Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells

Definitions

• This invention relates to mesenchymal stem cells. More particularly, this invention relates to novel uses for mesenchymal stem cells, including promoting angiogenesis in various tissues and organs, treating autoimmune diseases, treating allergic responses, treating cancer, treating inflammatory diseases and disorders, promoting would healing, treating inflammation, and repairing epithelial damage.
• MSCs Mesenchymal stem cells
• adipocytes a multipotent stem cells that can differentiate readily into lineages including osteoblasts, myocytes, chondrocytes, and adipocytes
• MSCs Mesenchymal stem cells
• In vitro studies have demonstrated the capability of MSCs to differentiate into muscle (Wakitani, et al., Muscle Nerve, vol. 18,, pg. 1417 (1995)), neuronal-like precursors (Woodbury, et al., J.
• MSCs have been shown to provide effective feeder layers for expansion of hematopoietic and embryonic stem cells (Eaves, et al., Ann. N. Y. Acad. Sci.. vol. 938, pg.
• MSCs may be useful in the repair or regeneration of damaged bone, cartilage, meniscus or myocardial tissues (DeKok, et al., Clin. Oral Implants Res., vol. 14, pg. 481 (2003)); Wu, et al., Transplantation, vol. 75, pg. 679 (2003); Noel, et al., Curr. Opin. Investig. Drugs, vol. 3, pg. 1000 (2002); Ballas, et al., J. Cell. Biochem. Suppl.. vol. 38, pg.
• MSCs express major histocompatibility complex (MHC) class I antigen on their surface but do not express MHC class II (Le Blanc, et al., Exp. Hematol.. vol. 31, pg. 890 (2003); Potian, et al., J. Immunol., vol. 171, pg. 3426 (2003)) and no B7 or CD40 co-stimulatory molecules (Majumdar, et al., J. Biomed. Sci., vol. 10, pg. 228 (2003)), suggesting that these cells have a low-immunogenic phenotype (Tse, et al., Transplantation, vol. 75, pg. 389 (2003)).
• MHC major histocompatibility complex
• MSCs also inhibit 1-cell proliferative responses in an MHC-independent manner (Bartholomew, et al., Exp. Hematol., vol. 30, pg. 42 (2002); Devine, et al., Cancer J.. vol. 7, pg. 576 (2001); DiNicola, et al., Blood, vol. 99, pg. 3838 (2002)).
• These immunological properties of MSCs may enhance their transplant engraftment and limit the ability of the recipient immune system to recognize and reject allogeneic cells following transplantation.
• MSCs The production of factors by MSCs, that modulate the immune response and support hematopoiesis together with their ability to differentiate into appropriate cell types under local stimuli make them desirable stem cells for cellular transplantation studies (Majumdar, et al., Hematother. Stem Cell Res., vol. 9, pg. 841 (2000); Haynesworth, et al., J. Cell. PhvsioL, vol. 166, pg. 585 (1996).
• mesenchymal stem cells may regulate the production of various factors that may regulate several steps in the immune response process.
• the mesenchymal stem cells may be employed in the treatment of disease conditions and disorders involving the immune system, or diseases, conditions, or disorders involving inflammation, epithelial damage, or allergic responses.
• Such diseases, conditions, and disorders include, but are not limited to, autoimmune diseases, allergies, arthritis, inflamed wounds, alopecia araeta (baldness), periodontal diseases including gingivitis and periodontitis, and other diseases, conditions or disorders involving an immune response.
• autoimmune diseases e.g., rhinitis, rhinitis, rhinitis, rhinitis, rhinitis, and others.
• VEGF vascular endothelial growth factor
• Mesenchymal stem cells also stimulate peripheral blood mononuclear cells (PBMCs) to produce VEGF.
• PBMCs peripheral blood mononuclear cells
• mesenchymal stem cells stimulate dendritic cells (DCs) to produce Interferon-Beta (IFN- ⁇ ), which promotes tumor suppression and immunity against viral infection.
• DCs dendritic cells
• IFN- ⁇ Interferon-Beta
• a method of treating a disease selected from the group consisting of autoimmune diseases and graft- versus-host disease in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to treat the disease in the animal.
• IL-10 Interleukin-10
• T Reg cells regulatory T-cells
• DC dendritic cells
• Autoimmune diseases which may 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, Graves' Disease, lupus, Crohn's disease, autoimmune lymphoproliferative disease (ALPS), demyelinating disease, autoimmune encephalomyelitis, autoimmune gastritis (AIG), and autoimmune glomerular diseases. Also, as noted hereinabove, graft-versus- host disease may 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.
• 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.
• the mesenchymal stem cell (MSC) therapy is based, for example, on the following sequence: harvest of MSC-containing tissue, isolation and expansion of MSCs, and administration of the MSCs to the animal, with or without biochemical or genetic manipulation.
• the mesenchymal stem cells that are administered may be a homogeneous composition or may be a mixed cell population enriched in MSCs.
• Homogeneous mesenchymal stem cell compositions may be obtained by culturing adherent marrow or periosteal cells, and the mesenchymal stem cell compositions may be obtained by culturing adherent marrow or periosteal cells, and the mesenchymal stem cells may be identified by specific cell surface markers which are identified with unique monoclonal antibodies.
• a method of 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.
• the mesenchymal stem cells may be administered by a variety of procedures.
• the mesenchymal stem cells may be administered systemically, such as by intravenous, intraarterial, or intraperitoneal administration.
• the mesenchymal stem cells may be from a spectrum of sources including autologous, allogeneic, or xenogeneic.
