WO2012076741A1

WO2012076741A1 - Mesenchymal stem cells isolated from peripheral blood

Info

Publication number: WO2012076741A1
Application number: PCT/ES2011/070844
Authority: WO
Inventors: Javier GARCÍA CASTRO; Luis Antonio MARIÑAS PARDO; Miguel Ángel RODRÍGUEZ MILLA; Isabel María CUBILLO MORENO; Armando BLANCO MORÓN; Fernando GARCÍA ALCALDE; Manuel MASIP ORDÓÑEZ; René RODRÍGUEZ GONZÁLEZ; Ruth Rubio Amador
Original assignee: Instituto De Salud Carlos Iii; Universidad De Granada; Fundación Progreso Y Salud
Priority date: 2010-12-10
Filing date: 2011-12-09
Publication date: 2012-06-14
Also published as: ES2384790A1; WO2012076741A8; ES2384790B1

Abstract

The present invention relates to mesenchymal stem cells (MSC) isolated from peripheral blood, characterized in that said cells express the interleukin 13 receptor alpha-2 (IL13RA2), and also to a method for isolating said MSCs, which comprises detecting expression of said IL13RA2 in cells of a peripheral-blood sample and, if required, isolating said cells that express IL13RA2.

Description

MOTHER CELLS ME SENOUIM ALE S ISOLATED FROM BLOOD

PERIPHERAL

FIELD OF THE INVENTION

The present invention relates to mesenchymal stem cells (CMM) isolated from peripheral blood, or from its blood products, characterized in that they express the interleukin 13 alpha-2 receptor (IL13RA2), and its applications. The invention also relates to a method for isolating said CMM based on the detection of the IL13RA2 marker.

BACKGROUND OF THE INVENTION

A type of adult stem cells especially promising in therapeutic applications is that formed by the so-called mesenchymal stem cells (CMM). CMMs are progenitor pluripotent stem cells that have been isolated from bone marrow, adipose tissue, cartilage, skin, muscle, bone, peripheral blood and other tissues. These cells have a potential medical use in tissue regeneration since isolated and expanded CMMs in culture are able to differentiate into osteoblasts, chondrocytes, myocytes and adipocytes, among other cell types, and can be subsequently reintroduced into the human body to repair lost or damaged tissues. Another even more valuable feature in clinical practice is their immunomodulation capacity, which makes them tremendously useful for immune-based diseases.

The differentiation of CMM is not only a fact described in vitro, but in various animal models of tissue damage it has been observed how, in the damaged area, non-hematopoietic cells of the bone marrow or CMM directly appeared, and that these cells they were able to differentiate into the cell lineage of the tissue where they nested (skin, brain, liver, etc.) (Prockop DJ et al. 2003. Proc Nati Acad Sci USA., 100: 11917-11923). This demonstrates the potential of CMMs to repair and regenerate multiple organs thanks to their ability to move from their natural reservoirs. to the peripheral blood, and from there extravacate towards the place where the damage is located. This mobilization implies the presence of CMM in the blood, at least temporarily. Indeed, several articles have shown the possibility of isolating CMM from peripheral blood, umbilical cord blood and even fetal blood (Zvaifler NJ et al. 2000. Arthritis Research, 2: 477-488; and He Q et al. 2007, Stem Cells; 25: 69-77).

A barrier to the study and therapeutic application of these cells is the difficulty of isolating CMM from peripheral blood with the methods currently available (Roufosse CA et al. 2004 International Journal Biochemistry Cell Biology, 36: 585-597). The culture of peripheral blood CMM is also poorly reproducible, since only in a small percentage of the samples it is possible, by traditional methods, to obtain and expand for months populations enriched in CMM (Ramírez M et al. 2006. British Journal Sports Medicine, 40: 719-22; García-Castro J et al. 2007, Journal Pediatric Hematology Oncology, 29: 388-392).

At present, the process of obtaining and purifying CMM from various solid tissues is based on the same principle, the innate adherence of CMM to plastic. In general, after disintegrating the tissue and obtaining a sample of cells in suspension, a fraction enriched in mononuclear cells is obtained, using density gradients, such as ficoll or percoll. This fraction is usually deposited directly on standard cell culture flasks with general means to which fetal bovine serum is added, and, the next day, the non-adhered cells are removed and the culture of adhered cells is maintained until it is enriched in CMM (Friedenstein AJ et al. 1974 Transplantation, 17: 331-340; WO 0183709; WO 0134775; WO 2005121317; WO 2005015151). With this methodology, CMM can be obtained from mobilized peripheral blood and with a much lower probability of non-mobilized peripheral blood (Zvaifler NJ et al. 2000, Arthritis Research, 2: 477-488; He Q et al. 2007. Stem Cells, 25 : 69-77; Lazarus HM et al. 1995. Bone Marrow Transplantation, 16: 557-564; Wexler SA et al. 2003. British Journal Haematology, 121: 368-374; WO 0022097; WO 9901145). Patent application WO 2009/040458 describes a method of obtaining CMM with pluripotent capacity that uses peripheral blood from mammals or their blood products as a cellular source, and which consists in the density gradient centrifugation of said peripheral blood and subsequent separation and washing by centrifugation of the obtained mononuclear cell fraction, then resuspended the sedimented cells and culturing them under certain conditions.

It is known that MMC undergo phenotypic and functional changes when they leave the bone marrow to go to the peripheral circulation, which translates into a different gene expression pattern in CMM from bone marrow and in CMM from peripheral blood (Conrad C et to 2008. Stem Cells Dev, 17: 23-27).

Tondreau T et al. (Tondreau T et al. 2005. Stem Cells, 23: 1105-1 112) present three methods for the isolation of CMM from peripheral blood and blood from the umbilical cord: two of them are based on the ability of these cells to adhere to the plastic, and, the third, in the selection of CD133 ⁺ cells consisting of isolating said cells by magnetic beads coated with anti-CD 133 antibody from the mononuclear fraction obtained after subjecting the peripheral blood samples to a density gradient. However, the separated fraction contains both CMM and hematopoietic stem cells, so it is necessary to subsequently cultivate the isolated cells in vitro.

Therefore, there is a need to develop a method of isolation of CMM from peripheral blood alternative to those described in the prior art, which allows CMM to be isolated from said cellular source in a specific, reliable, simple and reproducible manner. .

SUMMARY OF THE INVENTION The present invention is based on the identification of the alpha-2 receptor of interleukin 13 (IL13RA2) in peripheral blood mesenchymal stem cells (CMM), or their blood products, and the use of said IL13RA2 as a marker of said CMM. Therefore, the present invention relates to said CMMs isolated from peripheral blood, or their blood products, which express IL13RA2, and with a method for isolating said CMMs based on the detection of said IL13RA2. The invention also relates to the uses and applications of said isolated CMMs derived from peripheral blood or its blood products.

In one aspect, the invention relates to a mesenchymal stem cell, isolated, from peripheral blood, or its blood products, characterized in that it expresses IL13RA2.

In another aspect, the invention relates to an isolated cell population comprising CMM from peripheral blood, or from its blood products, which express IL13RA2, provided by the present invention.

