ES2708627T3 - Mesenchymal stem cells and their uses - Google Patents

Abstract

To provide novel uses of mesenchymal stem cells.SOLUTION: The present invention discloses methods for treating animal autoimmune diseases, allergic diseases, cancer, or inflammatory diseases, methods for promoting wound healing, and methods promoting angiogenesis in animal organs and tissues by administering an effective amount of animal mesenchymal stem cells.SELECTED DRAWING: None

Description

DESCRIPTION

Mesenchymal stem cells and their uses

This invention relates to mesenchymal stem cells for use in the treatment of coronary insufficiency and peripheral artery or for use in the treatment of blocked arteries in a human promoting or inducing angiogenesis. Mesenchymal stem cells (MSCs) are multipotent stem cells that can be differentiated easily in lineages that include osteoblasts, myocytes, chondrocytes, and adipocytes (Pittenger, et al., Science, vol 284, page 143 (1999), Haynesworth, et al., Bone, vol 13, page 69 (1992) Prockop, Science, vol.276, page 71 (1997)). In vitro studies have demonstrated MSC's ability to differentiate into muscle (Wakitani, et al., Muscle Nerve, vol.18, p.1417 (1995)), neuronal-type precursors (Woodbury, et al., J. Neurosci. ., vol 69, page 908 (2002), Sanchez-Ramos, et al., Exp. Neurol., vol 171, page 109 (2001)), cardiomyocytes (Toma, et al., Circulation, vol. 105, page 93 (2002), Fakuda, Artif. Organs, vol.25, page 187 (2001)), and possibly another type of cells. In addition, it has been shown that MSCs provide effective feeder layers for the expansion of hematopoietic and embryonic stem cells (Eaves, et al., Ann.N.A. Acad.Sci., Vol.938, page 63 (2001); Wagers, et al. al., Gene Therapy, vol.9, page 606 (2002)). Recent studies with a variety of animal models have shown that MSCs can be useful in the repair or regeneration of damaged bone, cartilage, meniscus, or myocardial tissues (DeKok et al., Clinical Implants Oral Res., Vol 481 (2003). )); Wu et al., Transplantation, vol. 75, pag. 679 (2003); Noel et al., Curr. Opin. Investig. Drugs, vol. 3, pag. 1000 (2002); Ballas, et al., J. Cell. Biochem. Suppl., Vol. 38, p. 20 (2002); Mackenzie, et al., Blood Cells Mol. Dis., Vol. 27 (2002)). Several researchers have used MSC with encouraging results for transplantation in models of animal diseases including osteogenesis imperfecta (Pereira, et al., Proc. Nat. Acad. Sci., Vol 95, page 1142 (1998)), parkinsonism (Schwartz , et al., Hum. Gene Ther., vol.10, page 2539 (1999)), spinal cord injury (Chopp, et al., Neuroreport, vol.11, page 3001 (2000); Wu, et al., J. Neurosci. Res., vol 72, page 393 (2003)) and cardiac disorders (Tomita, et al., Circulation, vol.100, page 247 (1999) .Shake, et al. , Ann, Thorac, Surg., Vol 73, p.

1919 (2002)). It is important to note that promising results have also been published in clinical trials for osteogenesis imperfecta (Horwitz et al., Blood, volume 97, page 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, page 307 (2000)).

MSCs express the class I antigen of the major histocompatibility complex (MHC) on their surface, but do not express MHC class II (Le Blanc et al., Exp. Hematol., Vol.31, page 890 (2003) , Potian, et al., J. Immunol., Vol.

171, p. 3426 (2003)) and no costimulatory B7 or CD40 molecule (Majumdar et al., J. Biomed, Sci., Vol 10, page (2003)), suggesting that these cells have a low immunogenicity phenotype (Tse). , et al., Transplantation, vol.

75, pag. 389 (2003)). MSCs also inhibit cell proliferative responses in an MHC-independent manner (Bartholomew et al., Exp. Hematol., Vol 30, p 42 (2002), Devine et al., Cancer J., vol. , page 576 (2001), DiNicola, et al., Blood, vol 99, page 3838 (2002)). These immunological properties of MSCs can improve their transplant graft and limit the ability of the recipient immune system to recognize and reject allogenic cells after transplantation. The production of factors by MSCs, which 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 cell transplantation studies (Majumdar, et al., Hematother, Stem Cell Res., Vol.9, page 841 (2000), Haynesworth et al., J. Cell. Physiol., Vol.166, page 585 (1996).

The international patent application publication number WO 03/059272 A2 discloses a therapeutic agent for use in the induction of angiogenesis and vasculogenesis, including the therapeutic agent factors that induceangiogenesis and vasculogenesis isolated from stem cells together with a pharmaceutically acceptable cell therapy.

Currently, applicants have examined the interactions of mesenchymal stem cells with populations of isolated immune cells, including dendritic cells (DC1 and DC2), effector T cells (Th1 and Th2) and NK cells. Based on such interactions, the applicants discovered that mesenchymal stem cells can regulate the production of various factors that can regulate several stages in the immune response process. Thus, mesenchymal stem cells can be used in the treatment of diseases, conditions and disorders involving the immune system, or diseases, conditions or disorders involving inflammation or allergic responses. Such diseases, conditions and disorders include, but are not limited to, autoimmune diseases, allergies, arthritis, inflamed wounds, araceous alopecia (baldness), periodontal diseases including gingivitis and periodontitis, and other diseases, conditions or disorders that involve an immune response. .

In addition, it is believed that mesenchymal stem cells stimulate peripheral blood mononuclear cells (PBMCs) to produce vascular endothelial growth factor, or VEGF, which promotes angiogenesis by stimulating the formation of new blood vessels.

In addition, it is believed that mesenchymal stem cells stimulate dendritic cells (DCs) to produce interferon-beta (IFN-p), which promotes the suppression of tumors and immunity against viral infection.