• the mesenchymal stem cells are administered in an amount effective to treat an autoimmune disease or graft-versus-host disease in an animal.
• the mesenchymal stem cells may be administered in an amount of from about IxIO 5 cells/kg to about 1 xlO 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 6 cells/kg.
• the amount of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the autoimmune disease to be treated, and the extent and severity thereof.
• the mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier.
• the mesenchymal stem cells may be administered as a cell suspension in a pharmaceutically acceptable liquid medium or gel for injection or topical application.
• a method of treating an inflammatory response in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to treat the inflammatory response in the animal.
• the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that the mesenchymal stem cells promote T-cell maturation to regulatory T-cells (T Reg ), thereby controlling inflammatory responses. It is also believed that the mesenchymal stem cells inhibit T helper 1 cells (ThI cells), thereby decreasing the expression of the Interferon- ⁇ (IFN- ⁇ ) in certain inflammatory reactions, such as those associated with psoriasis, for example.
• T Reg regulatory T-cells
• IFN- ⁇ Interferon- ⁇
• the inflammatory responses which may be treated are those associated with psoriasis.
• the mesenchymal stem cells may be administered to an animal such that the mesenchymal stem cells contact microglia and/or astrocytes in the brain to reduce inflammation, whereby the 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.
• the mesenchymal stem cells may 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 may occur in psoriasis, chronic dermatitis, and contact dermatitis.
• this embodiment is not to be limited to any theoretical reasoning, it is believed that the mesenchymal stem cells may contact the keratinocytes and Langerhans cells in the epidermis, and alter the expression of T-cell receptors and cytokine secretion profiles, leading to decreased expression of tumor necrosis factor-alpha (TNF- ⁇ ) and increased regulatory T-cell (T reg cell) population.
• TNF- ⁇ tumor necrosis factor-alpha
• T reg cell regulatory T-cell
• the mesenchymal stem cells may be used to reduce inflammation in the bone, as occurs in arthritis and arthritis-like conditions, including but not limited to, osteoarthritis and rheumatoid arthritis, and other arthritic diseases listed in the website www.arthritis.org/conditions/diseases.
• arthritis and arthritis-like conditions including but not limited to, osteoarthritis and rheumatoid arthritis, and other arthritic diseases listed in the website www.arthritis.org/conditions/diseases.
• the mesenchymal stem cells may be used to limit inflammation in the gut and liver during inflammatory bowel disease and chronic hepatitis, respectively.
• the scope of this aspect of the present invention is not intended to be limited to any theoretical reasoning, it is believed that the mesenchymal stem cells promote increased secretion of Interleukin-10 (IL-IO) and the generation of regulatory T-cells (T reg cells).
• IL-IO Interleukin-10
• T reg cells regulatory T-cells
• the mesenchymal stem cells may be used to inhibit excessive neutrophil and macrophage activation in pathological conditions such as sepsis and trauma, including burn injury, surgery, and transplants.
• pathological conditions such as sepsis and trauma, including burn injury, surgery, and transplants.
• the mesenchymal stem cells promote secretion of suppressive cytokines such as IL-10, and inhibit macrophage migration inhibitory factor.
• the mesenchymal stem cells may be used to control inflammation in immune privileged sites such as the eye, including the cornea, lens, pigment epithelium, and retina, brain, spinal cord, pregnant uterus and placenta, ovary, testes, adrenal cortex, liver, and hair follicles.
• immune privileged sites such as the eye, including the cornea, lens, pigment epithelium, and retina, brain, spinal cord, pregnant uterus and placenta, ovary, testes, adrenal cortex, liver, and hair follicles.
• the mesenchymal stem cells may be used to treat tissue damage associated with end-stage renal disease (ESRD) infections during dialysis and/or glomerulonephritis.
• ESRD end-stage renal disease
• mesenchymal stem cells may promote renal repair.
• Mesenchymal stem cells also express and secrete vascular endothelial growth factor, or VEGF, which stimulates new blood vessel formation, which should aid in the repair of damaged kidney tissue.
• VEGF vascular endothelial growth factor
• the mesenchymal stem cells may be used to control viral infections such as influenza, hepatitis C, Herpes Simplex Virus, vaccinia virus infections, and Epstein-Barr virus.
• viral infections such as influenza, hepatitis C, Herpes Simplex Virus, vaccinia virus infections, and Epstein-Barr virus.
• IFN- ⁇ Interferon-Beta
• the mesenchymal stem cells may be used to control parasitic infections such as Leishmania infections and Helicobacter infections.
• parasitic infections such as Leishmania infections and Helicobacter infections.
• the scope of this embodiment is not to be limited to any theoretical reasoning, it is believed that the mesenchymal stem cells mediate responses by T helper 2 (Th2) cells, and thereby promote increased production of Immunoglobulin E (IgE) by B-cells.
• Th2 T helper 2
• IgE Immunoglobulin E
• the mesenchymal stem cells may be administered to an animal to treat inflammation which results from a lung disease or disorder.
• lung diseases or disorders include, but are not limited to, Acute Respiratory Distress Syndrome (ARDS), Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), asthma, and pulmonary hypertension.
• the inflammatory response in the above-mentioned lung diseases or disorders involves the secretion of TNF-alpha and/or MCP-I. It is believed that the mesenchymal stem cells migrate to inflamed lung tissue due to increased production of TNF-alpha and/or MCP-I, which are chemoattractants for mesenchymal stem cells.
• the mesenchymal stem cells may be administered to a mammal, including human and non- human primates, as hereinabove described.
• the mesenchymal stem cells also may be administered systemically, as hereinabove described.
• the mesenchymal stem cells maybe administered directly to an arthritic joint.
