WO2010122206A1

WO2010122206A1 - Multipotent stem cells obtained from an adult thymus

Info

Publication number: WO2010122206A1
Application number: PCT/ES2010/070260
Authority: WO
Inventors: Francisco José TINAHONES MADUEÑO; Rajaa El Bekay; Julián SALAS MILLÁN; María Dolores MAYAS TORRES
Original assignee: Fundación Instituto Mediterráneo Para La Biotecnología Y La Investigación Sanitaria (Imabis)
Priority date: 2009-04-24
Filing date: 2010-04-26
Publication date: 2010-10-28
Also published as: ES2347121B1; ES2347121A1

Abstract

The present invention relates to multipotent stem cells derived from the thymus and, preferably, from the fatty tissue, capable of differentiating and producing cells with the characteristics of specialised cells, and to the methods for obtaining such cells. Said cells can be used for preparing drugs and pharmaceutical compositions for the prevention or treatment of lesions and degenerative or genetic diseases, as well as for assessing the cellular response to biological or pharmacological agents. Furthermore, the present invention relates to the use of fatty cells from the thymus of mammals for inducing angiogenesis in at least one isolated endothelial cell as well as to a method for achieving said induction.

Description

POTENTIAL MOTHER M U LTI CELLS OBTAINED FROM THE TIMO OF A

ADULT

The present invention relates to thymus-derived multipotential stem cells and, preferably, adipose tissue, with the ability to differentiate and give rise to cells with specialized cell characteristics, and methods of obtaining them. Said cells can be used in the preparation of drugs and pharmaceutical compositions for the prevention or treatment of lesions, degenerative or genetic diseases, as well as in the evaluation of the cellular response to biological or pharmacological agents. In addition, the present invention relates to the use of mammalian thymus fat cells to induce angiogenesis in at least one isolated endothelial cell as well as to the method to achieve said induction.

STATE OF THE PREVIOUS TECHNIQUE

Currently, the development of technology in the field of stem cells has made them be considered as a promising therapy for various human pathologies, including: chondral, bone and muscle lesions, neurodegenerative diseases, immune rejection, heart disease and disorders of Ia skin (US 5,811,094, 5,958,767, 6,328,960, 6,379,953, 6,497,875).

On the other hand, the stem cells can be a potential source of virtually unlimited amounts of cells, both undifferentiated and differentiated, for the performance of in vitro assays aimed at the search and development of new therapeutic compounds (US Patent 6,294,346), thus as to determine its activity, metabolism and toxicity.

Depending on the origin of the stem cells, we can differentiate between embryonic stem cells and adult stem cells. Embryonic stem cells they originate from the internal cell mass of the blastocysts and have as their main characteristic the fact that they are pluripotential, which means that they can give rise to any adult tissue derived from the three embryonic layers (US 6,200,806).

Despite the high pluripotentiality of embryonic stem cells, therapies based on the use of adult stem cells have a series of technical advantages over those based on embryonic stem cells. First, cells derived from ES cells are normally the object of rejection by the immune system while adult stem cells are not rejected by the immune system if they have been obtained by autologous transplantation or heterologous transplantation of immunocompatible cells. On the other hand, the use of this type of cells is not associated with any type of ethical or legal controversy. Finally, although this type of cells has a lower potential for differentiation than embryonic stem cells, most of them are multipotent, which means that they can be differentiated to more than one type of tissue that comes from the same embryonic layer.

Currently, the two sources most used to derive multipotent stem cells have been bone marrow and subcutaneous adipose tissue, mainly obtained from liposuctions. With multipotential cells derived from adipose tissue, obtained from liposuctions and subcutaneous fat, higher bypass yields have been obtained than in the case of cells from bone marrow. However, not all human adipose tissue has exactly the same endocrine functions and therefore its nature and cellular composition is variable.

Currently, multipotential stem cells have great success potential for use in cell repair therapies. In addition, they constitute a source of undifferentiated and differentiated cells of great utility for the performance of in vitro assays aimed at the development of therapeutic compounds. There is therefore a need to obtain stem cells multipotent with high plasticity and expansion capacity in cultivation for long periods.

EXPLANATION OF THE INVENTION

The inventors have developed multipotential (multipotent) stem cell lines derived from human thymus, and specifically, from adipose tissue. These cell lines from adipose tissue, in addition to their ability to be expanded for long periods of culture, have a high cellular plasticity, so they can be used for use in cell therapy of lesions, degenerative or genetic diseases, with the advantages techniques presented by adult stem cells, such as the simplification of the protocol for the induction of their differentiation or the reduction of the possibility of immunological rejection in transplantation. On the other hand, these cell lines can be used for the development of toxicological, pharmacogenomic or genetic pharmacological studies.

Experimental tests (see examples) demonstrate the presence of multipotential cells in the thymus adipose tissue. For example, the presence of the PPAR gamma marker is determined, a transcription factor that regulates Ia cell differentiation, development and metabolism, related to the differentiation of fat cells. In addition, the inventors determine the presence of other markers of differentiation of cells in adipocytes and their increase with the age of the person from whom the sample is extracted, such as C / EBPa, FABP4 to ADRP. In order for differentiation to fat cells (adiposites) to occur, there must be progenitor cells or multipotential cells. The analysis of the expression levels of the genes encoding CD73, CD29 and Thy-1 indicates that there is presence of multipotential cells.