The present invention is directed to mesenchymal stem cells for use in the treatment of coronary and peripheral artery insufficiency or for use in the treatment of blocked arteries in a human through the promotion or induction of angiogenesis, where the mesenchymal stem cells are genetically unmanipulated and allogenic, and where the mesenchymal stem cells stimulate the peripheral blood mononuclear cells (PBMCs) to produce the vascular endothelial growth factor (VEGF) and , therefore, promote or induce angiogenesis.

In one embodiment of the present invention, mesenchymal stem cells are used to treat coronary artery disease, ischemic heart disease and peripheral artery disease.

In an embodiment of the present invention, the blocked arteries are in the extremities or in the neck, where, for example, the extremities are selected between arms, legs, hands and feet.

In an embodiment of the present invention, the mesenchymal stem cells are for use in the treatment of blocked arteries supplying the brain, optionally in which the mesenchymal stem cells are for use in the treatment or prevention of apoplexy.

In an embodiment of the present invention, the mesenchymal stem cells are administered systemically or by direct administration to a tissue or organ that needs angiogenesis, optionally wherein the systemic administration is by intravenous, intraarterial or intraperitoneal administration.

Also disclosed herein is a method for treating autoimmune diseases in an animal. The method comprises administering to the animal the animal mesenchymal stem cells in an amount effective to treat the disease in the animal.

Although the scope of this aspect of the present disclosure is not limited to any theoretical reasoning, it is believed that at least one mechanism by which mesenchymal stem cells suppress autoimmune disease is causing the release of interleukin-10 (IL-10) from Regulatory T cells (TReg cells) and / or dendritic cells (DC). Autoimmune diseases that can be treated according to the present disclosure include, among others, multiple sclerosis, type 1 diabetes, rheumatoid arthritis, uveitis, autoimmune thyroid disease, inflammatory bowel disease, autoimmune lymphoproliferative disease (ALPS), demyelinating disease, autoimmune encephalomyelitis, autoimmune gastritis (AIG) and autoimmune glomerular diseases. It should be understood, however, that the scope of the present disclosure should not be limited to the treatment of the specific diseases mentioned in this document.

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

In general, mesenchymal stem cell therapy (MSC) is based, for example, on the following sequence: tissue collection containing MSC, isolation and expansion of the MSCs and administration of the MSCs to the animals, with or without biochemical manipulation or genetic

The mesenchymal stem cells that are administered can be a homogenous composition or can be a mixed cell population enriched in the MSCs. Homogeneous mesenchymal stem cell compositions can be obtained by culturing adherent medullary or periosteal cells and compositions of mesenchymal stem cells can be obtained by culturing adherent medullary or periosteal cells and mesenchymal stem cells can be identified by specific cell surface markers that 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, umbilical cord blood, muscle, fat, bone and perichondrium.

The mesenchymal stem cells can be administered by a variety of procedures. The mesenchymal stem cells can be administered systemically, such as by intravenous, intraarterial or intraperitoneal administration. The mesenchymal stem cells may be from a spectrum of sources including autologous, allogenic or xenogenic.

The mesenchymal stem cells are administered in an amount effective to treat an autoimmune disease in an animal. The mesenchymal stem cells can be administered in an amount of about 1x10 5 cells / kg to about 1 x 10 7 cells / kg, preferably from about 1x10 6 cells / kg to about 5x10 6 cells / kg. The amount of mesenchymal stem cells that will be administered depends on a variety of factors, including the age, weight and sex of the patient, the autoimmune disease to be treated and the extent and severity of the same.

The mesenchymal stem cells can be co-administered with an acceptable pharmaceutical carrier. For example, mesenchymal stem cells may be administered as a cell suspension in a pharmaceutically acceptable liquid medium for injection.

Also disclosed is a method for treating an inflammatory response in an animal. The method comprises administering animal mesenchymal stem cells in an effective amount to treat the inflammatory response in the animal.

Although the scope of this aspect of the present disclosure is not limited by any theoretical reasoning, it is believed that mesenchymal stem cells promote maturation of T cells to regulatory T cells (TReg), thereby controlling inflammatory responses. It is also believed that mesenchymal stem cells inhibit the T helper cells 1 (Th1 cells), thereby decreasing the expression of interferon-Y (IFN-y) in certain inflammatory reactions, such as those associated with psoriasis, for example.

In one disclosure, the inflammatory responses that can be treated are those associated with psoriasis.

In another disclosure mesenchymal stem cells can be administered to an animal in such a way that the mesenchymal stem cells come in contact with the microglia and / or the astrocytes in the brain to reduce inflammation, whereby the mesenchymal stem cells limit the neurodegeneration caused by glial cells activated in diseases or disorders such as Alzheimer's disease, Parkinson's disease, apoplexy or lesions of brain cells.

In yet another disclosure, mesenchymal stem cells can be administered to an animal in such a manner that the mesenchymal stem cells come in contact with keratinocytes and Langerhans cells in the epidermis of the skin to reduce inflammation, as may occur in psoriasis. , chronic dermatitis and contact dermatitis. Although this embodiment is not limited to any theoretical reasoning, it is believed that mesenchymal stem cells can come into contact with keratinocytes and Langerhans cells in the epidermis, and alter the expression of T cell receptors and secretion profiles of cytokines, which leads to a lower expression of the tumor necrosis factor alpha (TNF-a) and the increase in the population of regulatory T cells (Treg cells).

In a further disclosure, mesenchymal stem cells may be used to reduce inflammation in the bone, as occurs in arthritis and arthritis-like conditions, including, among others, osteoarthritis and rheumatoid arthritis and other arthritic diseases listed on the site. web www.arthritis.org/conditions/diseases. Although the scope of this embodiment 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 in the synovial fluid.