• the mesenchymal stem cells are administered in an amount effective to treat an inflammatory response in an animal.
• the mesenchymal stem cells may be administered in an amount of from about IxIO 5 cells/kg to about IxIO 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 6 cells/kg.
• the exact dosage of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the inflammatory response being treated, and the extent and severity thereof.
• the mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier, as hereinabove described.
• a method of treating inflammation and/or repairing epithelial damage in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to treat the inflammation and/or epithelial damage in the animal.
• the mesenchymal stem cells cause a decrease in the secretion of the pro-inflammatory cytokines TNF- ⁇ and Interferon- ⁇ 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. It is also believed that the mesenchymal stem cells cause a decrease in Interferon- ⁇ secretion by natural killer (NK) cells.
• NK natural killer
• the inflammation and/or epithelial damage which 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, rejection of transplanted organs, burns, cuts, lacerations, and ulcerations, including skin ulcerations and diabetic ulcerations.
• diseases and disorders including, but not limited to, autoimmune disease, rejection of transplanted organs, burns, cuts, lacerations, and ulcerations, including skin ulcerations and diabetic ulcerations.
• the mesenchymal stem cells are administered to an animal in order 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 (AIG), and autoimmune glomerular disease.
• the mesenchymal stem cells also may repair epithelial damage resulting from graft-versus-host disease (GVHD).
• GVHD graft-versus-host disease
• This aspect of the present invention is applicable particularly to the repair of epithelial damage resulting from graft-versus-host disease, and more particularly, to the repair of epithelial damage resulting from severe graft-versus-host disease, including Grades III and IV graft-versus- host disease affecting the skin and/or the gastrointestinal system.
• mesenchymal stem cells when administered to a patient suffering from severe graft- versus-host disease, and in particular, Grades III and IV gastrointestinal graft-versus-host disease, the administration of the mesenchymal stem cells resulted in repair of skin and/or ulcerated intestinal epithelial tissue in the patient.
• the mesenchymal stem cells are administered to an animal in order 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.
• the 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.
• the mesenchymal stem cells may be administered to a mammal, including human and non- human primates, as hereinabove described.
• the mesenchymal stem cells also may be administered systemically, as hereinabove described.
• the mesenchymal stem cells are administered in an amount effective to repair epithelial damage in an animal.
• the mesenchymal stem cells may be administered in an amount of from about 1 xlO 5 cells/kg to about 1x10 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about IxIO 6 cells/kg to about 5xlO 6 cells/kg.
• the exact dosage of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the type of epithelial damage being repaired, and the extent and severity thereof.
• a method of treating cancer in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to treat cancer in the animal.
• the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that the mesenchymal stem cells interact with dendritic cells, which leads to IFN- ⁇ secretion, which in turn acts as a tumor suppressor.
• Cancers which may be treated include, but are not limited to, hepatocellular carcinoma, cervical cancer, pancreatic cancer, prostate cancer, fibrosarcoma, medullablastoma, 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.
• the animal may be a mammal, including human and non-human primates, as hereinabove described.
• the mesenchymal stem cells are administered to the animal in an amount effective to treat cancer in the animal.
• the mesenchymal stem cells are administered in an amount of from about 1x10 5 cells/kg to about 1x10 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 6 cells/kg.
• the exact amount of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the type of cancer being treated, and the extent and severity thereof.
• the mesenchymal stem cells are administered in conjunction with an acceptable pharmaceutical carrier, and may be administered systemically, as hereinabove described. Alternatively, the mesenchymal stem cells may be administered directly to the cancer being treated.
• a method of treating an allergic disease or disorder in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to treat the allergic disease or disorder in the animal.
• mesenchymal stem cells when administered after an acute allergic response, provide for inhibition of mast cell activation and degranulation. Also, it is believed that the mesenchymal stem cells downregulate basophil activation and inhibit cytokines such as TNF- ⁇ , chemokines such as Interleukin-8 and monocyte chemoattractant protein, or MCP- 1, lipid mediators such as leukotrienes, and inhibit main mediators such as histamine, heparin, chondroitin sulfates, and cathepsin.
• cytokines such as TNF- ⁇
• chemokines such as Interleukin-8 and monocyte chemoattractant protein
• MCP- 1 lipid mediators
• leukotrienes lipid mediators
• main mediators such as histamine, heparin, chondroitin sulfates, and cathepsin.
• Allergic diseases or disorders which may 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.
• the mesenchymal stem cells are administered to the animal in an amount effective to treat the allergic disease or disorder in the animal.
• the animal may be a mammal.
• the mammal may be a primate, including human and non-human primates.
• the mesenchymal stem cells are administered in an amount of from about 1x10 5 cells/kg to about 1x10 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 cells/kg.
• the exact dosage is dependent upon a variety of factors, including the age, weight, and sex of the patient, the allergic disease or disorder being treated, and the extent and severity thereof.
• the mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier, as hereinabove described.
• the mesenchymal stem cells may be administered systemically, such as by intravenous or intraarterial administration, for example.
• a method of promoting wound healing in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to promote wound healing in the animal.
• the scope of the present invention is not to be limited to any theoretical reasoning, it is believed that, as mentioned hereinabove, the mesenchymal stem cells cause T reg cells and dendritic cells to release Interleukin-10 (IL-10).
• IL-10 Interleukin-10 limits or controls inflammation in a wound, thereby promoting healing of a wound.
• the mesenchymal stem cells may promote wound healing and fracture healing by inducing secretion factors by other cell types.
