 (IL-10) from regulatory T cells (Treg cells) and/or dendritic cells (DC).

[011] Autoimmune diseases that can be treated in accordance with the present invention include, but are not limited to, multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, uveitis, autoimmune thyroid disease, inflammatory bowel disease, scleroderma, Grave's disease , lupus, Crohn's disease, autoimmune lymphoproliferative disease (ALPS), demyelinating disease, autoimmune encephalomyelitis, autoimmune gastritis (AIG), and autoimmune glomerular diseases. Also, as noted above, graft versus host disease can be treated. It is to be understood, however, that the scope of the present invention is not to be limited to the treatment of the specific diseases mentioned herein.

[012] In one embodiment, the animal to which the mesenchymal stem cells are administered is a mammal. The mammal may be a primate, including human and non-human primates.

[013] In general, mesenchymal stem cell (MSC) therapy is based, for example, on the following sequence: collection of tissue containing the MSCs, isolation and expansion of the MSCs, and administration of the MSCs to the animal, with or without manipulation. biochemistry or genetics.

[014] The mesenchymal stem cells that are administered may be a homogeneous composition or may be a mixed cell population enriched in MSCs. Homogeneous compositions of mesenchymal stem cells can be obtained by culturing adherent marrow or periosteum cells, or mesenchymal stem cells can be identified by specific cell surface markers, which are identified with unique monoclonal antibodies. A method for obtaining a cell population enriched in mesenchymal stem cells is described, for example, in U.S. Patent. No. 5,486,359. Alternative sources for mesenchymal stem cells include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium.

[015] Mesenchymal stem cells can be administered by a variety of procedures. Mesenchymal stem cells can be administered systemically, such as by intravenous, intra-arterial, or intraperitoneal administration.

[016] Mesenchymal stem cells can be from a spectrum of sources, including autologous, allogeneic, or xenogenic.

[017] Mesenchymal stem cells are administered in an effective amount to treat an autoimmune disease or graft versus host disease in an animal. Mesenchymal stem cells can be administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The amount of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, the autoimmune disease being treated, and its proportion and severity.

[018] Mesenchymal stem cells can be administered in conjunction with an acceptable pharmaceutical carrier. For example, mesenchymal stem cells can be administered as a suspension of cells in a pharmaceutically acceptable liquid medium or a gel for injection or topical application.

[019] In accordance with another aspect of the present invention, there is provided a method of treating an inflammatory response in an animal. The method comprises administering to the animal mesenchymal stem cells in an amount effective to treat the inflammatory response in the animal.

[020] While the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells promote T cell maturation into regulatory T cells (Treg), thereby controlling inflammatory responses. . Mesenchymal stem cells are also believed to inhibit T1 helper cells (Th1 cells), thereby decreasing the expression of Interferon-y (IFN-y) in certain inflammatory reactions, such as those associated with psoriasis, for example. .

[021] In one embodiment, the inflammatory responses that can be treated are those associated with psoriasis.

[022] In another embodiment, mesenchymal stem cells can be administered to an animal in such a way that the mesenchymal stem cells contact microglia and/or astrocytes in the brain to reduce inflammation, whereby the mesenchymal stem cells contact microglia and/or astrocytes in the brain. Mesenchymal stem cells limit neurodegeneration caused by activated glial cells in diseases or disorders such as Alzheimer's Disease, Parkinson's Disease, stroke, or brain cell injuries.

[023] In yet another embodiment, mesenchymal stem cells can be administered to an animal such that the mesenchymal stem cells contact keratinocytes and Langerhans cells in the epidermis of the skin to reduce inflammation as it occurs. in psoriasis, chronic dermatitis, or contact dermatitis. Although this modality is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells can contact keratinocytes and Langerhans cells in the epidermis, and alter T cell receptor expression and cytokine secretion profiles. , resulting in decreased expression of tumor necrosis factor alpha (TNF-α) and increased population of regulatory T cells (Treg cell).

[024] In an additional embodiment, mesenchymal stem cells can be used to reduce inflammation in bone, as it occurs in arthritis and arthritis-like conditions, including but not limited to osteoarthritis and rheumatoid arthritis, and in others. arthritic diseases listed on the website www.arthritis.org/conditions/diseases. Although the scope of this modality is not intended to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells can inhibit the secretion of Interleukin-17 by memory T cells into synovial fluid.

[025] In another embodiment, mesenchymal stem cells can be used to limit inflammation in the gut and liver during inflammatory bowel disease and chronic hepatitis, respectively. While the scope of this aspect of the present invention is not intended to be limited by any theoretical reasoning, it is believed that mesenchymal stem cells promote the increased secretion of Interleukin-10 (IL-10) and the generation of regulatory T cells ( Treg).

[026] In another embodiment, mesenchymal stem cells can be used to inhibit excessive neutrophil and macrophage activation in pathological conditions such as sepsis and trauma, including burn injury, surgery, and transplants. Although the scope of this modality is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells promote the secretion of suppressor cytokines, such as IL-10, and inhibit macrophage migration inhibitory factor.

[027] In another embodiment, mesenchymal stem cells can be used to control inflammation at immune-privileged sites, such as the eye, including the cornea, lens, pigment epithelium, and retina, brain, cord. spinal cord, uterus and placenta of pregnant woman, ovary, testes, adrenal cortex, liver, and hair follicles. Although the scope of this modality is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells promote the secretion of suppressor cytokines, such as IL-10, and the generation of Treg cells.

[028] In yet another embodiment, mesenchymal stem cells can be used to treat tissue damage associated with end-stage renal disease infections (ESRDs) during dialysis and/or glomerulonephritis. Although the scope of this modality is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells can promote renal restoration. Mesenchymal stem cells also express and secrete vascular endothelial growth factor, or VEGF, which stimulates the formation of new blood vessels, which should aid in the restoration of damaged kidney tissue.

[029] In an additional embodiment, mesenchymal stem cells can be used to control viral infections such as influenza, hepatitis C, Herpes Simplex Virus, vaccinia virus, and Epstein-Barr virus infections. Although the scope of this modality is not to be limited to any theoretical reasoning, mesenchymal stem cells are believed to promote the secretion of Interferon-Beta (IFN-β).