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

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

In another disclosure mesenchymal stem cells can be used to control inflammation in privileged immune sites such as the eye, including the cornea, the lens, the pigment epithelium and the retina, the brain, the spinal cord, the pregnated uterus and the placenta, the ovary, the testicles, the adrenal cortex, the liver and the hair follicles. Although the scope of this embodiment is not limited to any theoretical reasoning, it is believed that mesenchymal stem cells promote the secretion of suppressive cytokines such as IL-10 and the generation of Treg cells. In yet another disclosure, mesenchymal stem cells can be used to control infections with end-stage renal disease (ESRD) during dialysis and / or glomerulonephritis. Although the scope of this embodiment is not limited to any theoretical reasoning, it is believed that mesenchymal stem cells induce peripheral blood mononuclear cells to express vascular endothelial growth factor, or VEGF, which stimulates glomerular structuring.

In a further disclosure, mesenchymal stem cells can be used to control viral infections such as influenza, hepatitis C, herpes simplex virus, vaccine virus infections and Epstein-Barr virus. Although the scope of this embodiment is not limited to any theoretical reasoning, it is believed that mesenchymal stem cells promote the secretion of interferon-beta (IFN-p).

In yet another disclosure, mesenchymal stem cells can be used to control parasitic infections such as Leishmania infections and Helicobacter infections. Although the scope of this embodiment is limited to no theoretical reasoning, it is believed that the mesenchymal stem cells mediate the responses of the auxiliary T 2 (Th2) cells, and thereby promote a greater production of immunoglobulin E (IgE) by the cells. p.

The mesenchymal stem cells can be administered to a mammal, including human and non-human primates, as described above.

The mesenchymal stem cells can also be administered systematically, as described above.

Alternatively, in the case of osteoarthritis or rheumatoid arthritis, the mesenchymal stem cells can be administered directly to an articular 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 about 1x10 5 cells / kg to about 1x10 7 cells / kg, preferably from about 1x10 6 cells / kg to about 5 x 10 6 cells / kg. The exact dosing of the mesenchymal stem cells to be administered depends on a variety of factors, including the age, weight and sex of the patient, the inflammatory response being treated and its extent and severity.

The mesenchymal stem cells can be co-administered with an acceptable pharmaceutical carrier, as described above.

In accordance with yet another disclosure of the present disclosure, a method for treating cancer in an animal is provided. The method comprises administering to the animal mesenchymal stem cells in an amount effective to treat the cancer in the animal.

Although the scope of this aspect of the present disclosure is not limited to any theoretical reasoning, it is believed that mesenchymal stem cells interact with the dendritic cells, which leads to the secretion of IFN-p, which in turn acts as a suppressant. of tumors. Cancers that can be treated include, but are not limited to, hepatocellular carcinoma, cervical cancer, pancreatic cancer, prostate cancer, fibrosarcoma, medulloblastoma, and astrocytoma. However, it should be understood that the scope of the present disclosure is not limited to any specific type of cancer.

The animal may be a mammal, including human and non-human primates, as described earlier in this document.

The mesenchymal stem cells are administered to the animal in an effective amount to treat the cancer in the animal. In general, mesenchymal stem cells are administered in an amount of about 1x10 5 cells / kg to about 1x10 7 cells / kg, preferably from about 1x10 6 cells / kg to about 5x10 6 cells / kg. The exact amount of mesenchymal stem cells to be administered depends on a variety of factors, including the age, weight and sex of the patient, the type of cancer being treated and the extent and severity of the same.

The mesenchymal stem cells are administered together with an acceptable pharmaceutical vetch, and can be administered systematically, as described hereinabove. Alternatively, mesenchymal stem cells can be administered directly to the cancer being treated.

According to yet another disclosure of the present disclosure, there is provided a method for treating an allergic disease or disorder in an animal. The method comprises administering to the animal mesenchymal stem cells in an amount effective to treat the allergic disease or disorder in the animal.

Although the scope of this aspect of the present disclosure is not limited to any theoretical reasoning, it is believed that mesenchymal stem cells, when administered after an acute allergic response, provide inhibition of mast cell activation and degranulation. In addition, it is believed that mesenchymal stem cells downregulate the activation of basophils and inhibit cytokines such as TNF-α, chemokines such as interleukin-8 and monocyte chemoattractant protein, or MCP-1, lipid mediators such as leukotrienes, and inhibit major mediators such as histamine, heparin, chondroitin sulfates and cathepsin. The allergic diseases or disorders that can be treated include, but are not limited to, asthma, allergic rhinitis, atopic dermatitis and contact dermatitis. However, it should be understood that the scope of the present disclosure is 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 can be a mairnfero. The mammal can be a primate, including human and non-human primates. In general, mesenchymal stem cells are administered in an amount of about 1x10 5 cells / kg to about 1x10 7 cells / kg, preferably from about 1x10 6 cells / kg to about 5x10 6 cells / kg. The exact dosage depends on a variety of factors, including the age, weight and sex of the patient, the disease or allergic disorder being treated and the extent and severity of the same.

The mesenchymal stem cells may be administered together with an acceptable pharmaceutical vehicle, as described hereinabove. The mesenchymal stem cells can be administered systematically, for example, by intravenous or intraarterial administration, for example.

According to a further aspect of the present disclosure, there is provided a method for promoting the healing of wounds in an animal. The method comprises administering to the animal mesenchymal stem cells in an amount effective to promote wound healing in the animal.

Although the scope of the present disclosure is not limited to any theoretical reasoning, it is believed that, as mentioned hereinabove, mesenchymal stem cells cause Treg cells and dendritic cells to release interleukin-10 (IL-10). IL-10 limits or controls inflammation in a wound, thus promoting the healing of a wound.

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

The mesenchymal stem cells are administered to the animal in an effective amount 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 of about 1x10 5 cells / kg to about 1x10 7 cells / kg, preferably from about 1x10 6 cells / kg to about 5x10 6 cells / kg. The exact amount of mesenchymal stem cells to be administered depends on 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 together with an acceptable pharmaceutical carrier, as described hereinabove. The mesenchymal stem cells can be administered systematically, as described earlier in this document. Alternatively, the mesenchymal stem cells can be administered directly to a wound, such as in a fluid in a dressing or reservoir containing the mesenchymal stem cells.