In another aspect, the invention relates to a composition of CMM from peripheral blood, or its blood products, in which at least 50% of the CMM from peripheral blood, or its blood products, comprising said composition , express IL13RA2. In a particular embodiment, said peripheral blood, or its blood products, comes from a mammal, for example, a human being.

In another aspect, the invention relates to a pharmaceutical composition comprising at least one of said CMM from peripheral blood, or its blood products, provided by the present invention, or the isolated cell population of CMM provided by the present invention, or the composition of CMM from peripheral blood, or its blood products, provided by the present invention in which at least 50% of the CMM from peripheral blood, or its blood products, comprising said composition, express IL13RA2, and a pharmaceutically acceptable vehicle.

In another aspect, the invention relates to an in vitro method for the identification and / or isolation of CMM from peripheral blood or its blood products which it comprises detecting the expression of IL13RA2 in cells of a peripheral blood sample or its blood products and, if desired, isolating said cells expressing IL13RA2. Said CMM, isolated, obtainable according to said method, as well as its applications, constitute additional aspects of the present invention.

In another aspect, the invention relates to the use of IL13RA2 as a marker of a CMM from peripheral blood, or its blood products.

In another aspect, the invention relates to the use of a reagent capable of detecting said IL13RA2 for the identification and / or isolation of CMM from a peripheral blood sample, or its blood products.

Finally, the uses of said CMM, cell population, CMM composition or pharmaceutical composition provided by this invention constitute additional inventive aspects, for example, its use in the preparation of a medicament to induce transplant tolerance, or to treat an autoimmune disease, or to treat an inflammatory disease, or to repair and regenerate tissues, or as a cellular vehicle of therapeutic agents. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Characterization of peripheral blood CMM according to the panel of markers established for the CMM in in vitro culture. A) Histograms showing the analysis of the markers CD105, CD73, CD90, CD-19 and HLA-DR. B) Histograms showing the analysis of the markers CD-106, CD-14 and CD-45.

Figure 2. Functional characterization of CMM of peripheral blood for its ability to differentiate adipocytes and osteocytes. A) Adipogenic differentiation control; B) Adipogenic differentiation; C) Osteogenic differentiation control; D) Osteogenic differentiation. Figure 3. Analysis by qRT-PCR of differential expression of IL13RA2 mRNA in peripheral blood CMM, in bone marrow CMM and in adipose tissue CMM.

Figure 4. Analysis of the expression of IL13RA2 in CMM obtained from different tissues: peripheral blood, bone marrow, adipose tissue and umbilical cord.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of IL13RA2 in CMM of peripheral blood, or of its blood products, and in the use of said IL13RA2 as a marker of said CMM. Indeed, the inventors have observed that the MMC of peripheral blood of a mammal (human) express IL13RA2, which allows to use said IL 13RA2 as a marker to identify and / or isolate CMM from peripheral blood, or its blood products, of a subject. Said inventors have also observed that, surprisingly, said marker (IL13RA2) is overexpressed in CMM of peripheral blood with respect to the expression of said marker in CMM of bone marrow and in CMM of adipose tissue. As shown in Example 1, the inventors isolated total CMM RNA from peripheral blood (SP-CMM), bone marrow (MO-CMM) and adipose tissue (TA-CMM), and subjected it to expression tests gene by microarrays to identify differentially expressed genes in each of said cell populations. Among the genes differentially expressed in SP-CMM with respect to MO-CMM and TA-CMM is IL13RA2. Based on this discovery, the inventive aspects that are explained in detail below have been developed.

CMM FROM PERIPHERAL BLOOD OR ITS HEMODERIVATES OF THE INVENTION (SP-CMM) In one aspect, the invention relates to an isolated CMM, from peripheral blood, or its blood products, hereinafter SP-CMM of the invention, characterized in that it expresses IL13RA2. In a particular embodiment, the SP-CMM of the invention further expresses one or more plasma membrane markers selected from the group consisting of CAMK2N1, CDH10, CLDN11, LSAMP, PSCA and SFRP1.

The term "stem cell", "stem cell" or stem cell, as used herein, refers to a totipotent, pluripotent or multipotent cell, capable of generating one or more differentiated cell types, and which also has the ability to regenerate itself, that is, to produce more stem cells. Totipotent stem cells can give rise to both the embryonic components (such as the three embryonic layers, the germ lineage and the tissues that will give rise to the yolk sac), as well as the extraembryonic (such as the placenta). That is, they can form all cell types and give rise to a complete organism. Pluripotent stem cells can form any type of cell corresponding to the three embryonic lineages (endoderm, ectoderm and mesoderm), as well as the germinal and yolk sac. They can, therefore, form cell lineages but from them a whole organism cannot be formed. Multipotent stem cells are those that can only generate cells of the same embryonic layer or lineage of origin.

As used in the present invention, the term "mesenchymal stem cell" (CMM) (in English, "mesenchymal stem cell" or "MSC") refers to a pluripotent mesenchymal stem cell originally derived from peripheral blood or its blood products , which has the ability to regenerate itself and differentiate itself to produce descendant cells with a wide phenotypic variety, including connective tissues, bone marrow stroma, adipocytes, dermis and muscle, among others. In general, CMMs have an expression profile of cellular markers characterized in that they are negative for the CD19, CD45, CD14 and HLA-DR markers, and are positive for the CD105, CD106, CD90 and CD73 markers. The SP-CMMs of the invention come from peripheral blood, or from a blood product of a subject. As used herein, the term "subject" includes any animal that presents blood circulation, preferably, a mammal, more preferably, a primate, and even more preferably, a human being, and may be cells of autologous, allogeneic or Xenogenic depending on the subject to be administered. In a particular embodiment, the SP-CMM of the invention is of human origin.

In the present invention, "peripheral blood" is understood as the blood that circulates through the organism of a subject. Likewise, as used herein, the expression "peripheral blood hemoderivatives" includes all those derived from peripheral blood but which do not have all its components, for example, a product resulting from an apheresis process, the layer leuco-platelet {buffy coat), platelet concentrates, etc.

As the person skilled in the art knows, peripheral blood progenitor cells (among which SP-CMMs are found) are found in low concentrations in peripheral blood which makes their collection difficult. However, this situation can be solved by the use of stimulatory factors, including granulocyte colony stimulating factor (G-CSF), which causes the release of stem cells from their natural environment in the bone marrow niches and their mobilization to the blood (what is called mobilized peripheral blood). Therefore, in a particular embodiment, the sample of peripheral blood or its blood products comes from a subject who has been given a stimulatory factor, such as G-CSF, the granulomachophageal colony growth factor (GM-CSF). , a CXCR4 receptor antagonist, for example, the CXCR4 receptor antagonist called AMD3100, or a catecholamine.

As used in the present invention, the term "interleukin alpha-2 receptor 13" or "IL13RA2" (hereafter "marker of the invention") refers to the membrane protein that acts as a receptor inactive, since once interleukin 13 (IL13) is attached to said receiver, it is unable to transduce the signal. At In the context of the present invention, this term includes all variants of IL13RA2, that is, those proteins present in different species that although they have different amino acid sequences perform the same function. For example, in a particular embodiment of the invention, the SP-CMM of the invention is derived from human peripheral blood, or from its blood products; thus, in a particular embodiment, said IL 13RA2 has the amino acid sequence shown in SEQ ID NO: 1 corresponding to human IL13RA2 (GenBank, accession No. AAH33705.1).