In addition, it is demonstrated that the thymus fat cells from which the multipotential cells are extracted, are capable of inducing angiogenesis in isolated endothelial cells such as human umbilical cord endothelial cells. As demonstrated in the examples of the present invention, thymus fat cells express the transcription factor related to VEGF angiogenesis.

A first aspect of the present invention, refers to an isolated multipotential stem cell, obtained from the thymus of a mammal, characterized by expressing messenger RNA of CD73, CD29 and Thy-1 with levels higher than the levels of the control cells. Control cells are differentiated cells of the thymus of a mammal.

The isolated multipotential stem cell is obtained from the thymus of a mammal. Preferably the mammal is a human. The thymus is a lymphoid organ, and constitutes one of the most important organs of the body's immune system in which the differentiation of undifferentiated lymphocytes from the bone marrow takes place, thus converting them into mature T cells. The thymus also secretes hormones and other soluble factors, which in addition to controlling the production and maturation of T lymphocytes in the thymus, regulate the activity and interactions of T cells in peripheral tissues. Stem cells can be classified according to their differentiation potential: totipotential stem cells are capable of producing embryonic and extra-embryonic tissue; pluripotential stem cells have the ability to differentiate into tissues from any of the three embryonic layers and, finally, multipotential stem cells, capable of differentiating into different cell types from the same embryonic layer (Weissman et al., 2001. Annu Rev CeII Dev Biol, 17: 387-403). Therefore, the term "multipotential stem cell" or "multipotential stem cells", as used in the present description, refers to cells / s capable of differentiating into different cell types from the same embryonic layer.

The thymus comes from the embryonic layer called endoderm. Endoderm cells give rise to the digestive system, except mouth, pharynx and the terminal portion of the rectum, and respiratory. It also forms the cells that cover the glands that drain into the digestive tract, including those of the liver and pancreas, the epithelium of the ear canal and the tympanic cavity. It also gives rise to the urinary bladder and part of the urethra and the epithelium that lines the follicles of the thyroid gland and the thymus. Therefore, a multipotential stem cell that is obtained from the thymus epithelium can give rise to any cell type that comes from the endoderm.

In a preferred embodiment, the multipotential stem cell is obtained from the stroma of the adipose tissue of the thymus of a mammal. The multipotential stem cell can come from, but not limited to, a primate or a human. Control cells are differentiated cells of adipose tissue of the type, that is, adipocytes. In a more preferred embodiment, the multipotential stem cell is obtained from the stroma of the adipose tissue of the thymus of a human having an age equal to or greater than 65 years.

The multipotential stem cell that is obtained from the stroma of the adipose tissue of the thymus of a mammal comes from the mesoderm. This stem cell Multipotential can give rise to any cell type that comes from the mesoderm. The cell type is selected from the list of cell tissues, but not limited to mesenchymal, connective or muscular tissue. The mesenchymal tissue is selected from the list comprising, but not limited to, bone, cartilaginous, adipose or vascular tissue (endothelial cells, lymphatic cells or blood cells). Muscle tissue is selected from the list comprising, but not limited to, smooth, striated or cardiac muscle tissue.

Hereinafter, to refer to the multipotential stem cells of the thymus or of the adipose tissue of the mammalian thymus, the terms "multipotential stem cell / s of the present invention" or "multipotential stem cell / s" will be used / is of the invention ".

Another preferred embodiment refers to the isolated differential cell derived from the multipotential stem cell that expresses at least one characteristic of a specialized cell. The partially or totally differentiated specialized cell includes, but not limited to, cell lines of the following tissues and organs: cartilage, bone, fat, muscle, nerve tissue, skin, liver or pancreas, for example the specialized cell is selected, but without limiting the list comprising chondrocyte, osteocyte, adipocyte, myocyte, cardiomyocyte, oligodendrocyte, keratinocyte, hepatocyte or pancreatic cell.

The cells included in the previous list come either from the endoderm or from the mesoderm, therefore, the multipotential stem cell of the present invention can be differentiated, but not limited, to any of the cells of the previous list.

A more preferred embodiment refers to the isolated differentiated cell derived from the multipotential stem cell, where the specialized cell is selected from the list comprising either chondrocyte, osteocyte or adipocyte. The ability of chondrogenic, osteogenic or adipogenic in vitro differentiation will be carried out through the use of suitable culture media. For example, the culture media that favor cell differentiation Multipotential mother may contain external signals for the differentiation of the cell such as for example chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules of the micro environment in which the cell types are found to which they want to differentiate said stem cells.

Hereinafter, to refer to the isolated differentiated cell derived from the multipotential stem cell of the invention, the terms "differentiated cell / s of the present invention" or "differentiated cell / s of the invention" will be used.

Another aspect of the invention refers to an isolated cell population comprising either multipotential stem cells of the invention or differentiated isolated cells derived from the multipotential stem cell, characterized by expressing messenger RNA of CD73, CD29 and Thy-1 with higher levels at the levels of the control cells. Control cells are differentiated cells. Preferably, the isolated cell population comprises multipotential stem cells from a primate and, more preferably, from a human. Therefore, this aspect of the present invention refers either to a cell population of the multipotential stem cells of the invention, or to a cell population of the differentiated cells (differentiated cell population) derived from the multipotential stem cell of the present invention.