• the mesenchymal stem cells may induce prostaglandin E 2 (PGE 2 )-mediated release of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs), as well as PGE 2 -mediated release of growth hormone, insulin, insulin-like growth factor 1 (IGF-I) insulin-like growth factor binding protein-3 (IGFBP-3), and endothelin-1.
• PGE 2 prostaglandin E 2
• IGF-I insulin-like growth factor 1
• IGFBP-3 insulin-like growth factor binding protein-3
• Wounds which may be healed include, but are not limited to, those resulting from cuts, lacerations, burns, and skin ulcerations.
• the mesenchymal stem cells are administered to the animal in an amount effective to promote wound healing in the animal.
• the animal may be a mammal, and the mammal may be a primate, including human and non-human primates.
• the mesenchymal stem cells are administered in an amount of from about IxIO 5 cells/kg to about IxIO 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 cells/kg.
• the exact amount of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, and the extent and severity of the wound being treated.
• the mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier, as hereinabove described.
• the mesenchymal stem cells may be administered systemically, as hereinabove described.
• the mesenchymal stem cells may be administered directly to a wound, such as in a fluid on a dressing or reservoir containing the mesenchymal stem cells.
• a method of treating or preventing fibrosis or fibrotic disorder in an animal comprises administering to the animal mesenchymal stem cells in an amount effective to treat or prevent fibrosis or a fibrotic disorder in an animal.
• the mesenchymal stem cells may be administered to the animal in order to treat or prevent any type of fibrosis or fibrotic disorder and in the animal, including, but not limited to, cirrhosis of the liver, fibrosis of the kidneys associated with end-stage renal disease, and lung disorders or diseases having fibrotic and may include in addition, inflammatory components, including, but not limited to, Acute Respiratory Distress Syndrome (ARDS), Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), asbestosis, and fibrosis resulting from pulmonary hypertension and asthma.
• ARDS Acute Respiratory Distress Syndrome
• COPD Chronic Obstructive Pulmonary Disease
• IPF Idiopathic Pulmonary Fibrosis
• asbestosis fibrosis resulting from pulmonary hypertension and asthma. It is to be understood that the scope of the present invention is not to be limited to any specific type of fibrosis or fibrotic disorder.
• the mesenchymal stem cells are administered to the animal in order to improve pulmonary function due to pulmonary diseases or diseases in other organs leading to pulmonary insufficiency or lung fibrosis.
• diseases have fibrotic, and may also have in addition, inflammatory and/or immunological components, and include but are not limited to, Acute Respiratory Distress Syndrome (ARDS), Chronic Obstructive Pulmonary Disease (COPD), asthma, pulmonary hypertension, asbestosis, and Idiopathic Pulmonary Fibrosis (IPF).
• Acute Respiratory Distress Syndrome is a life threatening lung disease having a variety of causes, including but not limited to ventilator injury and sudden blunt trauma to the chest.
• the disease is characterized by inflammation of the lung parenchyma, resulting in impaired gas exchange and concomitant expression and secretion of inflammatory mediators.
• These inflammatory mediators include TNF-alpha, IL-I, IL-8, and monocyte chemoattractant protein-1 or MCP-I .
• TNF-alpha and MCP-I are chemoattractants for mesenchymal stem cells.
• increased expression of TNF-alpha and MCP-I in the damaged lung will facilitate mesenchymal stem cell recruitment to the area of lung tissue damage.
• COPD Chronic Obstructive Pulmonary Disease
• COPD is a major cause of illness and death worldwide.
• COPD is characterized by airflow obstruction due to chronic bronchitis or emphysema.
• COPD is characterized by a thickening of the alveolar walls and inflammation, resulting in a loss of elasticity in and damage to the alveolar tissue, as well as clogging of the lung bronchi with mucus deposits.
• the inflammatory response in COPD includes the local secretion of IL-6, IL-I Beta, TNF- alpha, and MCP-I.
• mesenchymal stem cells migrate to the damaged lung tissue in COPD patients due to increased production in the damaged lung of the chemoattractants TNF-alpha and MCP-I.
• neutrophil infiltration is characteristic of COPD
• neutrophilic inflammation is resistant to current COPD treatments, such as corticosteroid therapy.
• treatment with mesenchymal stem cells inhibits neutrophilic inflammation by downregulation of factors that act as chemoattractants for neutrophils.
• Apoptotic death of lung cells is another result of COPD (Calabrese, et al., Respir. Res., vol. 6, pg. 14 (2005)).
• Mesenchymal stem cells secrete a variety of growth factors including hepatocyte growth factor (HFG) and fibroblast growth factors (FGFs), which have been shown to be beneficial for treatment of pulmonary emphysema (Shigemura, et al., Circulation, vol. I l l, pg. 1407 (2005); Morino, et al., Chest, vol. 128, pg. 920 (2005)).
• HFG hepatocyte growth factor
• FGFs fibroblast growth factors
• Asthma is a chronic or recurring inflammatory condition in which the airway develops increased responsiveness to various stimuli; characterized by bronchial hyper responsiveness, inflammation, increased mucus production, and intermittent airway obstruction.
• mesenchymal stem cells downregulate the inflammatory and immune reactions associated with asthma, as well as repair the fibrotic and scar tissue associated therewith.
• Idiopathic Pulmonary Fibrosis is marked by progressive scarring of the lungs.
• the scarring interferes with the patient's ability to breathe and obtain enough oxygen for vital organs to function normally.
• Injured lung epithelial cells subsequently initiate apoptosis and the production of excess TNF-alpha and MCP-I.
• the interstitium, or tissue surrounding the air sacs progressively becomes thickened and stiff as fibrosis continues. As the disease progresses, oxygen cannot pass effectively from the air sacs to the capillaries of the lung.