According to yet another aspect of the present disclosure, there is provided a method of treating or preventing fibrosis in an animal. The method comprises administering to the animal mesenchymal stem cells in an amount effective to treat or prevent fibrosis in an animal.

The mesenchymal stem cells may be administered to the animal for the purpose of treating or preventing fibrosis in the animal, including, but not limited to, liver cirrhosis, kidney fibrosis associated with end-stage renal disease and fibrosis of the lungs . It should be understood that the scope of the present disclosure is not limited to any specific type of fibrosis.

The 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 of about 1x10 5 cells / kg to about 1x10 7 cells / kg, preferably from about 1x10 6 cells / kg to about 5x10 6 cells / kg. The exact amount of mesenchymal stem cells that will be administered depends on a variety of factors, including the age, weight, and sex of the patient, and the extent and severity of the fibrosis that is being treated or prevented.

The mesenchymal stem cells may be administered together with an acceptable pharmaceutical carrier, as described hereinabove. The mesenchymal stem cells can be administered systematically, also as described above in this document.

Another object of the present invention is to promote angiogenesis in a tissue or organ of an animal, in which such tissue or organ is in need of angiogenesis.

Thus, in accordance with a further aspect of the present invention, there is provided a method for 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.

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

The induction of angiogenesis is used to treat coronary and peripheral artery insufficiency, and therefore it can be a non-invasive and curative approach for the treatment of coronary artery disease, ischemic heart disease and peripheral artery disease. Angiogenesis can play a role in the treatment of diseases and disorders in tissues 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 the implantation of embryos and placental growth, as well as in the development of the embryonic vasculature. Angiogenesis is also essential for the coupling of the reabsorption of the cartilage with bone formation, and is essential for the correct morphogenesis of the growth plate.

Additionally, angiogenesis is necessary for the successful engineering and maintenance of highly metabolic organs, such as liver, where a dense vascular network is needed to provide sufficient transport of nutrients and gases.

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 systematically, such as 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, allogenic or xenogenic.

Although the scope of the present disclosure is not 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 vascular endothelial growth factor, or VEGF. , which stimulates the formation of new blood vessels.

In one embodiment, the animal is a mammal. The mammal can be a primate, including human and non-human primates.

The mesenchymal stem cells, according to the present disclosure, can be used in the treatment, alleviation or prevention of any disease or disorder that can be alleviated, treated or prevented by angiogenesis. Thus, for example, mesenchymal stem cells can be administered to an animal to treat blocked arteries, including those of the extremities, i.e., arms, legs, hands and feet, as well as the neck or in various organs. For example, mesenchymal stem cells can be used to treat the blocked arteries that supply the brain, to treat or prevent apoplexy. In addition, mesenchymal stem cells can be used to treat blood vessels in embryonic and postnatal corneas and can be used to provide glomerular structuring. In another embodiment, the mesenchymal stem cells can be used in the treatment of wounds, both internal and external, as well as in the treatment of dermal ulcers that are 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.

Additionally, because angiogenesis is involved in embryo implantation and placental formation, mesenchymal stem cells can be used to promote embryo implantation and prevent spontaneous abortion.

In addition, mesenchymal stem cells can be administered to an unborn animal, including humans, to promote the development of the vasculature in the unborn animal.

In another embodiment, the mesenchymal stem cells can be administered to an animal, born or unborn, in order to promote the reabsorption of cartilage and bone formation, as well as promoting the correct morphogenesis of the growth plate.

The mesenchymal stem cells are administered in an effective amount to promote angiogenesis in an animal. The mesenchymal stem cells can be administered in an amount of about 1x10 5 cells / kg to about 1x10 7 cells / kg, preferably about 1x10 6 cells / kg to about 5x10 6 cells / kg. The amount of mesenchymal stem cells that are administered depends on 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 of the same.

The mesenchymal stem cells can be administered together with an acceptable pharmaceutical vehicle. For example, mesenchymal stem cells can be administered as a cell suspension in a pharmaceutically acceptable liquid medium for injection. The injection can be local, that is, directly into the tissue or organ that needs angiogenesis, or systemically.

Mesenchymal stem cells can be genetically engineered with one or more polynucleotides that encode a therapeutic agent. The polynucleotides can be delivered to the mesenchymal stem cells through an appropriate expression vein. Expression vehicles that can be used to genetically design the mesenchymal stem cells include, but are not limited to, retroviral vectors, adenoviral vectors and adeno-associated virus vectors.

The selection of an appropriate polynucleotide encoding a therapeutic agent depends on various factors, including the disease or disorder being treated, and the extent and severity thereof. Polynucleotides encoding the appropriate therapeutic agents and expression vehicles are further described in U.S. Pat. Do not.

6,355,239.

It should 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 antiinflammatory drugs and cells other than mesenchymal stem cells, such as dendritic cells, and can be administered with soluble carriers for cells such as hyaluronic acid or in combination with solid matrices such as collagen, gelatin, or other biocompatible polymers, as appropriate.

It should be understood that the methods described herein can be carried out in various ways and with various modifications and permutations thereof which are well known in the art. It can also be appreciated that any theory exposed as to the modes of action or interactions between types of cells should not be construed as limiting this disclosure in any way, but presented in such a way that the methods of disclosure can be understand more completely.

The invention will now be described with respect to the drawings, in which:

Figure 1. MSCs modulate the functions of the dendritic cells. (A) Cytomene analysis of flow of mature DC1 monodic cells using antibodies against HLA-DR and CD11c and DC2 plasmacytoid cells using antibodies against HLA-DR and CD123 (IL-3 receptor). (-): isotype control; (-): antibodies conjugated with FITC / PE. (B) MSCs inhibit the secretion of TNF-a (axis and primary) and increase the secretion of 1L-10 (secondary and secondary) from activated DC1 and DC2, respectively. (C) MSCs cultured with mature DC1 cells inhibit the secretion of IFN-y (axis and primary) by T cells and increase the levels of IL-4 (axis and secondary) in comparison with MSC or DC alone. The decrease in IFN- ^and proinflammatory production and the increase in the production of anti-inflammatory IL-4 in the presence of MSC indicated a shift of the T-cell population towards an anti-inflammatory phenotype.