Variants contemplated in the context of the present invention include proteins that show at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% identity with the SEQ ID NO: 1. The degree of identity between two proteins or peptides can be determined by conventional methods, for example, using computer-implemented algorithms and methods that are widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J., 1990, Mol. Biol. 215: 403-410).

The SP-CMMs of the invention are capable of expanding ex vivo, that is, after being isolated, said cells can be maintained and allowed to proliferate in a culture medium. In a particular embodiment, said culture medium comprises Dulbecco-modified Eagle medium (DMEM), antibiotics and L-glutamine, supplemented with fetal bovine serum (FBS). It is up to the skill of the person skilled in the art to modify or modulate media concentrations and / or media complements as required for the cells used. Serums often contain cellular factors and components that are necessary for cell viability and expansion. Illustrative, non-limiting examples of sera include FBS, bovine serum (BS), calf serum (CS), fetal ram serum (FCS), neonate ram serum (NCS), goat serum ( GS), horse serum (HS), pig serum, sheep serum, rabbit serum, rat serum (RS), etc. It is also contemplated, if the cells of the invention are of human origin, complement the cell culture medium with a human serum, preferably of autologous origin. It is understood that the sera can be heat inactivated at 55-65 ° C, if deemed necessary, to inactivate the components of the complement cascade. Modulation of serum concentrations, serum withdrawal from the culture medium can also be used to promote the survival of one or more desired cell types. The SP-CMM of the invention can be expanded in a culture medium of defined composition, in which the serum is replaced by a combination of serum albumin, serum transferrin, selenium and recombinant proteins including, but not limited to, insulin , platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF).

Many cell culture media already contain amino acids; however, some need to be supplemented before culturing the cells. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine and the like. Normally antimicrobial agents are also used in cell culture to mitigate microbial contamination. Usually, the antibiotic or antifungal compounds used include mixtures of penicillin and streptomycin, which may also include, but are not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromazine, kanamycin, mitomycin, etc. The hormones can also be used in cell culture and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, b-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH) , etc. The maintenance conditions of the SP-CMM of the invention may also include the use of cellular factors that allow the cells to remain in an undifferentiated form. Subsequently, as the person skilled in the art understands, when you want the cells to differentiate, supplements that inhibit cell differentiation must be removed from the culture medium.

If desired, the SP-CMMs of the invention can be clonally expanded using a suitable method for cloning cell populations. For example, a population Proliferated cells can be physically collected and seeded on a separate plate (or the wells of a "multi-well" plate). Another option is that the cells can be subcloned into a "multi-well" plate in a statistical relationship to facilitate the operation of placing a single cell in each well (eg, from about 0.1 to about one cell / well or even about 0.25 to 0.5 cells / well, such as 0.5 cells / well). Of course, the cells can be cloned at low density (for example, in a Petri dish or other suitable substrate) and isolated from other cells using devices such as cloning rings. The production of a clonal population can be expanded in any suitable culture medium. In any case, isolated cells can be cultured to a suitable point when their development phenotype can be evaluated.

Preferably, the culture of the SP-CMMs of the invention is carried out on an extracellular matrix, so that the cells can adhere to it easily and their growth is favored. Said extracellular matrix will preferably be composed of receptor ligands with the RGD (arginine-glycine-aspartic) binding motif; however, other receptor ligands with other different binding motifs can also be used. If desired, the SP-CMM of the invention can be genetically modified by any conventional method including, by way of illustration, not limitation, transgenesis processes, deletions or insertions in its genome that modify the expression of genes that are important for their properties. basic (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences that encode proteins of interest, such as proteins with therapeutic properties. Table 1 shows, by way of illustration, not limitation, examples of therapeutic proteins that can be produced by the SP-CMM of the invention and the disease that can be treated with them. Table 1

Therapeutic proteins and diseases for which they can be used

In another aspect, the invention relates to an isolated cell population, hereinafter, "cell population of the invention", which comprises mesenchymal stem cells from peripheral blood, or their blood products, which express IL13RA2, ie SP-CMM of the invention. The cell population of the invention can be preserved in the long term in an appropriate medium, compatible with SP-CMM of the invention, for example, isotonic solutions, optionally supplemented with serum; cell culture media or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, as mentioned below. Additionally, if desired, said medium may contain a cryopreservation agent, for example, dimethylsulfoxide (DMSO). The cell population of the invention can be kept frozen under conditions that do not affect or compromise its viability after reconstitution; also, if desired, the cell population of the invention can be found in a cell bank for transplantation. In another aspect, the invention relates to a composition of CMM from peripheral blood, or its blood products, hereinafter "CMM composition of the invention", in which at least 50% of the CMM from blood peripheral, or of its blood products, comprising said composition express IL13RA2, that is, they are SP-CMM of the invention.

In a particular embodiment, said CMM composition of the invention is a composition of CMM from peripheral blood, or its blood products, in which at least 60%, preferably 70%>, more preferably 80%> , even more preferably, 90%>, and, even more preferably, 95% of the CMMs derived from peripheral blood, or their blood products, comprising said composition express IL13RA2, that is, they are SP-CMM of the invention .

Said CMM composition of the invention may contain a medium in which the cells of said CMM of the invention are located; said medium must be compatible with said cells, in particular with the SP-CMM of the invention present in said CMM composition of the invention, for example, isotonic solutions, optionally supplemented with serum; cell culture media or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, as mentioned below.

PHARMACEUTICAL COMPOSITION OF THE INVENTION The SP-CMM of the invention, the cell population of the invention, as well as the CMM composition of the invention, can be part of a pharmaceutical composition for administration to a subject. Therefore, in another aspect, the invention relates to a pharmaceutical composition, hereinafter "pharmaceutical composition of the invention", which comprises a SP-CMM of the invention, a cell population of the invention, or a CMM composition of the invention, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable vehicle" refers to a vehicle that must be approved by a federal government or state government regulatory agency, or listed in the United States Pharmacopoeia or the European Pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more specifically in humans.

The term "vehicle", as used herein, includes diluents, adjuvants, excipients or carriers with which the SP-CMM or the CMM composition of the invention should be administered. Obviously, said vehicle must be compatible with said CMM. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (eg, 0.9% sterile saline NaCl, phosphate buffered saline (PBS), Ringer-lactate solution, etc.) , optionally supplemented with serum, preferably with autologous serum; cell culture media (eg, DMEM, etc.); or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, such as co-lagen, glycoamino-glycan-co-cholesterol, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, polyiorithin, etc., hydrogels, agarose, sulfate. silicone dextran. Also, if desired, the support medium may, in specific embodiments, contain growth factors or other agents. If the support is solid, semi-solid, or gelatinous, the CMM provided by this invention (SP-CMM of the invention and CMM composition of the invention) can be introduced into a liquid phase of the vehicle that is subsequently treated in such a way that It becomes a more solid phase. The pharmaceutical composition of the invention, if desired, may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the CMMs provided by the invention, which comprise said pharmaceutical composition, and / or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators. The stability of the CMMs provided by the invention in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, etc. Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions. Examples of suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin. Additional information on these vehicles can be found in any pharmaceutical technology manual (Galenic Pharmacy).