• TNF-alpha and MCP-I expression results in the recruitment of mesenchymal stem cells to damaged lung tissue.
• the mesenchymal stern cells improve pulmonary function in an animal having lung fibrosis by inhibiting inflammatory responses by downregulating pro-inflammatory cytokine and chemokine secretion, resulting in a subsequent decrease in recruitment of inflammatory cells to the site. It also is believed that the mesenchymal stem cells inhibit the immune response in those lung disorders which elicit an immune response, thereby preventing cell- mediated as well as soluble factor mediated tissue cell killing.
• the mesenchymal stem cells also facilitate tissue repair by protection of tissue cells from apoptosis, and stimulate cell proliferation and mobilization of tissue-specific stem cells via secretion of growth factors such as HGF, VEGF, and FGFs.
• the mesenchymal stem cells prevent pathological remodeling and scar formation in the lung tissue.
• the mesenchymal stem cells reduce local expression of TNF-alpha, which in turn leads to a reduction in TGF-beta expression and a reduction in a recruitment of fibroblasts, which are the major cells contributing to scar formation, hi addition, the mesenchymal stem cells remodel the existing lung scar tissue and/or prevent expansion of the scar though the expression and local secretion of matrix metalloproteinases (MMPs).
• MMPs matrix metalloproteinases
• the mesenchymal stem cells are administered to the animal in an amount effective to treat or prevent fibrosis or a fibrotic disorder in the animal.
• the animal may be a mammal, and the mammal may be a primate, including human and non-human primates.
• the mesenchymal stem cells are administered in an amount of from about 1x10 5 cells/kg to about 1x10 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 6 cells/kg.
• the exact amount of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, and the extent and severity of the fibrosis or fibrotic disorder being treated or prevented.
• the mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier, as hereinabove described.
• the mesenchymal stem cells may be administered systemically, also as hereinabove described.
• a method of promoting angiogenesis in an organ or tissue of an animal comprises administering to the animal mesenchymal stem cells in an amount effective to promote angiogenesis in an organ or tissue of the animal.
• Angiogenesis is the formation of new blood vessels from a pre-existing microvascular bed.
• angiogenesis may be used to treat coronary and peripheral artery insufficiency, and thus may be a noninvasive 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 the development of the embryonic vasculature.
• Angiogenesis also is essential for the coupling of cartilage resorption with bone formation, and is essential for correct growth plate morphogenesis.
• angiogenesis is necessary for the successful engineering and maintenance of highly metabolic organs, such as the liver, where a dense vascular network is necessary to provide sufficient nutrient and gas transport.
• the mesenchymal stem cells can be administered to the tissue or organ in need of angiogenesis by a variety of procedures.
• the mesenchymal stem cells may be administered systemically, such as by intravenous, intraarterial, or intraperitoneal administration, or the mesenchymal stem cells may be administered directly to the tissue or organ in need of angiogenesis, such as by direct injection into the tissue or organ in need of angiogenesis.
• the mesenchymal stem cells may be from a spectrum of sources including autologous, allogeneic, or xenogeneic.
• the mesenchymal stem cells when administered to an animal, stimulate peripheral blood mononuclear cells (PBMCs) to produce vascular endothelial growth factor, or VEGF, which stimulates the formation of new blood vessels.
• PBMCs peripheral blood mononuclear cells
• VEGF vascular endothelial growth factor
• the animal is a mammal.
• the mammal may be a primate, including human and non-human primates.
• the mesenchymal stem cells may be employed in the treatment, alleviation, or prevention of any disease or disorder which can be alleviated, treated, or prevented through angiogenesis.
• the mesenchymal stem cells may be administered to an animal to treat blocked arteries, including those in the extremities, i.e., arms, legs, hands, and feet, as well as the neck or in various organs.
• the mesenchymal stem cells may be used to treat blocked arteries which supply the brain, thereby treating or preventing stroke.
• the mesenchymal stem cells may be used to treat blood vessels in embryonic and postnatal corneas and may be used to provide glomerular structuring.
• the mesenchymal stem cells may be employed in the treatment of wounds, both internal and external, as well as the treatment of dermal ulcers found in the feet, hands, legs or arms, including, but not limited to, dermal ulcers caused by diseases such as diabetes and sickle cell anemia.
• the mesenchymal stem sells may be employed to promote embryo implantation and prevent miscarriage.
• the mesenchymal stem cells may be administered to an unborn animal, including humans, to promote the development of the vasculature in the unborn animal.
• the mesenchymal stem cells may be administered to an animal, born or unborn, in order to promote cartilage resorption and bone formation, as well as promote correct growth plate morphogenesis.
• the mesenchymal stem cells are administered in an amount effective in promoting angiogenesis in an animal.
• the mesenchymal stem cells may be administered in an amount of from about IxIO 5 cells/kg to about IxIO 7 cells/kg.
• the mesenchymal stem cells are administered in an amount of from about 1x10 6 cells/kg to about 5x10 6 cells/kg.
• the amount of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the disease or disorder to be treated, alleviated, or prevented, and the extent and severity thereof.
• the mesenchymal stem cells may be administered in conjunction with an acceptable pharmaceutical carrier.
• the mesenchymal stem cells may be administered as a cell suspension in a pharmaceutically acceptable liquid medium for injection. Injection can be local, i.e., directly into the tissue or organ in need of angiogenesis, or systemic.
• the mesenchymal stem cells may be genetically engineered with one or more polynucleotides encoding a therapeutic agent.
• the polynucleotides may be delivered to the mesenchymal stem cells via an appropriate expression vehicle.