The terms "differentiated cell" or "differentiated cell population" refer to the isolated cell or to the isolated cell population obtained by culturing the cell or the multipotent stem cells of the invention under conditions that favor the induction of their differentiation to specialized cells, and that as a consequence, they express at least one characteristic of a specialized cell. To carry out the cell differentiation, widely known techniques are used, such as, but not limited to, the one described in the examples of the present invention. The multipotent stem cell of the invention, or the cell population of multipotent stem cells of the invention, can be characterized by the identification of surface and / or intracellular proteins, genes, and / or other markers indicative of their undifferentiated state. The methods that can be used for the characterization include, but are not limited to: immunocytochemical analysis, northern blot analysis, RT-PCR, gene expression analysis in microarrays, proteomic studies or differential display analysis.

Another aspect of the invention relates to a pharmaceutical composition comprising the multipotential stem cell / s of the present invention, differentiated cell / s of the present invention, or any of the cell populations described hereinbefore. .

Another aspect of the invention relates to a pharmaceutical composition comprising the multipotential stem cell / s of the present invention, differentiated cell / s of the present invention, or any of the cell populations described hereinbefore. for use in somatic cell therapy.

The term "somatic cell therapy" as understood in the present invention refers to the use of live somatic cells, both autologous (from the patient itself), as allogeneic (from another human being) or xenogeneic (from animals), whose Biological characteristics have been substantially altered as a result of their manipulation, to obtain a therapeutic, diagnostic or preventive effect, by metabolic, pharmacological or immunological means. Among the pharmaceutical compositions of somatic cell therapy are, for example, but not limited to: cells manipulated to modify their immunological, metabolic or other functional properties in qualitative or quantitative aspects; classified cells, selected and manipulated, which are subsequently subjected to a manufacturing process; cells manipulated and combined with non-cellular components (e.g. matrices or medical devices biological or inert); autologous cell derivatives expressed ex vivo (in vitro) under specific culture conditions; or cells genetically modified or subjected to another type of manipulation to express homologous or non-homologous functional properties previously not expressed. In short, the objective of somatic cell therapy is to restore the function of damaged organs and tissues as a result of traumatic injuries or degenerative diseases. Somatic cell therapy can not only be used for tissue repair, but also as an innovative system for the delivery of therapies, carried by the cells implanted in the patient.

Another aspect of the invention relates to a pharmaceutical composition comprising the multipotential stem cell / s of the present invention, differentiated cell / s of the present invention, or any of the cell populations described hereinbefore. for use in the prevention or treatment of lesions, degenerative or genetic diseases of a cellular tissue that comes from the endodemo or mesoderm embryonic layer that is selected from the list comprising, but not limited to, cellular tissue from cartilage, bone, muscle, heart, skin, liver or pancreas. Preferably the cell tissue is selected from cartilaginous tissue, bone tissue or adipose tissue.

Another preferred embodiment refers to the pharmaceutical composition which also comprises a pharmaceutically acceptable excipient.

The term "excipient" refers to a substance that helps the absorption, distribution or adhesion of any of the cells of the present invention (active substance), stabilizes said active substance or helps the preparation of the drug in the sense of giving consistency or provide flavors that make it more enjoyable. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, function of protection of the medicine such as to isolate it from air and / or moisture, filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

The term "pharmaceutically acceptable" excipient refers to the excipient being allowed and evaluated so as not to cause damage to the organisms to which it is administered.

In addition, the excipient must be pharmaceutically suitable, that is, an excipient that allows the activity of the active principle or of the active principles, that is, that it is compatible with the active principle, in this case, the active principle is any of the compounds of the present invention.

Another preferred embodiment relates to the pharmaceutical composition which also comprises a pharmaceutically acceptable carrier.

A "pharmaceutically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.

The vehicle, like the excipient, is a substance that is used in the medicament to dilute any of the cells of the present invention to a certain volume or weight. The pharmaceutically acceptable carrier is an inert substance or action analogous to any of the cells of the present invention. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent. In another preferred embodiment, the pharmaceutical composition also comprises another active ingredient. This active substance must allow the activity of any of the cells of the invention, that is, it must be compatible.

The pharmaceutical compositions of the present invention can be formulated for administration in a variety of ways known in the state of the art.

In each case the form of presentation of the pharmaceutical composition will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in a therapeutically effective amount. .

Such compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal. , intraarticular, intrasynovial, intrathecal, intralesional, intraarterial, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular, topical, through transdermal patches, rectal, vaginal or urethral, through the administration of a suppository, percutanea, nasal spray , surgical implant, internal surgical paint, infusion pump, catheter, sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled.

The form adapted to parenteral administration refers to a physical state that can allow its injectable administration, that is, preferably in a liquid state. Parenteral administration can be carried out by intramuscular, intraarterial, intravenous, intradermal, subcutaneous or intraosseous administration but not limited to these types of parenteral administration routes. The pharmaceutical composition of the present invention can be associated, for example, but not limited to, with liposomes or micelles A liposome is a spherical vesicle with a phospholipid membrane. The liposome contains a core of aqueous solution. The micelle is a spherical lipid that contains non-aqueous material. Both liposomes and micelles can be used as carriers of various substances between the outside and inside of a cell.

The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex or tolerance of the mammal. In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of the pharmaceutical composition of the invention that produces the desired effect and, in general, will be determined, among other factors, by the characteristics of said pharmaceutical composition and the therapeutic effect to be achieved.