Figure 2. MSCs inhibit the function of proinflammatory effector T cells. (A) Cytomene flow analysis of TReg cell numbers (in%) by PBMC staining or non-adherent fraction in MSC culture + PBMC (MSC + PBMC) with CD4 conjugated FITC cone (x axis) and conjugated CD25 antibodies with PE. Gates based on isotype control antibodies were established as background. The graphics are representative of 5 independent experiments. (B) Th1 cells generated in the presence of MSC secreted reduced levels of IFN-y (primary Y-axis) and Th2 cells generated in the presence of MSC secreted higher amounts of IL-4 (axis and secondary) in cell culture supernatants. (C) MSCs inhibit the secretion of IFN-y from purified NK cells cultured for 0, 24 or 48 hours in a 24-well plate. The data shown are the mean ± SD of the cytokine secretion in one experiment and are representative of three independent experiments.

Figure 3. The MSCs lead to a greater number in the population of TReg cells and greater expression of the GITR. (A) A population of Cd4 + CD25 + TReg cells was isolated from PBMC or MSC + Pb Mc cultures (ratio of MSC to PBMC 1:10) (cultured without any additional stimulation for 3 days) using a magnetic isolation method. 2 stages These cells were irradiated (to block any further proliferation) and used as stimulators in a mixed lymphocyte reaction (MLR), in which the responders were allogenic PBMC (ratio of stimulator to responder 1: 100) in the presence of phytohemagglutinin (PHA) (2.5 mg / mL). The cells were cultured for 48 hours, after which 3H thymidine was added, and the incorporated radioactivity was counted after 24 hours. The results showed that the TReg population, generated in the presence of MSC (lane 3), was functionally similar to the TReg cells generated in the absence of MSC (lane 2). (B) PBMC were cultured for 3 days in the absence (upper plot) or presence (lower plot) of MSC (ratio of MSC to PBMC 1:10), after which the non-adherent fraction was collected and immunostained with GITR marked with FITC and CD4 marked with PE. The results show an increase of more than two times in the expression of GITR in cells cultured in the presence of MSC.

Figure 4. MSCs produce PGE2 and blockade of PGE2 reverses the immunomodulatory effects mediated by MSC. (A) secretion of PGE2 (mean ± SD) in culture supernatants obtained from MSCs cultured in the presence or absence of PGE2 blockers NS-398 or indomethacin (Indomet.) At various concentrations. The inhibitor concentrations are in pM and the data presented are values obtained after culturing for 24 hours (B) expression of COX-1 and COX-2 in MSC and PBMC using RT-PCR in real time. The MSCs expressed significantly higher levels of COX-2 in comparison with PBMC, and when MSCs were grown in the presence of PBMC, there was a> 3 fold increase in the expression of COX-2 in MSC. Representative data from 1 of 3 independent experiments are shown. The cultures of MSC + PBMC were prepared in a plate of migration chamber between wells where MSC was placed on the lower chamber and PBMC in the upper chamber. (C) The presence of PGE2 blockers indomethacin (Ind.) Or NS-398 increases the secretion of TNF-a from activated DC (□) and the secretion of IFN-y from T cells ^h 1 (□) compared with the controls. The data were calculated as the percentage change of the cultures generated in the absence of MSCs and PGE2 inhibitors (D) The presence of PGE2 blockers indomethacin (Indo) and NS-398 during the cocultivation of MSC-PBMC (1:10) reversed the anti-proliferative effects mediated by MSC in PBMC treated with PHA. The data shown are from one experiment and are representative of 3 independent experiments.

Figure 5. The constitutive secretion of MSC cytokines is elevated in the presence of allogenic PBMC. Using the previously characterized human MSCs, the levels of the cytokines 1L-6 and VEGF, the lipid mediator PGE2 and the matrix metalloproteinase 1 (pro MMP-1) in the culture supernatant of MSCs cultured for 24 hours in the presence were analyzed ( shaded bars) or absence (bars without color) of PBMC (relation of MSC with respect to PBMC 1:10). The MSCs produced 1L-6, VEGF and PGE2 constitutively, and the levels of these factors increased in the cocultivation with PBMC, suggesting that MSCs can play a role in the modulation of immune functions in an inflammatory environment.

Figure 6. The MSCs inhibit the proliferation of T cells induced by mitogen in a dose-dependent manner. Increasing amounts of allogenic PBMC were incubated with a constant number of MSCs (2,000 cells / well) in 96-well plates in the presence or absence of PHA (2.5 mg / mL) for 72 hours, and the incorporation of 3H thymidine was determined. (in counts per minute, or cpm). There was a dose-dependent inhibition of the proliferation of PBMC treated with PHA in the presence of MSC. Representative results from 1 of 3 independent experiments are shown. Similar results were reported by LeBlanc et al., Scand J. Immunol., Vol. 57, p. 11 (2003). Figure 7. Schematic diagram of the proposed mechanism of action of MSC. The MSCs mediate their immunomodulatory effects by affecting the cells of both the innate immune systems (routes 2 and 4 of DC, and route 6 of NK) and adaptive (routes 1 and 5 of T and route 7 of B). In response to an invading pathogen, immature DCs migrate to the site of potential entry, mature and acquire the ability to prime non-altered T cells (by specific signals and antigen costimulators) to become protective effector T cells (cell-mediated Th1 immunity or Th2 humoral). During the MSC-DC interaction, MSCs, through direct cell-cell contact or through a secreted factor, can alter the result of the immune response by limiting the ability of DCs to mount a cell-mediated response (path 2) or by promoting the ability to mount a humoral response (route 4). In addition, when mature effector T cells are present, MSCs can interact with them to shift the balance of T ^h 1 responses (path 1) towards T ^h 2 responses (path 5), and probably towards an increase in activity of IgE producing B cells (route 7), the desirable results for the suppression of GvHD and the symptoms of autoimmune disease. MSCs in their ability to result in a greater generation of TReg population (route 3) can result in a tolerant phenotype and can help a host host by buffering the inflammation of neighbors in their local microenvironment. The line of points (-) represents the proposed mechanism.