[030] In yet another modality, mesenchymal stem cells can be used to control parasitic infections such as Leishmania infections and Helicobacter infections. Although the scope of this modality is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells mediate responses by T 2 (Th2) helper cells, and thereby promote increased production of immunoglobulin E (IgE). ) by β cells.

[031] It is to be understood, however, that the scope of this aspect of the present invention is not to be limited to the treatment of any particular inflammatory response.

[032] Mesenchymal stem cells can be administered to a mammal, including human and non-human primates, as described above.

[033] Mesenchymal stem cells can also be administered systemically as described above. Alternatively, in the case of osteoarthritis or rheumatoid arthritis, mesenchymal stem cells can be administered directly to an arthritic joint.

[034] Mesenchymal stem cells are administered in an effective amount to treat an inflammatory response in an animal. Mesenchymal stem cells can be administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The exact dosage of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, the inflammatory response being treated, and its degree and severity.

[035] Mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier as described above.

[036] In accordance with another aspect of the present invention, there is provided a method of treating inflammation and/or repairing epithelial damage in an animal. The method comprises administering the mesenchymal stem cells to the animal in an amount effective to treat inflammation and/or epithelial damage in the animal.

[037] While the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells cause a decrease in the secretion of the pro-inflammatory cytokines TNF-α and Interferon-y by T cells. and an increase in the secretion of the anti-inflammatory cytokines Interleukin-10 (IL-10) and Interleukin-4 (IL-4) by T cells. Mesenchymal stem cells are also believed to cause a decrease in the secretion of Interferon-y by natural killer (NK) cells.

[038] Inflammation and/or epithelial damage that may be treated in accordance with this aspect of the present invention includes, but is not limited to, inflammation and/or epithelial damage caused by a variety of diseases and disorders, including, but not limited to autoimmune disease, organ transplant rejection, burns, cuts, lacerations, and ulcerations, including skin ulcers and diabetic ulcerations.

[039] In one embodiment, mesenchymal stem cells are administered to an animal to repair epithelial damage resulting from autoimmune diseases, including but not limited to rheumatoid arthritis, Crohn's Disease, Type 1 diabetes, multiple sclerosis, scleroderma, Graves disease, lupus, inflammatory bowel disease, autoimmune gastritis (AGA), and autoimmune glomerular disease. Mesenchymal stem cells can also repair epithelial damage resulting from graft versus host disease (GVHD).

[040] This aspect of the present invention is particularly applicable to the restoration of epithelial damage resulting from graft-versus-host disease, and more particularly, to the restoration of epithelial damage resulting from severe graft-versus-host disease, including graft-versus-host disease of Grades III and IV affecting the skin and/or gastrointestinal system. Applicants have found, in particular, that mesenchymal stem cells, when administered to a patient suffering from severe graft-versus-host disease, and in particular Grade-vs-host disease of Grades III and IV, administration of the mesenchymal stem cells resulted in restoration of the ulcerated skin and/or intestinal epithelial tissue in the patient.

[041] In another embodiment, mesenchymal stem cells are administered to an animal to repair epithelial damage to a transplanted organ or tissue, including but not limited to kidney, heart, and lung, caused by rejection of the transplanted organ or tissue. .

[042] In yet another embodiment, mesenchymal stem cells are administered to an animal to repair epithelial damage caused by burns, cuts, lacerations, and ulcerations, including but not limited to skin ulcerations and diabetic ulcerations.

[043] Mesenchymal stem cells can be administered to a mammal, including human and non-human primates, as described above.

[044] Mesenchymal stem cells can also be administered systemically, as described above.

[045] Mesenchymal stem cells are administered in an effective amount to repair epithelial damage in an animal. Mesenchymal stem cells can be administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The exact dosage of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, the type of epithelial damage being restored, and its degree and severity.

[046] In accordance with yet another aspect of the present invention, there is provided a method of treating cancer in an animal. The method comprises administering to the animal the mesenchymal stem cells in an amount effective to treat cancer in the animal.

[047] While the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells interact with dendritic cells, which results in the secretion of IFN-β, which in turn instead, acts as a tumor suppressor. Cancers that can be treated include, but are not limited to, hepatocellular carcinoma, cervical cancer, pancreatic cancer, prostate cancer, fibrosarcoma, medulloblastoma, and astrocytoma. It is to be understood, however, that the scope of the present invention is not to be limited to any specific type of cancer.

[048] The animal may be a mammal, including human and non-human primates, as described above.

[049] Mesenchymal stem cells are administered to the animal in an effective amount to treat cancer in the animal. In general, mesenchymal stem cells are administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The exact amount of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, the type of cancer being treated, and its grade and severity.

[050] Mesenchymal stem cells are administered in conjunction with an acceptable pharmaceutical carrier, and may be administered systemically, as described above. Alternatively, mesenchymal stem cells can be administered directly to the cancer being treated.

[051] In accordance with yet another aspect of the present invention, there is provided a method of treating an allergic disease or disorder in an animal. The method comprises administering to the animal the mesenchymal stem cells in an amount effective to treat the disease or allergic disorder in the animal.

[052] While the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells, when administered following an acute allergic response, provide inhibition of mast cell activation and degranulation. Also, mesenchymal stem cells are believed to down-regulate basophil activation and inhibit cytokines such as TNF-α, chemokines such as Interleukin-8, and monocyte chemotactic protein, or MCP-1, lipid mediators, such as leukotrienes, and inhibit major mediators, such as histamine, heparin, chondroitin sulfates, and cathepsin.

[053] Allergic diseases or disorders that can be treated include, but are not limited to, asthma, allergic rhinitis, atopic dermatitis, and contact dermatitis. It is to be understood, however, that the scope of the present invention is not to be limited to any specific allergic disease or disorder.

[054] Mesenchymal stem cells are administered to the animal in an amount effective to treat the disease or allergic disorder in the animal. The animal may be a mammal. The mammal may be a primate, including human and non-human primates. In general, mesenchymal stem cells are administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The exact dosage is dependent on a variety of factors, including the age, weight, and sex of the patient, the disease or allergic disorder being treated, and its degree and severity.