The pharmaceutical compositions of the present invention can be used in a treatment method in isolation or in conjunction with other pharmaceutical compounds.

Another aspect of the present invention relates to the use of any of the cells or isolated cell populations described above in this document for the preparation of a medicament.

A preferred embodiment refers to the use of any of the cells or cell populations described above in this document for the preparation of a somatic cell therapy drug. The somatic cell therapy is carried out in a cellular tissue that comes from the endoderm or mesoderm embryonic layer that is selected from the list comprising, but not limited to, cellular tissue from cartilage, bone, muscle, heart, skin, liver or pancreas. Preferably the cell tissue is selected from cartilaginous tissue, bone tissue or adipose tissue. Another aspect of the present invention refers to the use of any of the cells or cell populations described above in this document for the preparation of a medicament for the prevention or treatment of lesions, degenerative or genetic diseases. Lesions, degenerative or genetic diseases occur in a cellular tissue that comes from the endoderm or mesoderm embryonic layer that is selected from the list that includes, but is not limited to, cellular tissue from cartilage, bone, muscle, heart, skin, liver or pancreas Preferably the cell tissue is selected from cartilaginous tissue, bone tissue or adipose tissue.

Any of the cells or cell populations described above in this document may be applied to the development of in vitro and in vivo assays for the following industrial purposes: drug search, pharmacological studies, toxicological studies, pharmacogenomic studies and / or genetic studies. Such tests can be used for the identification and / or characterization of a multitude of biological targets, bioactive compounds and / or pharmacological agents.

The multipotential (multipotent) stem cell of the invention, or the cell population constituted by, or comprising, the multipotent stem cells of the invention, provides a unique system in which this cell or these cells can be differentiated to give rise to specific lineages. of the same individual. In addition, the multipotent stem cell of the invention, or the cell population constituted by, or comprising, the multipotent stem cells of the invention, provide a source of cells in culture from a potential variety of genetically diverse individuals that can respond from different way to various biological and pharmacological agents. By comparing the responses of cells from a statistically significant population of individuals, the effects of the biological or pharmacological agents tested on the specific cell type can be determined. Unlike most primary cultures, stem cells Multipotent of the invention can be maintained in culture and thus can be studied as time goes by. Therefore, multiple cell cultures from a single or different individuals can be treated with the agent of interest to determine if there are differences in the effect that said agent has on certain types of cells with the same genetic profile or, alternatively, in similar cell types from genetically distinct individuals.

The use of any of the cells or cell populations described hereinbefore in a high-throughput screening system allows a wide range of biological and / or pharmacological agents to be analyzed, as well as combinatorial libraries thereof. , in an effective way, to thereby elucidate its effects on human cells. Such agents include, but are not limited to: peptides, antibodies, cytokines, chemokines, growth factors, hormones, viral particles, antibiotics, inhibitory compounds, chemotherapeutic agents, cytotoxic agents, mutagens, food additives, pharmaceutical compositions or vaccine preparations.

Another aspect of the invention relates to the use of any of the cells or cell populations described above in this document to evaluate in vitro the cellular response to at least one biological or pharmacological agent.

A preferred embodiment of said method for evaluating in vitro the cellular response to biological or pharmacological agents, or combinatorial libraries of said agents, comprises the following: a) isolating the cells provided by the present invention from an individual or a population statistically significant of individuals; b) optionally differentiate isolated cells to a specific cell type; c) expand the cells in culture; d) optionally differentiate expanded cells to a specific cell type; e) contacting the culture with one or more biological or pharmacological agents or with a combinatorial library of said agents; and f) evaluate the possible biological effects of said agents on the culture cells.

In a preferred embodiment of this aspect of the invention, said biological or pharmacological agent is selected from the list comprising peptides, antibodies, cytokines, chemokines, growth factors, viral particles, hormones, antibiotics, inhibitory compounds, chemotherapeutic agents, cytotoxic agents , mutagens, food additives, pharmaceutical compositions or vaccine preparations.

Another aspect of the present invention relates to a method of obtaining a multipotent stem cell isolated from the invention, or a population of multipotential stem cells, characterized in that it comprises the following steps: a) obtaining an isolated biological sample of adipose tissue from a mammalian thymus, and b) select the multipotential stem cell from the biological sample obtained in (a).

Preferably, the isolated biological sample comes from a primate and, more preferably, from a human.

It is possible to obtain a biological sample isolated from the thymus and, preferably, from the thymus adipose tissue, for example, but not limited by open abdominal surgery or laparoscopic surgery. Open abdominal surgery is a surgical technique that allows the abdominal cavity to be left open temporarily, using various procedures known in the state of the art, to facilitate access to the abdomen; The advantage of this type of surgical intervention is that the accessibility to the tissue is complete and therefore an isolated biological sample of thymus and, preferably, of thymus adipose tissue can be obtained. The selection of multipotent stem cells of the present invention can be carried out by different procedures described in the state of the art, and which may include, for example, but not limited to, mechanical processing, enzymatic treatment (for example, but not limited to, with a collagenase), centrifugation, erythrocyte lysis, filtration, plastic culture without any other support or extracellular matrix, culture in media that favor the proliferation of these cells or immunocytometry. Some of these methods are explained in detail in the examples section of the present invention.