The invention will now be described with respect to the following examples; it should be understood, however, that the scope of the present invention should not be limited thereto.

Example 1

Materials and methods

Cultivation of human MSCs

Human MSCs were cultured as described in Pittenger et al., Science, vol. 284, p. 143 (1999). In summary, samples of marrow from the ilfaque crest of anonymous donors were collected after the informed consent of Poietics Technologies, Div. Of Cambrex Biosciences. The MSCs were cultured in a 1% antibiotico-antifungal solution (Invitrogen, Carlsbad, California) with low glucose content - complete Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, California) and 10% fetal bovine serum ( FBS, JRH BioSciences, Lenexa, Kansas). The MSCs grew as an adherent monolayer and were separated with trypsin / EDTA (0.05% trypsin at 37 ° C for 3 minutes). All the MSCs used were previously characterized by the potential of multilineage and retained the ability to differentiate into mesenchymal lineages (chondroditic, adipogenic and osteogenic) (Pittenger, et al., Science, volume 284, page 143 (1999)).

Dendritic cell isolation

Peripheral blood mononuclear cells (PBMC) were obtained through Poietics Technologies, Div. Of Cambrex Biosciences (Walkersville, MD). The dendritic cell (DC) precursors of monocytic lineage (CD1c +) were positively selected from PBMC using a 2-step magnetic separation method according to Dzionek et al. al., J. Immunol., vol. 165, p. 6037 (2000). Briefly, the amount of CD19 + cells in B cells expressing CD1c using magnetic beads was magnetically depleted, followed by labeling of the depleted fraction in B-cells with biotin-labeled CD1c (BDCA1 +) and anti-biotin antibodies and separating them from the fraction. Unmarked cellular using magnetic columns according to the manufacturer's instructions (Miltenyi Biotech, Auburn, California). DC precursors of plasmacytoid lineage were isolated from PBMC by immunomagnetic sorting of positively labeled antibody-coated cells (BDCA2 +) (Miltenyi Biotech, Auburn, California).

Cultivation of MSC-DC

In the majority of experiments, human MSCs and DCs were cultured in equal numbers for several periods of time and the cell culture supernatant was collected and stored at -80 ° C until further evaluation. In selected experiments, MSCs were cultured with mature DC1 or DC2 cells (ratio of MSC: DC of 1: 1) for 3 days and then the combined cultures were irradiated (MSC and DC) to avoid any proliferation. Then, purified allogenic T cells not altered with antibodies (CD4 +, CD45RA +) were added to the irradiated MSC / DC and cultured for 6 more days. The non-adherent cell fraction (purified T cells) was collected after the cultures, washed twice and re-stimulated with PHA for another 24 hours, after which supernatants were harvested from the cell culture and analyzed for IFN-y. secreted and IL-4 by ELISA.

Isolation of NK cells

Purified populations of NK cells were obtained by depleting non-NK cells that are magnetically labeled with a cocktail of monoclonal antibodies conjugated with biotin (anti-CD3, anti-CD14, anti-CD19, anti-CD36 and anti-IgE antibodies) as primary reagent and anti-biotin monoclonal antibodies conjugated with microbeads as secondary labeling reagent. The non-NK cells marked magnetically were retained in MACS columns (Miltenyi Biotech, Auburn, California) in a magnetic field, while the NK cells passed through and were collected.

Isolation of the TReg cell population

The TReg cell population was isolated using a two-step isolation procedure. First, non-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 on a MACS column (Miltenyi Biotech, Auburn, California). Next, the CD4 + CD25 + cells were directly labeled with CD25 microbeads and isolated by positive selection from the fraction of previously enriched CD4 + cells. The CD4 + CD25 + T cells were magnetically marked on the column and eluted after removing the column from the magnetic field.

In order to determine if the increase in the CD4 + CD25 + population generated in the presence of MSCs were of a suppressive nature, populations of TR4 CD4 + CD25 + cells were isolated from PBMC or MSC + PBMC cultures (MSC ratio with respect to a PBMC of 1:10) (cultured without any additional stimulation for 3 days) using a 2-stage magnetic isolation procedure. These cells were irradiated to block any further proliferation and used as stimulators in a mixed lymphocyte reaction (MLR), where the responders were allogenic PBMC (ratio of stimulator to responder 1: 100) in the presence of PHA (2). , 5 | jg / mL). The culture was carried out for 48 hours, after which thymidine 3H was added. The incorporated radioactivity was counted after 24 hours.

PBMC were cultured in the absence or presence of MSC (ratio of MSC to PBMC 1:10), after which the nonadherent fraction was collected and immunostained with the TNF receptor induced by FITC-labeled glucocorticoids, or GITR , and CD4 marked with PE.

Generation of Th1 / Th2 cells

Peripheral blood mononuclear cells (PBMC) were seeded at 2 x 106 cells / mL for 45 min at 37 ° C in order to eliminate the monocytes. The nonadherent fraction was incubated in the presence of anti-CD3 (5 jg / mL) and anti-CD28 (1 jg / mL) antibodies bound to the plate under T ^h 1 conditions (IL-2 (4 ng / mL) IL -12 (5 ng / mL) anti-IL-4 (1 jg / mL) or T ^h 2 (IL-2 (4 ng / mL) IL-4 (4 ng / mL) anti-IFN-Y (1 jg) / mL)) for 3 days in the presence or absence of MSC The cells were washed and then stimulated again with PHA (2.5 jg / mL) for another 24 or 48 hours, after which the IFN levels were measured. -y and IL-4 in culture supernatants by ELISA (R & D Systems, Minneapolis, Minnesota).