[055] Mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier as described above. Mesenchymal stem cells can be administered systemically, such as by intravenous or intraarterial administration, for example.

[056] In accordance with a further aspect of the present invention, there is provided a method of promoting wound healing in an animal. The method comprises administering to the animal the mesenchymal stem cells in an amount effective to promote wound healing in the animal.

[057] While the scope of the present invention is not to be limited to any theoretical reasoning, it is believed that, as mentioned above, mesenchymal stem cells cause Treg cells and dendritic cells to release Interleukin-10 (IL -10). IL-10 limits or controls inflammation in a wound, thereby promoting wound healing.

[058] In addition, mesenchymal stem cells can promote wound healing and fracture healing by inducing secretion factors by other cell types. For example, mesenchymal stem cells can induce prostaglandin E2 (PGE2)-mediated release of vascular endothelial growth factor (VEGF) from peripheral blood mononuclear cells (PBMCs), as well as PGE2-mediated release of growth hormone. , insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-3 binding protein (IGFBP-3), and endothelin-1.

[059] Wounds that can be healed include, but are not limited to, those resulting from cuts, lacerations, burns, and skin ulcerations.

[060] Mesenchymal stem cells are administered to the animal in an amount effective to promote wound healing in the animal. The animal can be a mammal, and the mammal can be a primate, including human and non-human primates. In general, mesenchymal stem cells are administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The exact amount of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, and the degree and severity of the wound being treated.

[061] Mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier as described above. Mesenchymal stem cells can be administered systemically, as described above. Alternatively, mesenchymal stem cells can be administered directly to a wound, such as in a fluid over a dressing or reservoir containing the mesenchymal stem cells.

[062] In accordance with yet another aspect of the present invention, there is provided a method of treating or preventing fibrosis in an animal. The method comprises administering to the animal the mesenchymal stem cells in an amount effective to treat or prevent fibrosis in an animal.

[063] Mesenchymal stem cells can be administered to the animal to treat or prevent any type of fibrosis in the animal, including but not limited to cirrhosis of the liver, fibrosis of the kidneys associated with end-stage kidney disease, and fibrosis of the lungs. , including but not limited to Acute Respiratory Diseases (ARDS) and Obstructive Pulmonary Disease (COPD). It is to be understood that the scope of the present invention is not to be limited to any specific type of fibrosis.

[064] Mesenchymal stem cells are administered to the animal in an amount effective to treat or prevent fibrosis in the animal. The animal can be a mammal, and the mammal can be a primate, including human and non-human primates. In general, mesenchymal stem cells are administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The exact amount of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, and the degree and severity of fibrosis being treated or prevented.

[065] Mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier as described above. Mesenchymal stem cells can be administered systemically, also as described above.

[066] It is another object of the present invention to promote angiogenesis in a tissue or organ of an animal, where such tissue or organ is in need of angiogenesis.

[067] Thus, in accordance with a further aspect of the present invention, there is provided a method of promoting angiogenesis in an organ or tissue of an animal. The method comprises administering to the animal the mesenchymal stem cells in an amount effective to promote angiogenesis in an organ or tissue of the animal.

[068] Angiogenesis is the formation of new blood vessels from a preexisting microvascular bed.

[069] Induction of angiogenesis can be used to treat coronary and peripheral artery insufficiency, and thus, may be a non-invasive and curative approach to the treatment of coronary artery disease, ischemic heart disease, and peripheral artery disease. Angiogenesis may play a role in the treatment of diseases and disorders in tissue and organs other than the heart, as well as in the development and/or maintenance of organs other than the heart. Angiogenesis may provide a role in the treatment of internal and external wounds as well as dermal ulcers. Angiogenesis also plays a role in embryo implantation, and placental growth, as well as in the development of embryonic vasculature. Angiogenesis is also essential for linking cartilage resorption with bone formation, and is essential for correct growth plate morphogenesis.

[070] Additionally, angiogenesis is required for the successful engineering and maintenance of highly metabolic organs such as the liver, where a dense vascular network is required to provide sufficient nutrient and gas transport.

[071] Mesenchymal stem cells can be delivered to the tissue or organ that is in need of angiogenesis by a variety of procedures. Mesenchymal stem cells can be administered systemically, such as by intravenous, intra-arterial, or intraperitoneal administration, or mesenchymal stem cells can be administered directly to the tissue or organ that is in need of angiogenesis, such as by direct injection. in the tissue or organ that required angiogenesis.

[072] Mesenchymal stem cells can be from a spectrum of sources, including autologous, allogeneic, or xenogenic.

[073] While the scope of the present invention is not to be limited to any theoretical reasoning, it is believed that mesenchymal stem cells, when administered to an animal, stimulate peripheral blood mononuclear cells (PBMCs) to produce growth factor. vascular endothelial tissue, or VEGF, which stimulates the formation of new blood vessels.

[074] In one embodiment, the animal is a mammal. The mammal may be a primate, including human and non-human primates.

[075] Mesenchymal stem cells, in accordance with the present invention, may be employed in the treatment, alleviation, or prevention of any disease or disorder that can be alleviated, treated, or prevented through angiogenesis. Thus, for example, mesenchymal stem cells can be administered to an animal to treat blocked arteries, including those in the extremities, i.e., arms, legs, hands, and feet, as well as in the neck or in various organs. For example, mesenchymal stem cells can be used to treat blocked arteries that supply the brain, thereby treating or preventing stroke. Also, mesenchymal stem cells can be used to treat blood vessels in the embryonic and postnatal corneas and can be used to provide glomerular structuring. In another embodiment, mesenchymal stem cells can be used to treat wounds, both internal and external, as well as in the treatment of dermal ulcers found on the feet, hands, legs, or arms, including but not limited to dermal ulcers caused by diseases such as diabetes and sickle cell anemia.

[076] In addition, because angiogenesis is involved in embryo implantation in placenta formation, mesenchymal stem cells can be employed to promote embryo implantation and prevent miscarriage.