• Expression vehicles which may be employed to genetically engineer the mesenchymal stem cells include, but are not limited to, retroviral vectors, adenoviral vectors, and adeno-associated virus vectors.
• mesenchymal stem cells when employed in the above- mentioned 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 may be administered with soluble carriers for cells such as hyaluronic acid, or in combination with solid matrices, such collagen, gelatin, or other biocompatible polymers, as appropriate.
• Fig. 1 MSCs modulate dendritic cell functions.
• A Flow cytometric analysis of mature monocytic DCl cells using antibodies against HLA-DR and CDl Ic and of plasmacytoid DC2 cells using antibodies against HLA-DR and CD123 (IL-3 receptor).
• B MSCs inhibit TNF- ⁇ secretion (primary y-axis) and increase IL- 10 secretion (secondary y-axis) from activated DCl and DC2 respectively.
• C MSCs cultured with mature DCl cells inhibit IFN- ⁇ secretion (primary y-axis) by T cells and increase IL-4 levels (secondary y-axis) as compared to MSC or DC alone.
• Fig. 2 MSCs inhibit pro-inflammatory effector T cell function.
• A Flow cytometric analysis of T Reg cell numbers (in %) by staining PBMCs or non-adherent fraction in MSC+PBMC culture (MSC+PBMC) with FITC-conjugated CD4 (x-axis) and PE conjugated CD25 (y-axis) antibodies. Gates were set based on isotype control antibodies as background. Graphs are representative of 5 independent experiments.
• C MSCs inhibit IFN- ⁇ secretion from purified NK cells cultured for 0, 24, or 48 hours in a 24-well plate. Data shown are mean ⁇ SD cytokine secretion in one experiment and are representative of 3 independent experiments.
• Fig. 3 MSCs lead to increased numbers of T Reg cell population and increased GITR expression.
• MLR mixed lymphocyte reaction
• PHA phytohemagglutinin
• PBMCs were cultured for 3 days in the absence (top plot) or presence (bottom plot) of MSCs (MSC to PBMC ratio 1 :10), following which the non-adherent fraction was harvested and immunostained with FITC-labeled GITR and PE-labeled CD4. Results show a greater than twofold increase in GITR expression in cells cultured in the presence of MSCs.
• Fig. 4 MSCs produce PGE 2 and blocking PGE 2 reverses MSC-mediated immunomodulatory effects .
• A PGE 2 secretion (mean ⁇ SD) in culture supernatants obtained from MSCs cultured in the presence or absence of PGE 2 blockers NS-398 or indomethacin (Indometh.) at various concentrations. Inhibitor concentrations are in ⁇ M and data presented are values obtained after 24 hour culture
• B COX-I and COX-2 expression in MSCs and PBMCs using real-time RT- PCR.
• MSCs expressed significantly higher levels of COX-2 as compared to PBMCs, and when MSCs were cultured in presence of PBMCs, there was a >3-fold increase in COX-2 expression in MSCs. Representative data from 1 of 3 independent experiments is shown. The MSC+PBMC cultures were setup in a trans-well chamber plate where MSCs were plated onto the bottom chamber and PBMCs onto the top chamber.
• C Presence of PGE 2 blockers indomethacin (Ind.) or NS-398 increases TNF- ⁇ secretion from activated DCs (D) and IFN- ⁇ secretion from T H I cells ( ⁇ ) as compared to controls.
• Fig. 5 Constitutive MSC cytokine secretion is elevated in the presence of allogeneic
• PBMCs PBMCs.
• cytokines 1L-6 and VEGF, lipid mediator PGE 2 , and matrix metalloproteinase 1 (pro MMP-I) were analyzed.
• the MSCs produced 1L-6, VEGF, and PGE 2 constitutively, and the levels of these factors increased upon co-culture with PBMCs, thereby suggesting that MSCs may play a role in modulating immune functions in an inflammatory setting.
• Fig. 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) plated on a 96-well plate in the presence or absence of PHA (2.5 mg/ml) for 72 hours, and 3 H thymidine incorporation determined (in counts per minute, or cpm).
• PHA 2.5 mg/ml
• FIG. 7 Schematic diagram of proposed MSC mechanism of action.
• MSCs mediate their immuno-modulatory effects by affecting cells from both the innate (DCs-pathways 2-4; and NK- pathway 6) and adaptive (T- pathways 1 and 5 and B-pathway 7) immune systems.
• immature DCs migrate to the site of potential entry, mature and acquire an ability to prime naive T cells (by means of antigen specific and co-stimulatory signals) to become protective effector T cells (cell-mediated T H I or humoral T H 2 immunity).
• MSCs may alter the outcome of immune response by limiting the ability of DCs to mount a cell-mediated response (pathway 2) or by promoting the ability to mount a humoral response (pathway 4). Also, when mature effector T cells are present, MSCs may interact with them to skew the balance of T H I (pathway 1) responses towards T H 2 responses (pathway 5), and probably towards an increased IgE producing B cell activity (pathway 7), desirable outcomes for suppression of GvHD and autoimmune disease symptoms.
• MSCs in their ability to result in an increased generation of T Reg population may result in a tolerant phenotype and may aid a recipient host by dampening bystander inflammation in their local micro-environment. Dashed line ( — ) represents proposed mechanism. [00110] Fig. 8. MSC treatment provides an improvement in percent of predicted forced expiratory volume in one second (Pred. FEVl %) in patients treated with mesenchymal stem cells as compared to patients who received a placebo.
• Fig. 9 Measurement of distance walked on a treadmill after six minutes. Patients who were treated with MSCs showed an increase in distance walked as compared to those who received a placebo.