The pharmaceutical composition of the invention will contain a prophylactic or therapeutically effective amount of the SP-CMM of the invention or of the CMM composition of the invention, preferably, a substantially homogeneous cell population, to provide the desired therapeutic effect. As used herein, the term "therapeutically or prophylactically effective amount" refers to the amount of CMM provided by the invention (SP-CMM of the invention or CMM composition of the invention) contained in the pharmaceutical composition of the invention that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, by the characteristics of said CMMs and the desired therapeutic effect that is sought. In general, the therapeutically effective amount of said CMM provided by the invention to be administered will depend, among other factors, on the subject's own characteristics, the severity of the disease, the form of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the expert in the matter, which should adjust that dose depending on the factors described above. By way of illustrative, non-limiting example, the pharmaceutical composition of the invention can be administered as a single dose, containing between about 10 x ⁵ O and about 10x10 ⁶ SP-CMM of the invention per kilogram (kg) of body weight of the recipient, preferably between about 5xl0 ⁵ and about 5xl0 ⁶ SP-CMM of the invention per kg of body weight of the recipient, more preferably between about lxl O ⁶ and about 2x10 ⁶ SP-CMM of the invention per kg of body weight of the recipient, depending on the factors described above. The dose of SP-CMM of the invention can be repeated, depending on the condition and evolution of the subject, in temporary intervals of days, weeks or months that the specialist must establish in each case.

The pharmaceutical composition of the invention will be formulated according to the chosen form of administration. The pharmaceutical composition of the invention can be prepared in a liquid or gel dosage mode, for example, in the form of a suspension, to be injected or perfused into a subject. Illustrative, non-limiting examples include the formulation of the pharmaceutical composition of the invention in a sterile suspension with a pharmaceutically acceptable excipient, such as an isotonic solution, for example, phosphate buffered saline (PBS), or any other pharmaceutically acceptable carrier. appropriate, for administration to a subject, for example, a human being, parenterally, for example, intravenously, intraperitoneally, subcutaneously, etc., although other alternative routes of administration are also possible.

The administration of the pharmaceutical composition of the invention to the subject will be carried out by conventional means. For example, said pharmaceutical composition can be administered to said subject intravenously using suitable devices, such as syringes, catheters (a standard peripheral intravenous catheter, a central venous catheter or a pulmonary arterial catheter, etc.), trocars, cannulas, etc. The flow of the cells can be controlled by inflating and deflating in series of distal and proximal balloons located within the vasculature of the subject, thus creating temporary areas without flow that promote cellular therapeutic action. In all cases, the pharmaceutical composition of the invention will be administered using the equipment, apparatus and devices suitable for the administration of cellular compositions and known to those skilled in the art.

As the person skilled in the art understands, sometimes direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit can be advantageous. Thus, the direct administration of the pharmaceutical composition of the invention to the desired organ or tissue can be achieved by direct administration (eg, by injection, etc.) on the external surface of the affected organ or tissue by means of insertion of a device. suitable, eg, an appropriate cannula, by arterial or venous perfusion (including retrograde flow mechanisms) or by other means mentioned in this description or known in the art.

The pharmaceutical composition of the invention, if desired, can be stored until the moment of its application by conventional procedures known to those skilled in the art. This pharmaceutical composition can also be stored together with additional medications, useful in the treatment of diseases, in an active form comprising a combination therapy. For short-term storage (less than 6 hours), the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container complementing it or not with a nutrient solution. Medium-term storage (less than 48 hours) is preferably carried out at 2-8 ° C, and the pharmaceutical composition of the invention will include an iso-osmotic and buffered solution in a container composed of, or coated with, material that prevents the cell adhesion Longer term storage is preferably carried out by means of adequate cryopreservation and storage under conditions that promote retention of cellular function.

The pharmaceutical composition of the invention can be used in a combination therapy as previously described useful in the prevention and / or treatment of the disease to be treated. Such additional compounds may be part of the same pharmaceutical composition or may alternatively be supplied in the form of a separate composition for simultaneous or successive administration. (sequential in time) with respect to the administration of the pharmaceutical composition of the invention. Another option is to mix any of said additional compounds in the same composition and administer them together. METHOD OF THE INVENTION

In another aspect, the invention relates to an in vitro method for the identification and / or isolation of a mesenchymal stem cell from peripheral blood or its blood products (SP-CMM of the invention), hereinafter "method of ", which comprises detecting the expression of IL13RA2 in cells of a peripheral blood sample or of its blood products and, if desired, isolating said cells expressing IL13RA2.

In the present invention, "identification" is understood as the process that allows to recognize a CMM present in a peripheral blood sample or its blood products and distinguish it from the rest of the cells present in said sample. Likewise, the term "isolation" of a CMM refers to the separation of said cell from the rest of the components that exist in a sample of peripheral blood or its blood products.

The term "isolated", as used herein, applied to an SP-CMM of the invention means that said CMM is substantially free (free) of other cells normally present in peripheral blood, or in a blood product thereof, of a subject from whom peripheral blood has been extracted for the isolation of said CMM. In general, a CMM from peripheral blood, or a blood product thereof, is essentially free of other cells normally present in said peripheral blood or blood product thereof when separated from at least 80%, preferably from, at least 90%>, more preferably of at least 95%, even more preferably of at least 96%>, 97%, 98% or even 99%, of other cells normally present in said blood peripheral or blood products thereof. The terms "stem cell", "mesenchymal stem cell" or "CMM", "peripheral blood" and "peripheral blood blood products" have been previously defined and explained in the description and are applicable to the method of the invention. The peripheral blood, or its blood products, for the isolation and identification of the SP-CMM of the invention comes from a subject. As used herein, the term "subject" includes any animal, in particular vertebrate animals, preferably mammals, such as mice, rats, horses, pigs, rabbits, cats, sheep, dogs, cows, humans, etc. . In a particular embodiment, the peripheral blood or its blood products is of human origin.

In a particular embodiment, the peripheral blood sample used is selected from the group consisting of fresh or cryopreserved peripheral blood, fresh or cryopreserved mobilized peripheral blood, mobilized and unmobilized peripheral blood obtained by fresh or cryopreserved apheresis techniques, CD34-fresh fraction or cryopreserved obtained from mobilized peripheral blood, "buffy coats" (fraction of a sample of anticoagulated blood, after a density gradient centrifugation, which contains the majority of leukocytes and platelets) and combinations thereof.

Once the peripheral blood sample has been obtained, it must be treated to isolate the mononuclear cells. Any of the methods existing in the state of the art for isolating mononuclear cells from peripheral blood or its blood products can be used in the practice of the present invention, for example, by centrifugation in Ficoll-Hypaque (GE Healthcare Bio-Science), Percoll, sucrose, etc.

As previously mentioned, peripheral blood progenitor cells (among which are the SP-CMM of the invention) are usually found in low concentrations which makes their collection difficult. To solve this situation, stimulatory factors that mobilize CMMs from bone marrow or adipose tissue to peripheral blood can be administered to the subject. Therefore, in a particular embodiment, the Peripheral blood sample or its blood products come from a subject who has been given a stimulating factor, for example, G-CSF, GM-CSF, AMD3100, etc.