[077] Furthermore, mesenchymal stem cells can be administered to an unborn animal, including humans, to promote vasculature development in the unborn animal.

[078] In another embodiment, mesenchymal stem cells can be administered to an animal, born or not, to promote cartilage resorption and bone formation, as well as promote correct epiphyseal plate morphogenesis.

[079] Mesenchymal stem cells are administered in an amount effective in promoting angiogenesis in an animal. Mesenchymal stem cells can be administered in an amount from about 1 x 10 5 cells/kg to about 1 x 10 7 cells/kg. In another embodiment, the mesenchymal stem cells are administered in an amount from about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg. The amount of mesenchymal stem cells to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient, the disease or disorder being treated, alleviated, or prevented, and its degree and severity. .

[080] Mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier. For example, mesenchymal stem cells can be administered as a suspension of cells in a pharmaceutically acceptable liquid medium for injection. The injection can be local, that is, directly into the tissue or organ in need of angiogenesis, or systemic.

[081] Mesenchymal stem cells can be genetically engineered with one or more polynucleotides encoding a therapeutic agent. Polynucleotides can be delivered to mesenchymal stem cells via an appropriate expression vehicle. Expression vehicles that can be employed to genetically engineer mesenchymal stem cells include, but are not limited to, retroviral vectors, adenoviral vectors, and adeno-associated virus vectors.

[082] The selection of an appropriate polynucleotide encoding a therapeutic agent is dependent on several factors, including the disease or disorder being treated, and its degree and severity. Polynucleotides encoding therapeutic agents, and appropriate expression vehicles are further described in U.S. Patent. No. 6,355,239.

[083] It is to be understood that mesenchymal stem cells, when employed in the aforementioned therapies and treatments, may be employed in combination with other therapeutic agents known to those skilled in the art, including, but not limited to, growth factors, cytokines, drugs, such as anti-inflammatory drugs, and cells other than mesenchymal stem cells, such as dendritic cells, and can be administered with cell-soluble carriers, such as hyaluronic acid, or in combination with solid matrices, such as collagen, gelatin, or other biocompatible polymers, as appropriate.

[084] It is to be understood that the methods described herein may be carried out in various modes and with various modifications and permutations thereof that are well known in the art. It can also be appreciated that any theories described as to modes of action or interactions between cell types should not be construed as limiting this invention in any way, but are presented in such a way that the methods of the invention can be more fully understood.

[085] The invention will now be described in relation to the drawings, in which:

[086] Figure 1 MSCs modulate dendritic cell functions. (A) Flow cytometric analysis of mature DC1 monocytic cells using antibodies against HLA-DR and CD11c and of plasmacytoid DC2 cells using antibodies against HLA-DR and CD123 (IL-3 receptor). (—): isotype control; (—): FITC/PE conjugated antibodies. (B) MSCs inhibit TNF-α secretion (primary y axis) and increase IL-10 secretion (secondary y axis) from activated DC1 and DC2, respectively. (C) MSCs cultured with mature DC1 cells inhibit IFN-γ secretion (primary y-axis) by T cells and increase IL-4 levels (secondary y-axis) compared to MSC or DC alone. Decreased production of pro-inflammatory IFN-γ and increased production of anti-inflammatory IL-4 in the presence of MSCs indicated a shift in the T cell population towards an anti-inflammatory phenotype.

[087] Figure 2 MSCs inhibit the function of pro-inflammatory effector T cells. (A) Flow cytometric analysis of TReg cell numbers (in %) by staining PBMCs or non-adherent fraction in MSC+PBMC culture (MSC+PBMC) with FITC-conjugated antibodies to CD4 (x-axis) and for PE-conjugated CD25 (y-axis). The ports are adjusted based on the isotype control antibodies as background. The graphs are representative of 5 independent experiments. (B) TH1 cells generated in the presence of MSCs secreted reduced levels of IFN-y (primary y axis) and TH2 cells generated in the presence of MSCs secreted increased amounts of IL-4 (secondary y axis) in cell culture supernatants . (C) MSCs inhibit IFN-γ secretion from purified NK cells cultured for 0, 24, or 48 hours in a 24-well plate. Data shown are the mean ± SD of cytokine secretion in one experiment and are representative of 3 independent experiments.

[088] Figure 3 MSCs result in increased numbers of Treg cell population and increased expression of GITR. (A) A population of CD4+ CD25+ Treg cells from cultures of PBMC or MSC + PBMC (ratio of MSC to PBMC 1:10) (cultured without any further stimulation for 3 days) was isolated using a magnetic isolation procedure from 2 steps. These cells were irradiated (to block any further proliferation) and used as stimulators in a mixed lymphocyte reaction (MLR) where the responders were allogeneic PBMCs (stimulator to responder ratio 1:100) in the presence of phytohaemagglutinin (PHA) ( 2.5 mg/ml). Cells were cultured for 48 hours, after which 3H thymidine was added, and incorporated radioactivity was counted after 24 hours. The results showed that the population of Treg generated in the presence of MSCs (path 3) was functionally similar to the Treg cells generated in the absence of MSCs (path 2). (B) PBMCs were cultured for 3 days in the absence (upper graph) or presence (lower graph) of MSCs (MSC to PBMC ratio of 1:10), after which the non-adherent fraction was collected and immunostained with FITC-labeled GITR and PE-labeled CD4. The results show a greater than two-fold increase in GITR expression in cells cultured in the presence of MSCs.