Analysis of the levels of VEGF, PGE2 and pro-MMP-1 in MSC culture supernatant.

Using the previously characterized human MSCs, the levels of interleukin 6 (IL-6), VEGF, prostaglandin E2 (PGE2) lipid mediator and matrix metalloproteinase 1 (pro-MMP-1) in culture supernatant of cultured MSCs were analyzed. 24 hours in the presence or absence of PBMC (ratio of MSC with respect to PBMC of 1:10).

Proliferation of PBMC

Purified PBMC were prepared by centrifugation of leukopack (Cambrex, Walkersville, Maryland) on Ficoll-Hypaque (Lymphoprep, Oslo, Norway). Separated cells were cultured (in triplicate) in the presence or absence of MSCs (seeded 3-4 hours before the addition of PBMC to allow them to settle) for 48 hours in the presence of the PHA mitogen (Sigma Chemicals, St. Louis, Missouri ). In selected experiments, PBMC were resuspended in medium containing PGE2 inhibitors Indometacin (Sigma Chemicals, St. Louis, Missouri) or NS-938 (Cayman Chemicals, Ann Arbor, Michigan). (3H) -thymidine (20 pL in a 200 pL culture) was added and the cells were harvested after an additional 24 hour culture using an automatic collector. The effects of MSCs or PGE2 blockers were calculated as the percentage of the control response (100%) in the presence of PHA.

Quantitative RT-PCR

Total RNA was prepared from cell pellets using a commercially available kit (Qiagen, Valencia, California) and according to the manufacturer's instructions. The contaminating genomic DNA was removed using the DNA-free kit (Ambion, Austin, Texas). The quantitative RT-PCR was performed in an MJ Research Opticon detection system (South San Francisco, California) using the QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, California) with primers at a concentration of 0.5 jM. Relative changes in expression levels in cells cultured under Different conditions were calculated by the difference in Ct values (crossing point) using p-actin as internal control. The sequences for specific primers of COX-1 and COX-2 were: COX-1: 5'-CCG GAT GCC AGT CAG GAT GAT G-3 '(direct), 5'-CTA GAC AGC CAG ATG CTG ACA G-3 '(inverse); COX-2: 5'-ATC TAC CCT CCT CAA GTC CC-3 '(direct), 5'-TAC CAG AAG GGC AGG ATA CAG-3' (inverse).

Increasing amounts of allogenic PBMC were incubated with a constant number of MSCs (2,000 cells / well) seeded in 96 well plates in the presence of PHA (2.5 pg / mL) for 72 hours, and the incorporation of 3H thymidine was determined ( counts per minute, cpm). The PBMC and MSC were cultivated in ratios of MSC: PBMC of 1: 1, 1: 3, 1:10, 1:30 and 1:81.

Results

In the present studies, the interaction of human MSCs with populations of isolated immune cells, including dendritic cells (DC1 and DC2), effector T cells (Th1 and Th2) and NK cells was examined. The interaction of MSCs with each type of immune cells has specific consequences, suggesting that MSCs can modulate several stages in the immune response process. The production of the factor or secreted factors that modulate and could be responsible for the immunomodulatory effects of MSC was evaluated and the synthesis of prostaglandins was implied.

Dendritic myeloid precursor cells (DC1) and plasmacytoid (DC2) were isolated by immunomagnetic classification 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, or IL-3 cells (10 ng / mL) for DC2 cells. Using cytomethane flow, the DC1 cells were HLA-DR + and CD11c +, while the DC2 cells were HLA-DR + and CD123 + (Figure 1A). In the presence of the bacterial lipopolysaccharide of the inflammatory agent (LPS, 1 ng / mL), the DC1 cells produced moderate levels of TNF-a, but when MSCs were present (relationships examined 1: 1 and 1:10), there was a reduction of> 50% in the secretion of TNF-a (Figure 1B). On the other hand, the DC2 cells produced IL-10 in the presence of LPS and their levels increased more than 2 fold after the cocultivation of MSC: DC2 (1: 1) (Figure 1B). Therefore, MSCs modified the cytokine profile of DCs activated in culture towards a more tolerogenic phenotype. In addition, activated DC, when cultured with MSC, were able to reduce IFN- ^y and increase the levels of IL-4 secreted by undated CD4 + T cells (Figure 1C), suggesting a MSC-mediated shift of the phenotype of T cells proinflammatory to anti-inflammatory.

As the increased secretion of IL-10 plays a role in the generation of regulatory cells (Kingsley et al., J. Immunol., Vol 168, page 1080 (2002)), the regulatory T cells were quantified (TReg ) by flow cytomethane in PBMC and MSC cocultures. After cultivation of PBMC with MSC for 3-5 days, there was an increase in the number of TReg cells determined by staining of the PBMC with anti-CD4 and anti-CD25 antibodies (Figure 2A), further supporting a tolerogenic response induced by MSC . The population of TReg CD4 + CD25 + cells, generated in the presence of MSC, expressed higher levels of the TNF receptor induced by glucocorticoids (GITR), a cell surface receptor expressed in populations of TReg cells, and was suppressive in nature, since it suppressed the proliferation of allogenic T cells (Figure 3A, B). Next, the MSCs were investigated for their direct ability to affect the differentiation of T cells. Using purified T cells selected from antibody (Th CD4 + cells), Th1 cells producing IL-4 producing IFN-y and Th2 were generated. in the presence or absence of MSC. When the MSCs were present during differentiation, there was a reduced secretion of IFN-y by TH1 cells and an increase in the secretion of IL-4 by TH2 cells (Figure 2B). No significant changes in the levels of IFN-y or IL-4 were observed when the MSCs were added to the culture after the Th cells had differentiated (after 3 days) in the effector types T ^h 1 or T ^h 2 ( data not revealed). These experiments suggest that MSCs can affect the differentiation of effector T cells directly and alter the secretion of cytokines from T cells to a humoral phenotype.