Another aspect of the present invention relates to a method of obtaining a differentiated isolated cell derived from the multipotent stem cell of the invention, or a population of differentiated isolated cells derived from said multipotent stem cell, characterized in that it comprises the following steps : a) Obtain an isolated biological sample of mammalian thymus adipose tissue, b) select the multipotential stem cell from the biological sample obtained in (a); and c) cultivate the multipotential stem cell of (b) under conditions that favor the induction of its differentiation to specialized cells.

The methods that can be used to induce the differentiation of the multipotent stem cells of the invention to various specific specialized cell types are known to those skilled in the art.

A preferred embodiment of the present invention refers to the method of obtaining an isolated multipotent stem cell of the invention, or a population of multipotent stem cells, or refers to the method of obtaining a differentiated isolated cell derived from the multipotent stem cell of the invention, or of a population of differentiated isolated cells derived from said multipotent stem cell, where the multipotent stem cell of Ia The invention is obtained from the stroma of the adipose tissue of the thymus of a human having an age equal to or greater than 65 years.

Another aspect of the present invention is the use of at least one thymus adipose cell of a mammal to induce angiogenesis in at least one isolated endothelial cell. Preferably the mammal is a primate. More preferably the mammal is a human.

Angiogenesis is a morphogenetic process in which new capillaries are generated from existing blood vessels. For this, vascular endothelial cells have to invade the surrounding tissue and proliferate at the apex of the new capillary. Both processes, invasion and proliferation, are repeated sequentially until the new capillary network is fully established. Initially, certain endothelial cells degrade their basement membrane and form tiny buds that penetrate the perivascular connective tissue. These buds are formed by migration of endothelial cells to their end and by proliferation of other endothelial cells below the end. At the same time that the yolks are elongated, a lumen gradually forms inside. In this way, hollow tubes are generated that anastomosed with each other forming capillary bonds. New buds may arise from the newly formed capillaries, eventually leading to the formation of a complete capillary network.

A preferred embodiment refers to the use of at least one thymus adipose cell of a mammal, where the isolated endothelial cell is obtained from a blood vessel, capillary or heart. According to a more preferred embodiment, the isolated endothelial cell comes from the umbilical cord.

Another aspect of the present invention refers to the use of at least one mammalian thymus adipose cell to evaluate in vitro the angiogenic cellular response of at least one isolated endothelial cell to at least one agent. Biological or pharmacological. Biological or pharmacological agents have been described throughout this description.

Another aspect of the present invention is a pharmaceutical composition comprising at least one thymus adipose cell of a mammal.

A preferred embodiment refers to the pharmaceutical composition for use in inducing angiogenesis in at least one isolated endothelial cell. According to a more preferred embodiment, the isolated endothelial cell is obtained from a blood vessel, capillary or heart. In an even more preferred embodiment, the isolated endothelial cell comes from the umbilical cord. Preferably the umbilical cord is human.

Another aspect of the present invention relates to a pharmaceutical composition for use in the prevention or treatment of lesions, degenerative or genetic diseases of a cellular tissue that is selected from vascular tissue or cardiac tissue. On the other hand, there are many pathologies dependent on angiogenesis. The lesion, degenerative or genetic disease is selected from the list comprising, but not limited to, rheumatic arthritis, diabetic retinopathy, psoriasis, bartonellosis, rejection of transplanted organs, hemorrhages or ocular neovascularization.

A preferred embodiment refers to the pharmaceutical composition which also comprises a pharmaceutically acceptable carrier or excipient. According to another more preferred embodiment, the pharmaceutical composition also comprises another active ingredient. The terms excipient, vehicle or active principle have already been defined in the present invention.

Another aspect of the present invention relates to a method for inducing angiogenesis in at least one isolated endothelial cell comprising: a) Obtaining an isolated biological sample of mammalian thymus, b) select at least one adipose cell from the biological sample obtained in (a), and c) cultivate at least one isolated endothelial cell and incubate it with the product obtained in section (b) under conditions that favor the migration and proliferation of said endothelial cell

A preferred embodiment refers to the method for inducing angiogenesis, where the isolated endothelial cell is obtained from a blood vessel, capillary or heart. According to a more preferred embodiment of the method, the isolated endothelial cell comes from the umbilical cord.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1. It shows that the thymus adipose tissue has an adipogenic capacity similar or superior to visceral fat.

FIG. 2. It shows that the human thymus adipose tissue increases its adipogenic capacity as the patient's age increases.

FIG. 3. It shows that a thymus extract has the capacity to induce the migration and proliferation of endothelial cells of the human umbilical cord (HUVEC). FIG. 4. It shows that the human thymus adipose tissue has markers of angiogenesis and lymphangiogenesis similar to subcutaneous and visceral fat.

FIG. 5. Shows the immunohistochemical identification of the expression of VEGF in the adipose tissue of the human thymus.

FIG. 6. Shows vascular growth in human thymus adipocytes by immunohistochemical analysis.

FIG. 7. Shows microscope images of differentiated osteocytes from multipotential human thymus fat cells.

FIG. 8. Shows microscope images of differentiated adipocytes from multipotential human thymus fat cells.

FIG. 9. Shows the expression of markers of adipogenesis (PPAR-γ2) and osteogenesis (SPARC) in the differentiation analysis of multipotential stem cells from both thymic fat and subcutaneous fat (SAT) of the same patient.

The figure shows the expression, relative to the endogenous cyclophilin (CyC) gene (the expression of the Cyc gene has the numerical value 1), of the messenger RNA for the adipogenesis marker PPAR-γ2. The expression, relative to the endogenous GADPH gene is also shown (the expression of the GADPH gene has the numerical value 1), of the messenger RNA for the SPARC osteogenesis marker.