[089] Figure 4 MSCs produce PGE2 and blocking PGE2 reverses the immunomodulatory effects mediated by MSCs. (A) PGE2 secretion (mean ± SD) in culture supernatants obtained from MSCs cultured in the presence or absence of the PGE2 blockers NS-398 or indomethacin (Indomet.) at various concentrations. Inhibitor concentrations are in μM and the data presented are values obtained after 24 hours of culture. (B) Expression of COX-1 and COX-2 in MSCs and PBMCs using real-time RT-PCR. MSCs expressed significantly higher levels of COX-2 compared to PBMCs, and when MSCs were cultured in the presence of PBMCs, there was a >3-fold increase in COX-2 expression in MSCs. Representative data from 1 of 3 independent experiments are shown. The MSC+PBMC cultures were fixed in a trans-well chamber plate where the MSCs were plated over the lower chamber and the PBMCs over the upper chamber. (C) The presence of the PGE2 blockers indomethacin (Ind.) or NS-398 increases TNF-α secretion from activated DCs (□) and IFN-γ secretion from TH1 cells (■) compared to with the controls. Data were calculated as % change from cultures generated in the absence of MSCs and PGE2 inhibitors. (C) The presence of the PGE2 blockers indomethacin (Indo) and NS-398 during MSC-PBMC co-culture (1:10) reverses the antiproliferative effects mediated by MSCs on PHA-treated PBMCs. The data shown is from one experiment and is representative of 3 independent experiments.

[090] Figure 5 Cytokine secretion in constitutive MSCs is elevated in the presence of allogeneic PBMCs. Using previously characterized human MSCs, we analyzed the levels of the cytokines IL-6 and VEGF, lipid mediator PGE2, and matrix metalloproteinase 1 (pro-MMP-1) in the culture supernatant of MSCs cultured for 24 hours in the presence ( hatched bars) or in the absence (open bars) of PBMCs (MSC to PBMC ratio of 1:10). MSCs produced IL-6, VEGF, and PGE2 constitutively, and levels of these factors increased in co-culture with PBMCs, thereby suggesting that MSCs may play a role in modulating immune functions in an inflammatory indication.

[091] Figure 6 MSCs inhibit mitogen-induced T cell proliferation in a dose-dependent manner. Increasing numbers of allogeneic PBMCs were incubated with constant numbers of MSCs (2,000 cells/well) prepared on a 96-well plate, in the presence or absence of PHA (2.5 mg/mL), for 72 hours, and incorporation of 3H thymidine determined (in counts per minute, or cpm). There was a dose-dependent inhibition of proliferation of PHA-treated PBMCs in the presence of MSCs. Representative results from 1 of 3 independent experiments are shown. Similar results have been reported by LeBlanc, et al., Scand J. Immunol., Vol. 57, p. 11 (2003). Figure 7 Schematic diagram of the proposed mechanism of action of MSCs

[092] MSCs mediate their immunomodulatory effects by affecting cells of both innate (DCs - pathways 2-4; and NK - pathway 6) and adaptive (T - pathways 1 and 5 and B - pathway 7) immune systems. In response to an invading pathogen, immature DCs migrate to the potential entry site, mature, and acquire an ability to provide simple T cells (through antigen-specific and costimulatory signals) to become protective effector T cells (immunity). cell-mediated TH1 or humoral TH2). During the interaction between MSC-DC, MSCs, through direct cell-cell contact or via a secreted factor, can alter the outcome of the immune response by limiting the ability of DCs to organize a cell-mediated response (pathway 2) or by promoting the ability to organize a humoral response (pathway 4). Also, when mature effector T cells are present, MSCs can interact with them to distort the balance of TH1 responses (pathway 1) to TH2 responses (pathway 5), and probably to an IgE-producing B cell activity. increased (lane 7), desirable results for GvHD suppression and symptoms of autoimmune diseases. MSCs in their ability to result in an increased generation of TReg population (pathway 3) may result in a tolerant phenotype and may assist a recipient host by weakening the surrounding inflammation in its local microenvironment. The dashed line ( ) represents the proposed mechanism.

[093] The invention will now be described in relation to the following examples; it is to be understood, however, that the scope of the present invention is not to be limited thereto.

Example 1 Materials and methods Culture of human MSCs

[094] Human MSCs were cultured as described by Pittenger et al., Science, Vol. 284, p. 143 (1999). Briefly, marrow samples were collected from the iliac crest of anonymous donors after informed consent by Poietics Technologies, Div of Cambrex Biosciences. MSCs were cultured in Dulbecco's Modified Eagle's Medium Complete-Low Glucose (Life Technologies, Carlsbad, Calif.) containing 1% antibiotic-antimiotic solution (Invitrogen, Carlsbad, Calif.) and 10% fetal bovine serum (FBS). , JRH BioSciences, Lenexa, Kansas). MSCs grew as an adherent monolayer and were removed with trypsin/EDTA (0.05% trypsin at 37°C for 3 minutes). All MSCs used were previously characterized for the potential for multiple lineages and maintained the ability to differentiate into mesenchymal lineages (chondrocytic, adipogenic, and osteogenic) (Pittenger, et al., Science, Vol. 284, p. 143 (1999)) . Isolation of Dendritic Cells

[095] Peripheral blood mononuclear cells (PBMCs) were obtained from Poietics Technologies, Div of Cambrex Biosciences (Walkersville, MD). Dendritic cell (DC) precursors of the monocytic lineage (CD1c+) were positively selected from the PBMCs, using a 2-step magnetic separation method, according to Dzionek, et al., J. Immunol., Vol. 165, p. 6037 (2000). Briefly, CD1c-expressing B cells were magnetically depleted from CD19+ cells using magnetic globules, followed by labeling the depleted fraction of B cells with biotin-labeled CD1c (BDCA1+) and antibiotin antibodies and their separation from the unlabeled cell fraction using magnetic columns. , according to the manufacturer's instructions (Miltenyi Biotech, Auburn, California). Plasmacytoid lineage DC precursors were isolated from PBMCs by immunomagnetic screening of cells coated with positively labeled antibodies (BDCA2+) (Miltenyi Biotech, Auburn, California).

MSC-DC Culture

[096] In most experiments, human MSCs and DCs were cultured in equal numbers for various periods of time and the cell culture supernatant collected and stored at -80°C until further evaluation. In selected experiments, MSCs were cultured with mature DC1 or DC2 cells (MSC:DC ratio 1:1) for 3 days, and then the combined cultures (MSCs and DCs) were irradiated to prevent any proliferation. Next, antibody-purified allogeneic, single T cells (CD4+, CD45RA+) were added to the irradiated MSCs/DCs and cultured for an additional 6 days. The non-adherent cell fraction (purified T cells) was then collected from the cultures, washed twice and stimulated again with PHA for another 24 hours, after which the cell culture supernatants were collected and analyzed for IFN-y and IL-4 secreted by ELISA.