Once the mononuclear cells are isolated, the expression of IL13RA2 that acts as a marker of the SP-CMM of the invention, present in peripheral blood or in its blood products, is detected, the identification of said cells and their subsequent isolation being possible.

Therefore, in another aspect, the invention relates to the use of IL13RA2 as a marker for CMM from peripheral blood, or its blood products, in particular, from the SP-CMM of the invention. Therefore, said IL 13RA2 can be used for the in vitro identification and / or isolation of SP-CMM of the invention from peripheral blood or its blood products. The term "interleukin alpha-2 receptor 13" or "IL13RA2" ("marker of the invention") has been previously defined.

In the present invention, "marker" is understood as a protein that distinguishes a cell (or group of cells) from another cell (or group of cells). For example, a protein that is expressed on the surface of precursor cells but not in other cells of a cell population acts as a marker protein for precursor cells. Typically, the marker is a surface cell antigen, so that antibodies that bind to the marker protein can be used in cell selection methods, for example, to produce a cell population rich in cells that express the marker protein. Additionally, the SP-CMM of the invention may contain other cellular markers characteristic of said SP-CMM of the invention. In a particular embodiment, the SP-CMM of the invention further expresses one or more plasma membrane markers selected from the group consisting of CAMK2N1, CDH10, CLDN11, LSAMP, PSCA and SFRP1.

The detection of the marker of the invention and the subsequent isolation of the cells expressing said marker, that is, the isolation of the SP-CMM of the invention, it can be performed by any method that allows cell separation based on a given phenotypic characteristic.

In the identification and / or isolation assays, the cell population is contacted with a specific reagent, labeled or not, depending on whether the assay is performed by a direct or indirect detection method, respectively.

Therefore, in another aspect, the invention relates to the use of a reagent capable of detecting the expression of IL13RA2 for the identification and / or isolation of SP-CMM of the invention.

The term "specific reagent" refers to a member of a specific binding partner. Members of a specific binding partner include, in addition to binding partners composed of antigens and antibodies, partners composed of MHC antigens and T cell receptors, complementary nucleotide sequences, as well as pairs of peptide ligands and their receptor. Specific binding partners include analogs, fragments and derivatives of a specific member of the binding partner. Of particular interest is the use of antibodies as affinity reagents. The production of specific monoclonal antibodies will be apparent to any average expert in the field. In experiments of identification or separation of cell populations, the antibody is mapped. For this, labels are used that include but are not limited to: magnetic particles, biotin and fluorochromes that will allow the identification or separation of that cell type to which the antibody has been bound. Thus, for example, the analysis of the cell population by flow cytometry allows the use of different fluorochrome labeled antibodies that emit at a different wavelength in the same sample. In this way, it is possible to know the specific profile of the cell population for these surface markers, as well as to carry out a separation by the set of markers used. The separation of populations presenting the phenotype of interest can be carried out by affinity separation techniques, including: magnetic separation (using magnetic particles coated with specific antibodies), affinity chromatography, cytotoxic agents bound to antibodies monoclonal or used together with monoclonal antibodies, and "panning" with the antibody associated with a solid support, as well as by other techniques that are suitable. A more precise separation can be obtained by flow cytometry, a technique that allows cell populations to be separated based on the intensity of the staining, together with other parameters such as cell size and cell complexity.

Flow cytometry is a cell analysis technique that involves measuring the characteristics of light scattering and fluorescence that cells possess as they are passed through a laser beam. In the flow cytometric analysis, cells are placed in the presence of a compound that specifically binds to said marker, such as an antibody, and depending on the compound the cells are classified. If the compound is fluorescently labeled, we speak strictly of flow cytofluorometers, known as "cytometers" or "FACS" (for "Fluorescence Analyzer Cell Sorter"). Flow cytometers can analyze particles based on their fluorescence and size. Those known as separators or sorters can also purify populations based on certain characteristics.

In flow cytometry, for a marker to be considered positive, the specific signal observed must be stronger than the background signal intensity, typically it must be at least 10%, preferably 20%, 30%, 40%, 50 %, 60%, 70%, 80%, 90%, or even 100%) higher than the background signal strength, using conventional methods and devices. The background signal is defined as the signal strength given by a non-specific antibody of the same isotype as the specific antibody used to detect the surface marker in conventional FACS analyzes. Once the method of the invention has been put into practice, a CMM isolated from peripheral blood, or its blood products, expressing IL13RA2 (ie SP-CMM of the invention) is obtained. Therefore, in another aspect, the invention relates to an isolated CMM from peripheral blood, or its blood products, which expresses IL13RA2 (SP-CMM of the invention), obtainable by the method of the invention.

Also, in another aspect, the invention relates to an isolated CMM from peripheral blood, or its blood products, which expresses IL13RA2 (SP-CMM of the invention), obtainable by a method comprising detecting IL13RA2 on the surface of said CMM from a sample of peripheral blood or its blood products and isolate said CMM expressing IL13RA2. In another aspect, the invention relates to an isolated cell population comprising said CMMs expressing IL13RA2 (SP-CMM of the invention), obtainable by the method of the invention, or by a method comprising detecting IL13RA2 on the surface of said CMM from a sample of peripheral blood or its blood products and isolate those CMMs that express IL13RA2.

In another aspect, the invention relates to a composition of CMM from peripheral blood, or its blood products, in which at least 50% of the CMM from peripheral blood, or its blood products, comprising said composition are CMMs expressing IL13RA2 (SP-CMM of the invention), obtainable by the method of the invention, or by a method comprising detecting IL13RA2 on the surface of said CMMs from a sample of peripheral blood or its blood products and isolating said CMM expressing IL13RA2. In a particular embodiment, said CMM composition is a composition of CMM from peripheral blood, or its blood products, in which at least 60%, preferably 70%, more preferably 80%, even more preferably, 90%, and, even more preferably, 95% of the CMMs coming from blood peripheral or its blood products, which comprises said composition express IL13RA2.

USES OF THE INVENTION CELLS

The use of CMM in the treatment of diseases is being studied, among other reasons, based on its anti-inflammatory properties in vitro, its efficacy in animal models and some isolated results achieved in clinical trials in humans. Phase I / II clinical trials are being developed to study its use in the treatment of Crohn's disease and multiple sclerosis and it is planned to begin them in the treatment of systemic lupus erythematosus, systemic sclerosis, systemic vasculitis, type 1 diabetes and others. Many immune based diseases.

It is also known from the state of the art the ability of CMMs to interact with cells of the immune system to control an immune immune response, which in the case of autoimmune diseases, is responsible for the destruction of different tissues or specific cells causing its deterioration In these cases, the use of CMM manages to energize the T, B and NK lymphocytes achieving an asymptomatic state free of immunosuppressive medications.

Therefore, within the scope of the present invention, the use of the SP-CMM of the invention is contemplated for the treatment of an autoimmune disease or an inflammatory disease, in the induction of transplant tolerance, in the repair and tissue regeneration, and in the cellular vehiculization of drugs or biologically active compounds to sites of interest, for example, to tumors or areas of tissue damage.

Thus, in another aspect, the invention relates to a composition of matter selected from an SP-CMM of the invention, a cell population of the invention, and a CMM composition of the invention for use in the treatment of an autoimmune disease. ; or, expressed alternatively, with the use of an SP-CMM of the invention, or of a cell population of the invention, or of a composition of CMM of the invention in the preparation of a medicament for preventing and / or treating an autoimmune disease.