Control: Refers to the expression of the differentiation markers of thymus cells or subcutaneous adipose tissue (SAT) that are not multipotential cells from the fat of said tissues.

SAT: Subcutaneous adipose tissue.

PPAR-γ2: Peroxisome Proliferator-Activated Gamma Receptor, isoform 2.

SPARC: Osteonectin. EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1. Characterization and isolation of multipotential cells obtained from adipose tissue of the human thymus. Detection of cell differentiation markers.

1.1. Real-time PCR analysis of the gene expression of the genes that code for different markers of multipotential cells in the adipose tissue of the human thymus.

The gene expression of messenger RNA (mRNA) of the following markers was analyzed: - CD31: Endothelial cell marker and endothelial progenitors.

- CD73 and CD29: multipotential cell markers of adipose tissue.

- Thy-1 (also known as CD90): marker of multipotential cells and preadipocytes.

It was found that the thymus adipose tissue expresses considerable levels of all these markers, which clearly indicates the presence of multipotential cells in said tissue.

1.2. Isolation of multipotential cells from the adipose tissue of the thymus of an adult human.

Despite the multiple existing protocols to isolate and expand multipotential cells and the different approaches to characterize the cells, the

Committee of Mesenchymal and Tissue Stem Cells of the International Society for Cell Therapy has established minimum criteria. First, multipotential cells exhibit adhesion to culture plastic when they are maintained under standard culture conditions. Furthermore, this population expresses markers such as CD105, CD73 and CD90 in a percentage greater than 95% and does not express CD45, CD 34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules, or not being present in more than 2 % of the population.

Once the patient's tissue was removed, the patient was processed, which has already been optimized in a short period of time (<2 hours).

The tissue was washed on a petri dish with a solution composed of Hank ^' s Balancee! Saline Solution (HBSS) with 2% antibiotic / antifungal (PAA) and was carefully minced in order to digest and isolate the stroma, the main source of multipotential stem cells from adipocytes in the thymus adipose tissue. After a couple of washes with the same solution, the mass fat thymus was incubated at 37 ⁰ C for 30 minutes with a solution of collagenase I (GIBCO) at 0.075% which released the stroma.

After three minutes of incubation the action of the collagenase I was quenched by adding a culture medium (Dulbecco ^'s Modified Eagle Medium F12 - DMEM F12) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic / antifungal (PAA). By centrifuging all this at 1200 rpm for 10 minutes, a pellet was obtained, corresponding to the stroma.

The pellet was resuspended in erythrocyte lysis buffer (GIBCO) and incubated at room temperature for 10 minutes. After the incubation time, the cell suspension was filtered with a 100 μm nylon mesh that removed the remains and was centrifuged again at 1200 rpm, this time for 5 minutes.

The pellet obtained, which corresponded with the multipotential cells, was resuspended in a culture medium consisting of DMEM F12 + 10% FBS + 1% antibiotic / antifungal. In this case, a medium without glutamine was used, which was supplemented with stable L-glutamine (PAA). This cell suspension in culture medium were seeded in flasks that were left place overnight at 37 ⁰ C in an oven with 5% CO2 in a humid atmosphere. After the indicated time, it was washed with PBS (Phosphate Buffered Saline) twice, in order to remove the cells that adhered to the plastic in the bottle. When the cells grew in the bottle and it was found that they have reached a confluence greater than 70% of the surface and that they had a typical phenotype, the pass or passage was carried out. In this case Accutase-Solution (PAA) was used to detach the cells from the bottle. The multipotential expansion medium used is MesenCult Human Basal Medium supplemented with MesenCult Human Supplement (PAA). In this way the cells were maintained during subsequent culture passes

1.3. Adipogenic capacity of the thymus adipose tissue.

In FIG. 1 shows the analysis of mRNA expression and ligand-activated proliferative receptor proteins (PPAR-γ, of the acronym for peroxisome proliferator activated receptor) in thymus adipose tissue (TAT) and compared with expression in human visceral (VAT) and subcutaneous (SAT) adipose tissue. PPAR-γ is a transcription factor that regulates cell differentiation, development and metabolism. This factor has been widely related to the differentiation of fat cells and, therefore, seems to act as an inhibitor of angiogenesis.

FIG.1A shows the results of real-time PCR of the isoforms of PPAR-γ (PPAR-γ1 & PPAR-γ2) in thymic (TAT), subcutaneous (SAT) and visceral (VAT) human adipose tissue. Real-time PCR is carried out using Taq Man probes. Gene expression has been normalized with the expression of the endogenous cyclophilin gene (Cyc). The results are expressed as the mean ± SEM of experiments done in triplicate with a n = 26. In FIG. 1 B e shows the westerblotting analysis of PPAR-γ. The graph below the protein gel represents the densitometric analysis of the PPAR-γ / β-actin ratio. ^* means that p <0.05.

1.4. Determination of the presence of differentiation markers of cells in adipocytes and their increase with age.

In FIG. 2 the results of the real-time PCR of the genes indicating the presence of adipogenesis mechanisms in the adipose tissue of the human thymus (c / EBPa, PPARY, FABP4 and ADRP) from three groups of patients separated by age are shown . Gene expression was normalized with the expression of the endogenous cyclophilin gene (Cyc). The results are expressed as the mean ± SEM of experiments done in triplicate with a n = 8 in each group.