Isolation of NK cells

[097] Purified populations of NK cells were obtained by depleting non-NK cells that are magnetically labeled with a cocktail of biotin-conjugated monoclonal antibodies (anti-CD3, -CD14, -CD19, -CD36 and anti-IgE antibodies), as a primary reagent, and antibiotin monoclonal antibodies conjugated to the microspheres, as a secondary labeling reagent. Magnetically labeled non-NK cells were retained on MACS columns (Miltenyi Biotech, Auburn, California) in a magnetic field, while NK cells crossed over and were collected.

Isolation of the population of TReg cells

[098] The TReg cell population was isolated using a 2-step isolation procedure. First, T cells that were not CD4+ were indirectly magnetically labeled with a cocktail of biotin-labeled antibodies and antibiotin microspheres. The labeled cells were then depleted by sorting over a MACS column (Miltenyi Biotech, Auburn, California). Next, CD4+CD25+ cells were directly labeled with CD25 microspheres and isolated by positive selection from the pre-enriched CD4+ T cell fraction. Magnetically labeled CD4+CD25+ T cells were retained on the column and eluted after removing the column from the magnetic field.

[099] To determine whether the increased population of CD4+CD25+ generated in the presence of the MSCs was suppressive in nature, populations of CD4+CD25+ Treg cells were isolated from PBMC or MSC+PBMC cultures (MSC to PBMC ratio of 1: 10) (cultured without any further stimulation, for 3 days) using a 2-step magnetic isolation procedure. These cells were irradiated to block any proliferation and used as stimulators in a mixed lymphocyte reaction (MLR), where the responders were allogeneic PBMCs (stimulator to responder ratio 1:100) in the presence of PHA (2.5 μg/ mL). The culture was carried out for 48 hours, after which 3H thymidine was added. Incorporated radioactivity was counted after 24 hours.

[0100] PBMCs were cultured in the absence or presence of MSCs (MSC to PBMC ratio of 1:10), after which the non-adherent fraction was collected and immunostained with FITC-labeled glucocorticoid-induced TNF receptor, or GITR, and PE-labeled CD4.

Generation of TH1/TH2 cells

[0101] Peripheral blood mononuclear cells (PBMCs) were plated at 2 x 10 6 cells/mL for 45 min at 37°C to remove monocytes. The non-adherent fraction was incubated in the presence of anti-CD3 (5 μg/mL) and anti-CD28 (1 μg/mL) antibodies bound to the plates, under TH1 conditions (IL-2 (4 ng/mL) + IL -12 (5 ng/mL) + anti-IL-4 (1 μg/mL)) or TH2 (IL-2 (4 ng/mL) + IL-4 (4 ng/mL) + anti-IFN-γ ( 1 μg/mL)) for 3 days, in the presence or absence of MSCs. Cells were washed and then restimulated with PHA (2.5 μg/mL) for another 24 or 48 hours, after which IFN-γ and IL-4 levels were measured in culture supernatants by ELISA (R&D Systems, Minneapolis, Minnesota). Analysis of VEGF, PGE2, and pro-MMP-1 levels in the culture supernatant of MSCs.

[0102] Using the previously characterized human MSCs, the levels of Interleukin-6 (IL-6), VEGF, lipid mediator prostaglandin E2 (PGE2), and matrix metalloproteinase 1 (pro-MMP-1) were analyzed in the supernatant of culture of MSCs grown for 24 hours, in the presence or absence of PBMCs (MSC to PBMC ratio of 1:10).

Proliferation of PBMCs

[0103] Purified PBMCs were prepared by centrifuging the leukopack (Cambrex, Walkersville, Maryland) over Ficoll-Hypaque (Lymphoprep, Oslo, Norway). Separated cells were cultured (in triplicates) in the presence or absence of MSCs (plated 3-4 hours before the addition of PBMCs to allow them to settle) for 48 hours in the presence of PHA mitogen (Sigma Chemicals, St. . Louis, Missouri). In selected experiments, PBMCs were resuspended in medium containing the PGE2 inhibitors Indomethacin (Sigma Chemicals, St. Louis, Missouri) or NS-938 (Cayman Chemicals, Ann Arbor, Michigan). (3H)-thymidine was added (20 μl in a 200 μl culture) and the cells collected after an additional 24 h cultivation using an automatic harvester. The effects of MSCs or PGE2 blockers were calculated as the percentage of control response (100%) in the presence of PHA.

Quantitative RT-PCR

[0104] Total RNA from the cell precipitates was prepared using a commercially available kit (Qiagen, Valencia, California) and according to the manufacturer's instructions. Contaminant genomic DNA was removed using the DNA-free kit (Ambion, Austin, Texas). Quantitative RT-PCR was performed on an MJ Research Opticon detection system (South San Francisco, California) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, California) with primers at 0.5 µM concentration. Relative changes in expression levels in cells grown under different conditions were calculated by the difference in Ct values (cross point) using β-actin as an internal control. The sequences for the COX-1 and COX-2 specific primers were: COX-1: 5'-CCG GAT GCC AGT CAG GAT GAT G-3' below, 5'-CTA GAC AGC CAG ATG CTG ACA G-3' previous; COX-2: 5'-ATC TAC CCT CCT CAA GTC CC-3' below, 5'-TAC CAG AAG GGC AGG ATA CAG-3' above.

[0105] The increasing numbers of allogeneic PBMCs were incubated with the constant numbers of MSCs (2,000 cells/well) prepared on a 96-well plate, in the presence of PHA (2.5 μg/mL), for 72 hours, and the 3 H thymidine incorporation (counts per minute, cpm) was determined. PBMCs and MSCs were cultured at MSC:PBMC ratios of 1:1, 1:3, 1:10, 1:30, and 1:81.