In another aspect, the invention relates to a composition of matter selected from an SP-CMM of the invention, a cell population of the invention, and a CMM composition of the invention for use in the treatment of an inflammatory disease; or, expressed alternatively, with the use of an SP-CMM of the invention, or of a cell population of the invention, or of a CMM composition of the invention in the preparation of a medicament for preventing and / or treating a inflammatory disease

Illustrative, non-limiting examples of inflammatory diseases and autoimmune diseases include, Addison's disease, alopecia areata, ankylosing spondylitis, hemolytic anemia, pernicious anemia, thrush, stomatitis, arthritis, arteriosclerosis, osteoarthritis, rheumatoid arthritis, aspermiogenesis, bronchial asthma autoimmune asthma, autoimmune hemolysis, Bechet's disease, Boeck's disease, inflammatory bowel disease, Burkitt's lymphoma, Crohn's disease, chorioiditis, ulcerative colitis, celiac disease, cryoglobulinemia, dermatitis herpetiformis, dermatomyositis, insulin-dependent diabetes, juvenile diabetes, diseases autoimmune demielinizers, Dupuytren's contracture, encephalomyelitis, allergic encephalomyelitis, endophthalmia, allergic enteritis, autoimmune enteropathy syndrome, leprosy nodal erythema, idiomatic facial paralysis, chronic fatigue syndrome, rheumatic fever, glomerulonephritis, Goodpasture syndrome, syn Graves drome, Harnman-Rich disease, Hashimoto's disease, sudden hearing loss, chronic hepatitis, Hodgkin's disease, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, iritis, leukopenia, disseminated lupus erythematosus, lupus erythematosus, cutaneous lupus, erythematosus lymphogranuloma, infectious mononucleosis, myasthenia gravis, traverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, pemphigus vulgaris, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradicultis, psoriasis, purpuraderma, psoriasis purpura, puri syndrome sarcoidosis, ataxic sclerosis, progressive systemic sclerosis, scleritis, scleroderma, multiple sclerosis, sclerosis disseminated, infertility due to anti-sperm antibodies, thrombocytopenia, thymoma, acute anterior uveitis, vitiligo, diseases associated with AIDS, SCID and Epstein Barr virus such as Sjorgren's syndrome, B-cell lymphoma associated with AIDS or Epstein's virus -Barr, parasitic diseases such as leishmaniasis and immune suppressed states such as viral infections after transplants, AIDS, cancer, chronic active hepatitis, transplant rejection as a result of tissue or organ transplantation and graft versus host disease that may result of bone marrow transplantation or ethical hematopoy stem cells. The immunomodulatory capacity of CMMs is not only important in the treatment of autoimmune diseases, but they are also outlined as an indispensable treatment element to promote tolerance towards solid organs such as the heart, lung and kidney; by way of illustration, co-infusion of CMM is possible at the time of transplantation.

Therefore, in another aspect, the invention relates to a composition of matter selected from an SP-CMM of the invention, a cell population of the invention, and a CMM composition of the invention for use in inducing tolerance to a transplant; or, expressed alternatively, with the use of an SP-CMM of the invention, or of a cell population of the invention, or of a CMM composition of the invention in the preparation of a medicament for inducing tolerance to a transplant.

Once the CMM enters the bloodstream, they are able to detect molecules that are secreted by damaged or dying tissues in what is known as the "homing" or nesting phenomenon. Once in close proximity to damaged tissue, the CMM adheres to the surface of the organ through molecular receptors that it expresses on the surface of the cell membrane. This starts a series of events that allow the CMM to integrate into the damaged organ and begin to secrete growth factors that locally stimulate the resident stem cells of the affected organ itself, in addition to changing the inflammatory microenvironment to give rise to an allowable microenvironment. to cell regeneration, in where a process of cell fusion or differentiation begins, then becoming a physiologically mature cell, in addition to promoting the formation of new blood vessels. Therefore, in another aspect, the invention relates to a composition of matter selected from an SP-CMM of the invention, a cell population of the invention, and a CMM composition of the invention for use in the repair and regeneration of tissues; or, expressed alternatively, with the use of an SP-CMM of the invention, or of a cell population of the invention, or of a CMM composition of the invention in the preparation of a medicament for tissue repair and regeneration .

In another aspect, the invention relates to the use of an SP-CMM of the invention, or a cell population of the invention, or of a CMM composition of the invention as a transport system or vehicle (delivery system) of a biologically active compound at a site of interest, that is, at the site where it is desired that the biologically active compound exert its effect, such as a tumor or an area of tissue damage. A "biologically active compound" as used herein refers to a compound that exerts an effect on the organism of a subject that receives it, for example, a therapeutic effect, and includes proteins, peptides, hormones, enzymes, chemical compounds of low molecular weight, etc. Illustrative, non-limiting examples of biologically active compounds capable of being vehicularized by the SP-CMM of the invention include therapeutic proteins such as erythropoietin (EPO), adrenocorticotropic hormone releasing hormone (CRH), somatotropic hormone releasing hormone (GHRH), Gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), prolactin-releasing hormone (PRH), melatonin-releasing hormone (MRH), prolactin-inhibiting hormone (PIH), somatostatin, adrenocorticotropic hormone (ACTH), somattapa hormone or growth (GH) hormone luteinizing (LH), follicle stimulating hormone (FSH), thyrotropin (TSH or thyroid stimulating hormone), prolactin, oxytocin, antidiuretic hormone (ADH or vasopressin), melatonin, Müllerian inhibitory factor, calcitonin, paratyphoid hormone, gastrin, quinine cholecid ( CCK), secretin, type I insulin growth factor (IGF-I), type II insulin growth factor (IGF-II), atrial natriuretic peptide (PNA), human chorionic gonadotrophin (GCH), insulin, glucagon , somatostatin, pancreatic polypeptide (PP), leptin, neuropeptide Y, renin, angiotensin I, angiotensin II, factor VIII, factor IX, tissue factor, factor VII, factor X, thrombin, factor V, factor XI, factor XIII, interleukin 1 (IL-1), Tumor Necrosis Factor Alpha (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8 and chemokines), interleukin 12 (IL-12), interleukin 16 (IL-16) , alpha, beta, gamma interferons, neuronal growth factor (NGF), growth factor Platelet-derived (PDGF), transforming growth factor beta (TGF-beta), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGF and KGF), epidermal growth factor (EGF and related), factor of vascular endothelial growth (VEGF), granulocyte colony stimulating factor (G-CSF), glial growth factor, keratinocyte growth factor, endothelial growth factor, 1 alpha antitrypsin, tumor necrosis factor, colony stimulating factor granulocytes and macrophages (GM-CSF), cyclosporine, fibrinogen, lactoferrin, tissue type plasminogen activator (tPA), chymotrypsin, immunoglobins, hirudin, superoxide dismutase, imiglucerase, etc.