Similarly, when the MSCs were cultured with purified NK cells (CD3-, CD14-, CD19-, CD36-) in a 1: 1 ratio for different periods of time (0-48 hours), the secretion of IFN was reduced -and in the culture supernatant (Figure 2C), suggesting that MSCs can also modulate the functions of NK cells.

Previous work has indicated that MSCs modify the functions of T cells by a factor or soluble factors (LeBlanc et al., Exp. Hematol., Vol.31, page 890 (2003), Tse et al., Transplantation, vol 75, page 389 (2003) It was observed that the MSCs segregated several factors, including IL-6, prostaglandin E2, VEGF and proMMP-1 constitutively, and the levels of each increased after the culture with PBMC (Figure 5 In order to investigate the factors derived from MSCs that lead to the inhibition of TNF-a and the increase in the production of IL-10 by DCs, the potential role of prostaglandin E2 was investigated, since it has been demonstrated which inhibits the production of TNF-a by activated DCs (Vassiliou, et al., Cell Immunol., vol 223, page 120 (2003)). Conditioned media from the MSC culture (24-hour culture). 0.5 x 106 cells / mL) account for approximately 1,000 pg / mL of PGE2 (Figure 4A) There was no detectable presence of known inducers of PGE secretion 2, for example, TNF-a, IFN-y or IL-1p (data not shown) in the culture supernatant indicating a constitutive secretion of PGE2 by the MSCs. The secretion of PGE2 by hMSCs was inhibited 60-90% in the presence of known inhibitors of the production of PGE2, NS-398 (5 pM) and indomethacin (4 pM) (Figure 4A). As the release of PGE2 secretion occurs as a result of the enzymatic activity of constitutively active cyclooxygenase enzyme 1 (COX-1) and inducible cyclooxygenase enzyme 2 (COX-2) (Harris, et al., Trends Immunol., Vol.23, page 144 (2002)) mRNA expression was analyzed for COX-1 and COX-2 in MSC and PBMC using an inter-well migration culture system. The MSCs expressed significantly higher levels of COX-2 compared to PBMC and expression levels increased> 3 fold after co-cultivation of MSCs and PBMCs (ratio of MSC to P ^b M ^c of 1:10) for 24 hours (Figure 4B) ). Modest changes in COX-1 levels were observed, suggesting that the increase in PGE2 secretion after MSC-PBMC cocultivation (Figure 5) is mediated by COX-2 upregulation. To investigate whether the immunomodulatory effects of MSC in DC and T cells were mediated by PGE2, MSCs were grown 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 the secretion of TNF-a by DC1 and the secretion of IFN-y from Th1 cells (Figure 4C), respectively, suggesting that the effects of MSC on immune cell types may be mediated by secreted PGE2. Recent studies have shown that MSCs inhibit the proliferation of T cells induced by various stimuli (DeNicola et al., Blood, vol 99, page 3838 (2002), LeBlanc et al., Scand. J. Immunol., Vol. , P.

11 (2003)). It was observed that the MSCs inhibit the proliferation of T cells induced by mitogens in a dose-dependent manner (Figure 6) and when the PGE2 inhibitors NS-398 (5 j M) or indomethacin (4 j M) were present, there were an increase> 70% in the incorporation of (3H) thymidine by PBMC treated with PHA in cultures containing MSC compared to controls without inhibitors (Figure 4D).

In summary, an interaction model of 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 IFN- and proinflammatory (route 1) and promote the phenotype of regulatory T cells (pathway 3) and anti-inflammatory Th2 cells (pathway 5). In addition, MSCs can alter the result of the immune response of T cells through DC secreting PGE2, inhibiting proinflammatory DC1 cells (pathway 2) and promoting anti-inflammatory DC2 cells (pathway 4) or regulatory DCs (pathway 3). ). A change towards the immunity of TH2, in turn, suggests a change in the activity of B cells towards the increase in the generation of IgE / IgG1 subtype antibodies (route 7). MSCs, due to their ability to inhibit the secretion of IFN- ^and NK cells, probably modify the function of NK cells (route 6). This model of interactions of MSC: immune cells is consistent with the experiments carried out in other laboratories (LeBlanc et al., Exp. Hematol., Vol.31, page 890 (2003), Tse et al., Transplantation, vol. , page 389 (2003), DiNicola et al., Blood, vol 99, page 3838 (2002)). A more detailed examination of the proposed mechanisms is underway and animal studies are now needed to examine the in vivo effects of MSC administration.

Claims (8)

1. Mesenchymal stem cells for use in the treatment of coronary insufficiency and peripheral artery or for use in the treatment of blocked arteries in a human by promoting or inducing angiogenesis, wherein the mesenchymal stem cells are genetically unmanipulated and allogenic, and where mesenchymal stem cells stimulate peripheral blood mononuclear cells (PBMCs) to produce vascular endothelial growth factor (VEGF) and, therefore, promote or induce angiogenesis. 2. The mesenchymal stem cells for use according to claim 1, wherein the mesenchymal stem cells are for use in the treatment of coronary artery disease, ischemic cardiac disease and peripheral artery disease. 3. The mesenchymal stem cells for use according to claim 1, wherein the blocked arteries are in the extremities or in the neck. 4. The mesenchymal stem cells for use according to claim 3, wherein the limbs are selected between arms, legs, hands and feet. 5. The mesenchymal stem cells for use according to claim 1, wherein the mesenchymal stem cells are for use in the treatment of blocked arteries supplying the brain. 6. The mesenchymal stem cells for use according to claim 5, wherein the mesenchymal stem cells are for use in the treatment or prevention of apoplexy. 7. The mesenchymal stem cells for use according to any one of claims 1 to 6, wherein the mesenchymal stem cells are administered systematically or by direct administration to a tissue or organ in need of angiogenesis. 8. Mesenchymal stem cells for use according to claim 7, wherein the systemic administration is by intravenous, intraarterial or intraperitoneal administration.