Results

[0106] In the present studies, we examined the interaction of human MSCs with isolated populations of immune cells, including dendritic cells (DC1 and DC2), effector T cells (TH1 and TH2) and NK cells. The interaction of MSCs with each type of immune cell had specific consequences, suggesting that MSCs can modulate several steps in the immune response process. The production of secreted factor(s) that modulate(m) and may be responsible for the immunomodulatory effects of MSCs was evaluated and prostaglandin synthesis was involved.

[0107] Myeloid precursor (DC1) and plasmacytoid (DC2) dendritic cells were isolated by immunomagnetic screening of BDCA1+ and BDCA2+ cells, respectively, and matured by incubation with GM-CSF and IL-4 (1 x 103 IU/mL and 1 x 10 3 IU/ml, respectively) for DC1 cells, or IL-3 (10 ng/ml) for DC2 cells. Using flow cytometry, DC1 cells were HLA-DR+ and CD11c+, while DC2 cells were HLA-DR+ and CD123+ (Figure 1A). In the presence of the inflammatory agent bacterial lipopolysaccharide (LPS, 1 ng/mL), DC1 cells produced moderate levels of TNF-α, however, when MSCs were present (ratios examined 1:1 and 1:10), there was a reduction of >50% in TNF-α secretion (Figure 1B). On the other hand, DC2 cells produced IL-10 in the presence of LPS and its levels were increased more than 2-fold in MSC:DC2 co-culture (1:1) (Figure 1B). Therefore, MSCs modified the cytokine profile of activated DCs in culture to a more tolerogenic phenotype. Additionally, activated DCs, when cultured with MSCs, were able to reduce IFN-γ and increase IL-4 levels secreted by CD4+ single T cells (figure 1C), suggesting an MSC-mediated change in T cell phenotype pro-inflammatory to anti-inflammatory.

[0108] As increased secretion of IL-10 plays a role in the generation of regulatory cells (Kingsley, et al., J. Immunol., Vol. 168, p. 1080 (2002 )), regulatory T cells (TReg) have been quantified by flow cytometry in cocultures of PBMCs and MSCs. In culturing the PBMCs with the MSCs for 3-5 days, there was an increase in the numbers of TReg cells as determined by staining the PBMCs with the anti-CD4 and anti-CD25 antibodies (Figure 2A), additionally supporting an MSC-induced tolerogenic response. . The CD4+CD25+ TReg cell population, generated in the presence of MSCs, expressed increased levels of glucocorticoid-induced TNF receptor (GITR), a cell surface receptor expressed on TReg cell populations, and was suppressive in nature as it suppressed allogeneic T cell proliferation (Figure 3A,B). Next, MSCs were investigated for their direct ability to affect T cell differentiation. Using antibody-selected purified T cells (CD4+ Th cells), IFN-y-producing TH1 and IL-4-producing TH2 cells were analyzed. generated in the presence or absence of MSCs. When MSCs were present during differentiation, there was reduced secretion of IFN-γ by TH1 cells and increased secretion of IL-4 by TH2 cells (Figure 2B). No significant change in IFN-γ and IL-4 levels was seen when MSCs were added to the culture after Th cells had differentiated (within 3 days) into effector TH1 or TH2 types (data not shown). These experiments suggest that MSCs can affect effector T cell differentiation directly and alter cytokine secretion by T cells to a humoral phenotype.

[0109] Similarly, when MSCs were cultured with purified NK cells (CD3-, CD14-, CD19-, CD36-) in a 1:1 ratio for different time periods (0-48 h), there was a secretion decreased IFN-γ in the culture supernatant (Figure 2C), thus suggesting that MSCs can modulate NK cell functions as well.

[0110] Previous work had indicated that MSCs modify T cell functions by soluble factor(s) (LeBlanc, et al., Exp. Hematol., Vol. 31, p. 890 (2003); Tse, et al., Transplantation, Vol. 75, p. 389 (2003)). MSCs were observed to secrete several factors, including IL-6, prostaglandin E2, VEGF, and proMMP-1 constitutively, and levels of each increased in culture with PBMCs (Figure 5). To investigate the factors derived from MSCs that result in the inhibition of TNF-α and the increase in IL-10 production by DCs, the potential role of prostaglandin E2 was investigated, as it had been shown to inhibit the production of TNF-α by the DCs. Activated DCs (Vassiliou, et al., Cell. Immunol., Vol. 223, p. 120 (2003)). MSC culture conditioned media (24 hour culture of 0.5 x 10 6 cells/ml) contained approximately 1000 pg/ml PGE2 (Figure 4A). There was no detectable presence of known inducers of PGE2 secretion, e.g., TNF-α, IFN-γ or IL-1β (data not shown) in the culture supernatant, indicating a constitutive secretion of PGE2 by MSCs. PGE2 secretion by hMSCs was inhibited 60-90% in the presence of the known inhibitors of PGE2 production, NS-398 (5 μM) and indomethacin (4 μM) (Figure 4A). As the release of PGE2 secretion occurs, as a result of the enzymatic activity of the constitutively active cyclooxygenase 1 (COX-1) enzyme and the inducible cyclooxygenase 2 (COX-2) enzyme (Harris, et al., Trends Immunol., Vol. 23, p 144 (2002)), mRNA expression for COX-1 and COX-2 in MSCs and PBMCs was analyzed using the transcavity culture system. MSCs expressed significantly higher levels of COX-2 compared to PBMCs and expression levels increase > 3-fold in co-culture of MSCs and PBMCs (ratio of MSC to PBMC 1:10) for 24 hours (Figure 4B). Modest changes were seen in COX-1 levels, suggesting that the increase in PGE2 secretion in the MSC-PBMC coculture (Figure 5) is mediated by COX-2 upregulation. To investigate whether the immunomodulatory effects of MSC on DCs and T cells were mediated by PGE2, MSCs were cultured with activated dendritic cells (DC1) or TH1 cells, in the presence of PGE2 inhibitors NS-398 or indomethacin. The presence of NS-398 or indomethacin increased TNF-α secretion by DC1s and IFN-y secretion from TH1 cells (figure 4C), respectively, suggesting that the effects of MSCs on immune cell types may be mediated by secreted PGE2. Recent studies have shown that MSCs inhibit T cell proliferation induced by various stimuli (DeNicola, et al., Blood, Vol. 99, p. 3838 (2002); LeBlanc, et al., Scand. J. Immunol., Vol. 57, p.11 (2003)). MSCs were observed to inhibit mitogen-induced T cell proliferation in a dose-dependent manner (Figure 6) and when PGE2 inhibitors NS-398 (5 μM) or indomethacin (4 μM) were present, there was a >70% increase in (3H) thymidine incorporation by PHA-treated PBMCs in cultures containing MSCs, compared to controls without the inhibitors (Figure 4D).