• Fig. 10 Heart Rate Recovery in Patients Subjected to Treadmill Test. A greater percentage of the patients subjected to the treadmill test showed heart rate recovery to baseline values in 15 minutes or less than those who were treated with a placebo.
• MSCs were cultured in complete Dulbecco's Modified Eagle's Medium-Low Glucose (Life Technologies, Carlsbad, California) containing 1% antibiotic ⁇ antimyotic solution (Invitrogen, Carlsbad, California) and 10% fetal bovine serum (FBS, JRH BioSciences, Lenexa, Kansas). MSCs grew as an adherent monolayer and were detached with trypsin/EDTA (0.05% trypsin at 37°C for 3 minutes).
• PBMCs Peripheral blood mononuclear cells
• DCs dendritic cells
• CDIc+ monocytic lineage
• BDCAl + biotin-labeled CDIc
• BDCA2+ positively labeled antibody coated cells
• MSCs and DCs were cultured in equal numbers for various time periods and cell culture supernatant collected and stored at -80°C until further evaluation.
• MSCs were cultured with mature DCl or DC2 cells (1 :1 MSC:DC ratio) for 3 days, and then the combined cultures (MSCs and DCs) were irradiated to prevent any proliferation.
• antibody purified, na ⁇ ve, allogeneic 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 re-stimulated with PHA for another 24 hours, following which cell culture supernatants were harvested and analyzed for secreted IFN- ⁇ and IL-4 by ELISA.
• NK cells 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, - CDl 4, -CDl 9, -CD36 and anti-IgE antibodies) as a primary reagent and anti-biotin monoclonal antibodies conjugated to Microbeads as secondary labeling reagent.
• the magnetically labeled non- NK cells were retained in MACS (Miltenyi Biotech, Auburn, California) columns in a magnetic field, while NK cells passed through and were collected.
• the T Reg cell population was isolated using a 2-step isolation procedure.
• CD4 + T cells were indirectly magnetically labeled with a cocktail of biotin labeled antibodies and anti-biotin microbeads. The labeled cells were then depleted by separation over a MACS column (Miltenyi Biotech, Auburn, California). Next, CD4 + CD25 + cells were directly labeled with CD25 microbeads and isolated by positive selection from the pre-enriched CD4 + . T cell fraction. The magnetically labeled CD4 + CD25 + T cells were retained on the column and eluted after removal of the column from the magnetic field.
• CD4+CD25+ T Reg cell populations were isolated from PBMC or MSC+PBMC (MSC to PBMC ratio 1 :10) cultures (cultured without any further stimulation for 3 days) using a 2-step magnetic isolation procedure. These cells were irradiated to block any further proliferation and used as stimulators in a mixed lymphocyte reaction (MLR), where responders were allogeneic PBMCs (stimulator to responder ratio 1 : 100) in the presence of PHA (2.5 ⁇ g/ml). The culture was carried out for 48 hours, following which 3 H thymidine was added. Incorporated radioactivity was counted after 24 hours.
• MLR mixed lymphocyte reaction
• PBMCs were cultured in the absence or presence of MSCs (MSC to PBMC ratio
• PBMCs Peripheral blood mononuclear cells
• Non-adherent fraction was incubated in the presence of plate-bound anti-CD3 (5 ⁇ g/ml) and anti-CD28 (1 ⁇ g/ml) antibodies under T H 1 (IL-2 (4 ng/ml) IL- 12 (5 ng/ml) + anti-IL-4 (1 ⁇ g/ml)) or T H 2 (IL-2 (4 ng/ml) + IL-4 (4 ng/ml) + anti-IFN- ⁇ (1 ⁇ g/ml)) conditions for 3 days in the presence or absence of MSCs.
• T H 1 IL-2 (4 ng/ml) IL- 12
• T H 2 IL-2 (4 ng/ml) + IL-4 (4 ng/ml) + anti-IFN- ⁇ (1 ⁇ g/ml)
• the cells were washed and then re-stimulated with PHA (2.5 ⁇ g/ml) for another 24 or 48 hours, following which levels of IFN- ⁇ and IL-4 were measured in culture supernatants by ELISA (R&D Systems, Minneapolis, Minnesota).
• VEGF vascular endothelial growth factor 1
• PGE 2 lipid mediator prostaglandin E 2
• pro-MMP-1 matrix metalloproteinase 1
• Purified PBMCs were prepared by centrifuging leukopack (Cambrex, Walkersville,
• RNA from cell pellets were prepared using a commercially available kit
• COX-I and COX-2 specific primers were: COX-I: 5'-CCG GAT GCC AGT CAG GAT GAT G-3 '(forward), 5'-CTA GAC AGC CAG ATG CTG ACA G-3' (reverse); COX-2: 5 1 - ATC TAC CCT CCT CAA GTC CC-3 '(forward), 5'-TAC CAG AAG GGC AGG ATA CAG-3' (reverse).
• MSCs 2,000 cells/well
• PHA 2.5 ⁇ g/ml
• H thymidine incorporation counts per minute, cpm
• the PBMCs and MSCs were cultured at ratios of MSC:PBMC of 1:1, 1:3, 1:10, 1 :30, and 1:81.
• DCl and DC2 precursor dendritic cells were isolated by immuno-magnetic sorting of BDCAl + and BDC A2 + cells respectively and matured by incubation with GM-CSF and IL-4 (IxIO 3 IU/ml and IxIO 3 IU/ml, respectively) for DCl cells, or IL-3 (10 ng/ml) for DC2 cells.