-C / EBPa and PPARY are two transcription factors indicating adipogenesis and the generation of new adipocytes.

-FABP4 (fatty acid binding protein 4, adipocyte) is a specific marker of preadipocytes and adipocytes

-ADRP (Adipose Differentiation Related Protein) Indicator marker of the principle of adipocyte differentiation.

In the FIG. 2A-D demonstrates that adipogenesis increases as age progresses. This indicates that the degeneration of the thymic gland in the adult causes the replacement of the immunogenic cells of the thymus with adipocytes. For there to be cell differentiation there must be progenitor cells or multipotential cells.

EXAMPLE 2. Use of thymus fat cells to induce angiogenesis.

By definition, angiogenesis is the process of extension of blood vessels already formed by budding of new capillaries through the migration and proliferation of previously differentiated endothelial cells.

2.1. Demonstration that the thymus extract induces the angiogenesis (migration and proliferation) of umbilical cord endothelial cells.

In FIG. 3 shows the ability of a human thymus extract to induce the proliferation and migration of HUVEC cells (Human umbilical vein endothelial cells).

FIG. 3A shows the microscopic view of the HUVEC cells at the beginning of the cell culture in the first 24 hours. FIG. 3B shows the HUVEC cells cultured after 72 hours. The semiconfluent cells were used for the analysis of migration and proliferation. FIG. 3C shows the effect of thymus adipose tissue extract on the migration of HUVEC cells. The cells of the upper half of the chamber were allowed to migrate through a porous membrane into the lower chamber. The control corresponds to a sample of HUVEC cells with a culture medium without the addition of thymus adipose tissue extract. The sample called "serum" corresponds to a sample of HUVEC cells with a culture medium supplemented with serum. FIG. 3D shows the effect of thymus adipose tissue extract on the proliferation of HUVEC cells. Subconfluent cells were maintained in serum-free medium and incubated in the absence or in the presence of increasing amounts of thymus adipose tissue extract (T1, T2, T3). ^* p <0.05 versus control, p <0.05 versus T2. ^** p <0.01.

2.2. Determination of mRNA and VEGF protein levels in the thymus adipose tissue.

In FIG. 4 shows the expression of mRNA and proteins of the different subtypes of VEGF (Vascular Endothelial Growth Factor) in the adipose tissue of the thymus compared with two other types of adipose tissue, the human visceral and subcutaneous.

In FIG. 4A shows the results of real-time PCR of the VEGF-A, VEGF-B, VEGF-C and VEGF-D isoforms in human thymus (TAT), subcutaneous (SAT) and visceral (VAT) adipose tissue using probes Taq Man. Gene expression was normalized with the expression of the endogenous cyclophilin gene (Cyc). The results are expressed as the mean ± SEM of experiments done in triplicate with a n = 26. In FIG. 4B shows the Westerblotting analysis of VEGF isoforms using specific antibodies. The graph below the protein gel represents the densitometric analysis of the VEGF / β-actin ratio. ^* means that p <0.05.

2.3. Immunohistochemical tests that demonstrate the presence of angiogenic factors (VEGF) or vascular growth in thymic adipocytes.

In FIG. 5 the results of immunohistochemical tests indicating the presence of VEGF factors type A, B, C or D in samples of thymus adipose tissue, subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT) are shown. The cells were labeled with specific antibodies against each of the types of VEGF factor. The negative control corresponds to the nuclei marked with hematoxylin. It has been shown that VEGF type A, B, C or D factors have angiogenic and lymphangiogenic capacity.

In FIG. 6 shows vascular growth in human thymus adipocytes.

EXAMPLE 3. Differentiation of multipotential thymic fat stem cells in osteocytes and adipocytes.

In FIG. 7 and 8 differentiated osteocytes and adipocytes are observed from thymic fat multipotential stem cells of an adult human over 65 years.

Likewise, the differentiation of said thymic fat multipotential stem cells in osteocytes and adipocytes from the subcutaneous fat extracted from the same patients was analyzed and compared with the differentiation obtained from thymic fat multipotential stem cells, both types of cells mother comes from adult patients with an age over 65 years.

No adipocytes were obtained when starting from multipotential stem cells of subcutaneous fat, however, it was possible to observe the differentiation in adipocytes from thymic fat cells. On the other hand, the differentiation in osteocytes was achieved both from multipotential stem cells of thymus fat and subcutaneous fat (FIG. 9). In said FIG. 9 the expression of adipogenesis markers (PPAR-γ2) and osteogenesis (SPARC; a marker of the presence of osteoblasts during bone formation) in the differentiation analysis of multipotential stem cells from both thymic fat and fat is represented. subcutaneous (SAT) of the same patient, with an age over 65 years. The results show the mRNA expression relative to the endogenous cyclophilin gene (Cyc) in the case of the representation of the mRNA expression of adipogenesis markers (PPAR-γ2) and the expression relative to the endogenous GADPH gene in the case of the representation of The mRNA expression of adipogenesis markers respective internal controls. In the case of the osteogenesis marker (SPARC). When the differentiation of the multipotential thymus fat stem cells was compared with the multipotential stem cells of subcutaneous fat at different ages, it was observed that at ages 65 and older, the multipotential stem cells of subcutaneous fat had less ability to differentiate into adipocytes and osteocytes that the multipotential stem cells of thymic fat, which continued to maintain the ability to differentiate.