[0111] In summary, a model of interaction of the MSC with other types of immune cells is proposed (figure 7). When mature T cells are present, MSCs can interact with them directly and inhibit the production of pro-inflammatory IFN-y (pathway 1) and promote the phenotype of regulatory T cells (pathway 3) and anti-inflammatory TH2 cells ( route 5). Furthermore, MSCs can alter the outcome of the T cell immune response through DCs by secreting PGE2, inhibiting pro-inflammatory DC1 cells (pathway 2) and promoting anti-inflammatory DC2 cells (pathway 4) or regulatory DCs (pathway 2). route 3). A shift to TH2 immunity, in turn, suggests a shift in B cell activity towards increased generation of antibodies of the IgE/IgG1 subtypes (pathway 7). MSCs, by their ability to inhibit IFN-γ secretion from NK cells, probably modify NK cell function (pathway 6). This model of interactions between MSCs:Immune cells is consistent with experimentation performed in several other laboratories (LeBlanc, et al., Exp. Hematol., Vol. 31, p. 890 (2003); Tse, et al., Transplantation, Vol. 75, p.389 (2003); DiNicola, et al., Blood, Vol.99, p.3838 (2002)). Further examination of the proposed mechanisms is ongoing and animal studies are now needed to examine the in vivo effects of MSC administration.

Example 2

[0112] Mesenchymal stem cells were given to a 33-year-old female patient suffering from Grade IV gastrointestinal graft versus host disease (GVHD). The patient was resistant to all other GVHD treatments. Endoscopic examination of the patient's colon showed areas of ulceration and inflammation prior to treatment. Histology of the patient's colon showed that graft versus host disease had destroyed the vast majority of the patient's intestinal crypts prior to treatment.

[0113] The patient received an intravenous infusion of allogeneic mesenchymal stem cells in 50 mL of Plasma Lyte A, in an amount of 3 X 106 cells per kilogram of body weight.

[0114] The patient was evaluated at two weeks after the infusion. In the two weeks after the infusion, an endoscopic examination of the patient's colon showed that the areas of inflammation and ulceration, visible before treatment, had resolved. In addition, a biopsy of the patient's colon showed significant regeneration of the intestinal crypts. Thus, the administration of the mesenchymal stem cells to the patient resulted in a significant reduction in the inflammatory component of gastrointestinal graft versus host disease, and resulted in the regeneration of new functional intestinal tissue.

[0115] The descriptions of all patents, publications, including published patent applications, depositary accession numbers, and database accession numbers are hereby incorporated by reference in the same proportion as if each patent, publication , depositary accession number, and database accession number were specifically and individually incorporated by reference.

[0116] It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced in a different manner as particularly described and still be within the scope of the accompanying claims.

Claims (22)

1. Use of mesenchymal stem cells, characterized in that it is for the preparation of a combination/composition/drug to repair epithelial damage and modulate an immune response in a human suffering from an autoimmune or inflammatory disease. 2. Use according to claim 1, characterized in that the repair comprises repairing injured intestinal crypts in a human in need of it. 3. Use according to claim 2, characterized in that said intestinal crypts have been damaged as a result of graft versus host disease. 4. Use according to claim 3, characterized in that said graft-versus-host disease is Grade IV gastrointestinal graft-versus-host disease. 5. Use according to claim 2, characterized in that said intestinal crypts have been damaged as a result of an autoimmune disease. 6. Use according to claim 5, characterized in that said autoimmune disease is selected from the group consisting essentially of rheumatoid arthritis, Type 1 diabetes, multiple sclerosis, scleroderma, Grave's Disease, lupus, autoimmune gastritis, and autoimmune glomerular disease. 7. Use according to claim 5, characterized in that said autoimmune disease is Crohn's disease. 8. Use according to claim 5, characterized in that said autoimmune disease is inflammatory bowel disease. 9. Use according to claim 2, characterized in that said damaged intestinal crypts comprise ulcerated intestinal epithelial tissue. 10. Use according to claim 2, characterized in that mesenchymal stem cells promote increased secretion of anti-inflammatory cytokines. 11. Use according to claim 10, characterized in that the anti-inflammatory cytokines are interleukin-4 (IL-4) and interleukin-10 (IL-10). 12. Use according to claim 2, characterized by the fact that mesenchymal stem cells promote production or decreased secretion of pro-inflammatory cytokines. 13. Use according to claim 12, characterized in that the pro-inflammatory cytokines are IFN-Y, IL-17 and TNF-alpha. 14. Use according to claim 2, characterized in that the mesenchymal stem cells are not genetically manipulated. 15. Use according to claim 1, characterized in that said animal is suffering from graft versus host disease. 16. Use according to claim 1, characterized in that said autoimmune disease is selected from the group consisting essentially of rheumatoid arthritis, Type 1 diabetes, multiple sclerosis, scleroderma, Grave's Disease, lupus, autoimmune gastritis, and autoimmune glomerular disease. 17. Use according to claim 1, characterized in that said autoimmune disease is Crohn's disease. 18. Use according to claim 1, characterized in that said autoimmune disease is inflammatory bowel disease. 19. Use according to claim 1, characterized in that said graft-versus-host disease is Grade IV gastrointestinal graft-versus-host disease. 20. Use according to claim 1, characterized in that the mesenchymal stem cells are genetically unmanipulated. 21. Use according to claim 1, characterized in that mesenchymal stem cells promote increased secretion of anti-inflammatory cytokines. 22. Use according to claim 1, characterized by the fact that mesenchymal stem cells promote production or decreased secretion of pro-inflammatory cytokines.

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