• GM-CSF and IL-4 IxIO 3 IU/ml and IxIO 3 IU/ml, respectively
• IL-3 10 ng/ml
• DCl cells In the presence of the inflammatory agent bacterial lipopolysaccharide (LPS, 1 ng/ml), DCl cells produced moderate levels of TNF- ⁇ but when MSCs were present (ratios examined 1:1 and 1 :10), there was >50% reduction in TNF- ⁇ secretion (Fig. IB).
• DC2 cells produced IL-IO in the presence of LPS and its levels were increased greater than 2-fold upon MSC:DC2 co-culture (1 :1) (Fig. IB). Therefore, the MSCs modified the cytokine profile of activated DCs in culture towards a more tolerogenic phenotype.
• activated DCs when cultured with MSCs, were able to reduce IFN- ⁇ and increase IL- 4 levels secreted by na ⁇ ve CD4 + T cells (Fig. 1C) suggesting a MSC-mediated shift from proinflammatory to anti-inflammatory T cell phenotype.
• T Reg T-regulatory cells
• GITR gluocorticoid-induced TNF receptor
• IL-4 a cell surface receptor expressed on T Reg cell populations
• MSCs inhibit T-cell proliferation induced by various stimuli (DeNicola, et al., Blood, vol. 99, pg. 3838 (2002); LeBlanc, et al., Scand. J. Immunol., vol. 57, pg. 11 (2003)). It was observed that MSCs inhibit mitogen-induced T cell proliferation in a dose-dependent manner (Fig. 6) and when PGE 2 inhibitors NS-398 (5 ⁇ M) or indomethacin (4 ⁇ M) were present, there was a >70% increase in ( 3 H) thymidine incorporation by PHA-treated PBMCs in MSC containing cultures as compared to controls without inhibitors (Fig. 4D).
• a model of MSC interaction with other immune cell types (Fig. 7) is proposed.
• MSCs may interact with them directly and inhibit the pro-inflammatory IFN- ⁇ production (pathway 1) and promote regulatory T cell phenotype (pathway 3) and anti-inflammatory T H 2 cells (pathway 5).
• MSCs can alter the outcome of the T cell immune response through DCs by secreting PGE 2 , inhibiting pro-inflammatory DCl cells (pathway 2) and promoting anti-inflammatory DC2 cells (pathway 4) or regulatory DCs (pathway 3).
• a shift towards T H 2 immunity suggests a change in B cell activity towards increased generation of IgE/IgGl subtype antibodies (pathway 7).
• MSCs by their ability to inhibit IFN- ⁇ secretion from NK cells likely modify NK cell function (pathway 6).
• This model of MSC:Immune cell interactions is consistent with the experimentation performed in several other laboratories (LeBlanc, et al., Exp. Hematol., vol. 31, pg. 890 (2003); Tse, et al., Transplantation, vol. 75, pg. 389 (2003); DiNicola, et al., Blood, vol. 99, pg. 3838 (2002)). Further examination of the proposed mechanisms is underway and animal studies are now necessary to examine the in vivo effects of MSC administration.
• the patient was given an intravenous infusion of allogeneic mesenchymal stem cells in 50 ml of Plasma Lyte A (Baxter) in an amount of 3 X 10 6 cells per kilogram of body weight.
• Plasma Lyte A Plasma Lyte A
• the patient was evaluated at two weeks post-infusion. At two weeks post-infusion, an endoscopic view of the patient's colon showed that the areas of inflammation and ulceration visible prior to treatment were resolved. In addition, a biopsy of the patient's colon showed significant regeneration of 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.
• Forced expiratory volume is the maximal volume of air exhaled in the first second of a forced expiration from a position of full inspiration, expressed in liters, at body temperature (37°C), ambient pressure, saturated with water vapor (BTPS).
• the predicted FEVl values were calculated for each patient based on age, sex, height, and race.
• the predicted FEVl for men was calculated as follows (Crapo, et al., Am. Rev. Respir. Pis., vol. 123, pgs.
• predicted FEVl 0.0414 x height (cm) - 0.0244 x age (years) - 2.190 [00139]
• FEVl values for all patients were measured using a spirometer, which was connected to a mouthpiece or tube which was inserted into the patient's mouth. The height and weight of each patient was measured, a nose clip was placed on each patient. Each patient was instructed to inhale completely and rapidly, with a pause of less than one second at total lung capacity, and to exhale maximally until no more air can be expelled. The procedure is repeated in triplicate, and the FEVl values for each patient were measured. From the FEVl values, the percent of predicted FEVl (Pred. FEV 1%) values were calculated. The percent of predicted FEVl (Pred. FEVl %) values for each patient were calculated as follows:
• MSCs showed a greater improvement in Pred. FEVl %, relative to baseline (pre-treatment) values, from three days through six months post-infusion. At both 10 and 30 days post-infusion, the difference in improvement in Pred. FEVl % values observed for MSC-treated and placebo patients was significant statistically (p ⁇ 0.05).
• MSC-treated and placebo patients also were subjected to a treadmill test in which the patients walked on a treadmill in which the distance walked by each patient was measured in six minute intervals. Distance measurements were taken after treatment (baseline), and at one month, three months, and six months post-treatment. The test was performed according to ATS (American Thoracic Society) guidelines (Am. J. Respir. Crit. Care Med., vol. 166, pg. I l l (2002)).
• the heart rate recovery results are shown in Figure 10. As shown in Figure 10, at six months after the treatment, the difference in the percentage of patients who received the MSC treatments showing heart rate recovery to baseline values within 15 minutes after the cessation of the treadmill walking as compared to the patients who received the placebo treatments was significant statistically.

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