Therefore, according to the results shown, the clinical application of multipotential stem cells from thymus fat is more effective than the current clinical application of multipotential stem cells of subcutaneous fat, especially when said clinical application is carried out in patients With a very advanced age, over 65 years. Therefore, thymic fat is a source of multipotential stem cells more suitable for the regeneration, for example, of ischemic tissues and in this case of cardiac ischemia. The regeneration of said tissue can be carried out with multipotential stem cells from thymic fat of the patient who manifests the cardiac ischemia.

Claims

1. Isolated multipotential stem cell obtained from the thymus of a mammal, characterized by expressing messenger RNA of CD73, CD29 and Thy-1 with levels higher than the levels of control cells.

2. Multipotential stem cell according to claim 1, wherein the cell is obtained from the stroma of the adipose tissue of the thymus.

3. Multipotential stem cell according to any one of claims 1 or 2, wherein the mammal is a human.

4. Multipotential stem cell according to claim 3, wherein the human has an age equal to or greater than 65 years.

5. Differentiated isolated cell derived from the multipotential stem cell according to any of claims 1 to 4, which expresses at least one characteristic of a specialized cell.

6. Differentiated isolated cell derived from the multipotential stem cell according to claim 5, wherein the specialized cell is selected from the list comprising chondrocyte, osteocyte or adipocyte.

7. Isolated cell population comprising cells according to any of claims 1 to 6.

8. Pharmaceutical composition comprising the cell according to any one of claims 1 to 7 or the cell population according to claim 7.

9. Pharmaceutical composition according to claim 8 for use in somatic cell therapy.

10. Pharmaceutical composition according to any of claims 8 or 9 for use in the prevention or treatment of lesions, degenerative or genetic diseases of cartilaginous tissue, bone tissue or adipose tissue.

11. Pharmaceutical composition according to any of claims 8 to 10 further comprising a pharmaceutically acceptable excipient.

12. Pharmaceutical composition according to any of claims 8 to 11, further comprising a pharmaceutically acceptable carrier.

13. Pharmaceutical composition according to any of claims 8 to 12 further comprising another active ingredient.

14. Use of the cell according to any of claims 1 to 6 or of the cell population according to claim 7 for the preparation of a medicament.

15. Use of the cell or cell population according to claim 14 for the preparation of a somatic cell therapy drug.

16. Use of the cell or cell population according to any of claims 14 or 15, wherein the medicament is used for the prevention or treatment of lesions, degenerative or genetic diseases of cartilaginous tissue, bone tissue or adipose tissue.

17. Use of the cell according to any of claims 1 to 6 or of the cell population according to claim 7 to evaluate in vitro the cellular response to at least one biological or pharmacological agent.

18. Method of obtaining the multipotential stem cell according to any of claims 1 to 4 comprising: a) Obtain an isolated biological sample of mammalian thymus adipose tissue, and b) select the multipotential stem cell from the biological sample obtained in (a).

19. Method of obtaining the cell according to any of claims 5 or 6, comprising: d) Obtaining an isolated biological sample of adipose tissue of mammalian thymus, e) selecting the multipotential stem cell from the biological sample obtained in (a ); and f) cultivate the multipotential stem cell of (b) under conditions that favor the induction of its differentiation to specialized cells.

20. Method according to any of claims 18 or 19, wherein the human is aged 65 years or older.

21. Use of at least one mammalian thymus adipose cell to induce angiogenesis in at least one isolated endothelial cell.

22. Use according to claim 21, wherein the isolated endothelial cell is obtained from a blood vessel, capillary or heart.

23. Use according to any of claims 21 or 22, wherein the isolated endothelial cell comes from the umbilical cord.

24. Use of at least one mammalian thymus adipose cell to evaluate in vitro the angiogenic cellular response of at least one isolated endothelial cell to at least one biological or pharmacological agent.

25. Pharmaceutical composition comprising at least one mammalian thymus fat cell.

26. Pharmaceutical composition according to claim 25 for use in inducing angiogenesis in at least one isolated endothelial cell.

27. Pharmaceutical composition according to claim 26 wherein the isolated endothelial cell is obtained from a blood vessel, capillary or heart.

28. Pharmaceutical composition according to any of claims 26 or 27, wherein the isolated endothelial cell comes from the umbilical cord.

29. Pharmaceutical composition according to any of claims 25 to 28 for use in the prevention or treatment of lesions, degenerative or genetic diseases of vascular tissue or cardiac tissue.

30. Pharmaceutical composition according to any of claims 25 to 29, further comprising a pharmaceutically acceptable carrier or excipient.

31. Pharmaceutical composition according to any of claims 25 to 30, further comprising another active ingredient.

32. Method for inducing angiogenesis in at least one isolated endothelial cell comprising: d) Obtaining an isolated biological sample of mammalian thymus, e) selecting at least one fat cell from the biological sample obtained in (a), and f ) cultivate at least one isolated endothelial cell and incubate it with the product obtained in section (b) under conditions that favor the migration and proliferation of said endothelial cell.

33. Method for inducing angiogenesis according to claim 30, wherein the isolated endothelial cell is obtained from a blood vessel, capillary or heart.

34. Method for inducing angiogenesis according to any of claims 32 or 33, wherein the isolated endothelial cell comes from the umbilical cord.