Additionally, the invention also relates to:

- a method for the prevention and / or treatment of an autoimmune or inflammatory disease comprising the administration of a therapeutically effective amount of a SP-CMM of the invention, or of a cell population of the invention, or of a CMM composition of the invention to a subject suffering from said disease;

- a method for inducing tolerance to a transplant comprising the administration of a therapeutically effective amount of an SP-CMM of the invention, or of a cell population of the invention, or of a composition of CMM of the invention to a subject in need of said treatment, for example, a subject who is to receive or has received a transplant;

- a method for tissue repair and regeneration which comprises the administration of a therapeutically effective amount of an SP-CMM of the invention, or of a cell population of the invention, or of a composition of

CMM of the invention to a subject in need of said treatment; or

- a method for vehiculizing a biologically active compound to a site of interest, such as a tumor or an area of tissue damage, which comprises the administration of a therapeutically effective amount of an SP-CMM of the invention, or a cell population of the invention, or a composition of

CMM of the invention comprising said biologically active compound to a subject in need of said treatment.

In general, for application in said methods, said SP-CMM of the invention, said cell population of the invention, or said CMM composition of the invention, will be administered in the form of a pharmaceutical composition of the invention.

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 features of the invention will be derived partly from the description and partly from the practice of the invention.

The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

EXAMPLE 1

Hierarchical study of differentially expressed genes in SP-CMM versus

MO-CMM and SP-CMM versus TA-CMM I. MATERIALS AND METHODS

Isolation and culture of Mesenchymal Stem Cells (CMM) Human peripheral blood (SP) samples were obtained in heparinized tubes, with prior informed consent, from donors of the Niño Jesús Hospital in Madrid. The mononuclear cells were isolated by centrifugation (400 g, 25 minutes, 20 ° C) of the heparinized samples in a density gradient of Ficoll-Hypaque (GE Healthcare Bio-Sciences). The mononuclear cell fraction was washed twice with PBS and settled (600 g, 5 minutes, room temperature). Next, the cells were resuspended in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 20% fetal calf serum.

Mononuclear cells obtained from human peripheral blood were seeded in fibronectin-coated culture bottles in order to obtain mesenchymal stem cells (CMM) from human peripheral blood. After 24 hours of culture, the non-adherent cells were removed from the culture plate and fresh medium was added. When the culture reached semiconfluence, the cells were trypsinized, washed with culture medium, sedimented (600 g, 10 minutes, room temperature) and replanted at a concentration of 4x10 ³ cells / cm ² . From the third pass a homogeneous CMM population was obtained, which was maintained in MesenPRO Medium (Gibco) commercial culture medium for the experiments.

CMM from bone marrow (MO-CMM) as well as CMM from adipose tissue (TA-CMM) were purchased from Inbiobank (San Sebastián, Spain) (http://www.inbiomed.org/Index.php/development technology / mbiobank / INB / mb MAIN? Pg = index).

All CMMs were characterized based on their morphology, membrane markers and in vitro differentiation potential.

Phenotypic and functional characterization of CMM The CMM obtained were characterized based on the following criteria:

- Morphological characterization: cells with fibroblast, elongated and narrow morphology are observed, large, with a lot of nuclear euchromatin and in the cytoplasm abundant lysosomes.

- Flow cytometry characterization: Receptor expression was analyzed by multiparameter flow cytometry (FACS) using human monoclonal antibodies conjugated with fluorochromes. The cells were resuspended in 200 PBS with 5% human immunoglobulin to favor specific binding. LxlO ⁵ cells were used in each measurement, incubating said cells with defined amounts of antibodies for 30 minutes at room temperature (TA) (18-22 ° C), in the dark, and resuspended in PBS before analysis by flow cytometry. As a negative control the cells were incubated with the same concentrations of isotype antibodies. The analysis was carried out with a FACS Canto cytometer.

- Functional characterization: CMMs are characterized by having the ability to differentiate, with specific means, towards cell types such as adipocytes and osteocytes. In order to assess this capacity, confluent cultures of CMM were incubated for two weeks in defined media for each type of differentiation consisting of:

• Osteogenic differentiation: IMDM medium (Iscove's Modified Dulbecco's Medium) supplemented with 0.1 μΜ dexamethasone, 10 mM beta-glycerol phosphate and 0.2 mM ascorbic acid.

• Adipogenic differentiation: IMDM medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μΜ hydrocortisone and 0.1 mM indomethacin. Subsequently, differentiated cultures were analyzed by histological techniques. The cells were stained with specific dyes, such as "Alizarin Red" for osteocytes and "Oil Red O" for adipocytes. Isolation, dizziness and hybridization of RNA.

Human CMMs were obtained from several donors and expanded in cell culture. Three biological replicates of each sample were prepared, which were independently expanded in vitro. The cell pellets of each sample were stabilized with PrepProtect ™. In total, 24 microarray experiments were performed.

RNA was isolated using a standard protocol (NucleoSpin R RNA II, Macherey-Nagel), and its quality was checked using an Agilent 2100 Bioanalyzer analyzer (Agilent Technologies). The analyzer results were visualized by running a gel. In addition, the software generated the "RNA Integrity Number (RIN)" to certify the purity and quality of RNA samples. The RIN (International Normalized Ratio) value is calculated based on an algorithm that takes into account various parameters, many of them based on ribosomal RNA (rRNA). In short, an RIN of 10 indicates high RNA quality, while a RIN of 1 would indicate a low quality RNA. The samples used obtained RIN values between 7.3 and 10, indicating their high quality.

To label the RNA with Cy3 (fluorescent marker), the RNA samples were amplified and labeled using the "Agilent Low RNA Input Linear Amp Kit" (Agilent Technologies) following the manufacturer's instructions. The amount of cRNA and the incorporation rate of the fluorescent marker (Cy3) were evaluated with an ND-1000 spectrophotometer (NanoDrop Technologies).

The hybridization procedure was performed using the "Agilent Gene Expression Hybridization Kit" (Agilent Technologies). Finally, the fragmented cRNAs labeled with Cy3 were hybridized with "Whole Human Genome Oligo Microarrays 4x44K" using Agilent technology. The microarrays were washed with buffer 6x SSPE containing 0.005% N-laurylsarcosine for 1 minute at room temperature followed by another wash with 0.06x SSPE buffer containing 0.005%) of N-laurylsarcosine for 1 minute at 37 ° C. Finally, a final wash with acetonitrile was performed for 30 seconds.

Image and data analysis

The detection of the fluorescence signal of the hybridization of the Agilent microarrays was carried out by means of the "Agilent Technologies Scanner G2505C" scanner (Agilent Technologies). The "Agilent Feature Extraction Software" (FES) program was used to read and process the microarray image files. This program determines the intensity of each element (including the correction of the background noise), eliminates the extreme values and calculates the statistical significance. Features of the arrays

DNA microarray experiments are very powerful for the investigation of large-scale transcriptional changes. For this study, the RNA obtained using the "Whole Human Genome Oligo Microarray Kit" was hybridized. These microarrays contain approximately 44,000 probes, which include more than 41,000 elements of the human genome. The sequences were obtained from the RefSeq, Ensembl and GenBank databases. Each oligonucleotide probe (60-mer) was printed directly on the array using the "Agilent SurePrint" technology. All hybridization procedures were carried out following the instructions provided by the manufacturer (http://www.agilent.com). The fluorescence intensity was obtained by means of the Agilent Technologies Scanner G2505C scanner and the values were processed by the "Feature Extraction Software" Version 10.2.1.3. Gene expression data Gene expression data consists of microarray data from 24 CMM samples. The samples were separated into three groups depending on their origin:

- MO-CMM: bone marrow derivatives;

- TA-CMM: derived from adipose tissue; Y

- SP-CMM: derived from mobilized peripheral blood.

For the purposes of the study, the MO-CMM and TA-CMM groups were considered as the reference groups. Both are composed of three different samples. On the other hand, the SP-CMM group was considered as the study or trial group. This group was composed of two samples. In order to ensure good quality results and minimize methodological biases, all samples were hybridized in triplicate.

Data preprocessing

The preprocessing of the crude intensities of the probes is a key procedure in the analysis of gene expression data from microarrays. Briefly, background noise correction and normalization are necessary to minimize the effect of possible variations in experimental conditions. The "Feature Extraction Software" program provides, in addition to the raw intensity values, the values obtained by correcting the background noise. However, in order to obtain robust results, more specific preprocessing is necessary. Thus, for the preprocessing process, the raw data was used and the existing methods were applied in the widely known p "R" Limma (Linear Models for Microarray Data) [http://bioinf.wehi.edu .au / limma / or http://www.bioconductor.org]. Because single-color microarrays were used, the preprocessing process for each of the two experiments consisted of three differentiated steps:

Background noise correction of initial data. For this task the "normexp + offset" method was applied, calculating the maximum probability by means of the chair point approach. A displacement value of 50 was selected to stabilize the calculations with low intensities. ii) Standardization Process. The data from the previous step were normalized by applying the quantile normalization method. iii) Generation of the expression matrix. For this task, the average of the three replicates of each sample was calculated, in order to obtain a unique intensity value for each gene in each sample.

Microarray analysis

Gene expression

As a first step in the analysis of preprocessed data, differentially expressed genes present in the experiments were identified. These genes were identified by applying the "two-class unpaired" method of the Microarray Significance Analysis Program (SAM) [Tusher, V. et al. 2001. Proceedings of the National Academy of Sciences, 98: 51 16- 5121]. SAM orders the genes based on a modified t-test statistic. Higher values imply a more significant differential expression of the genes in question. This statistic improves the traditional "fold-change" or t-test approaches [Jones, JO and Arvin, AM 2003. Journal of Virology, 77: 1268-1280; Smyth, GK et al. 2003. Statistical issues in cDNA microarray data analysis. In Functional Genomics: Methods and Protoco ls. Edited by Brownstein MJ et al. USA: Humana Press: 111-136]. One of the main advantages of using SAM is that an overall false positive discovery rate (FDR) is estimated, defined as the fraction of genes that are likely to be identified incorrectly significantly regulated. On the other hand, SAM provides a parameter (called "delta") that allows to vary the relationship between true and false positives. For each experiment, a delta value that optimizes this relationship was selected. Each experiment consisted of 24 microarrays (9 of bone marrow / 9 of adipose tissue and 6 of peripheral blood). As a result, SAM was able to find significantly regulated genes even if they had low variations in expression levels. In order to obtain results with a biologically significant impact, a minimum level was set for 2-fold expression variation throughout the analysis of all experiments.

II. RESULTS

Identification of differentially expressed genes

As a first step, the differences between the gene expression of SP-CMM versus MO-CMM were characterized. For this, a microarray experiment was designed consisting of 15 arrays (9 of MO-CMM and 6 of SP-CMM). The data were pre-processed and standardized as indicated in the Materials and Methods section. For the identification of differentially expressed genes, the widely known "two-class unpaired" method of significance analysis program (SAM) was applied. Thus, a list of genes whose differential expression was significant was obtained, many of which presented very low changes in the level of expression. Because the biological impact of genes with small differences in expression is not clear, an additional threshold was used for 2-fold expression changes in the analysis of all experiments. In this way, 582 genes were found that showed at least a 2-fold change in their expression. Table 2 shows the 10 genes with the highest overexpression and the 10 genes with the highest under-expression.

Table 2

The 10 most over- / under-expressed genes for the SP-CMM experiment versus

MO-CMM

Gene Name Times Description

MMP3 5.28181 matrix metallopeptidase 3

EPB41L3 4,27615 Erythrocyte 3 membrane protein band

-, actor s m ar a receiver e c to qu nas

Next, differences between gene expression in SP-CMM versus TA-CMM were characterized. For this microarray experiment, 15 arrays were used (9 for TA-CMM and 6 for SP-CMM), applying the procedure described above for pre-processing and normalization. On the other hand, the list of genes whose differential expression was significant was also obtained by applying the "two-class unpaired" SAM method. As previously mentioned, those genes that showed at least a 2-fold change in expression were selected for later analysis. Of the 492 genes that meet this criterion, 103 were also found in the case of SP-CMM versus MO-CMM. Table 3 shows the 10 genes with the highest overexpression and the 10 genes with the highest under-expression. Table 3

The 10 most over- / under-expressed genes for the SP-CMM experiment versus

TA-CMM

Finally, CMM biomarker candidates were investigated. 217 and 209 over-expressed biomarkers were found for the SP-CMM versus MO-CMM and SP-CMM versus TA-CMM experiments, respectively. This study has focused on proteins located in the plasma membrane, which are the easiest to use biomarkers to detect and isolate SP-CMM by cell classification or magnetic methods. Thus, 42 and 40 biomarkers located in the plasma membrane were obtained for cases of SP-CMM versus MO-CMM and SP-CMM versus TA-CMM, respectively. Table 4 shows the common biological markers for the two experiments. Table 4

Common biomarkers located in the plasma membrane

III. CONCLUSIONS

Human CMMs have been generated and characterized from peripheral blood (SP-CMM) and the gene expression profiles of these cells have been compared with other human CMMs obtained from bone marrow (MO-CMM) or adipose tissue (TA-CMM) .

High similarity in expression profiles has been found between SP-CMM, MO-CMM and TA-CMM, suggesting that any CMM population derived from any tissue can be defined with similar molecular characteristics. As expected, most of the genes that had differential expression encoded extracellular proteins, indicating that circulating CMMs change their gene expression as an adaptation to the new non-solid environment, that is, peripheral blood. In particular, there are numerous genes that are differentially expressed in SP-CMM with respect to MO-CMM and TA-CMM.

Claims

1. An isolated mesenchymal stem cell, derived from peripheral blood or its blood products, characterized in that it expresses the alpha-2 receptor of interleukin 13 (IL13RA2).

2. An isolated mesenchymal stem cell according to claim 1, obtainable by a method comprising detecting the alpha-2 receptor of interleukin 13 (IL13RA2) on the surface of said cell and isolating said cell expressing IL13RA2.

3. Mesenchymal stem cell according to claim 1, characterized in that it also expresses one or more plasma membrane markers selected from the group consisting of CAMK2N1, CDH10, CLDN11, LSAMP, PSCA and SFRP1.

4. Mesenchymal stem cell according to any one of claims 1 to 3, wherein said peripheral or blood-derived blood is of human origin.

5. Mesenchymal stem cell according to any one of claims 1 to 4, wherein the peripheral blood is selected from the group consisting of fresh or cryopreserved peripheral blood, fresh or cryopreserved peripheral blood, mobilized and unmobilized peripheral blood obtained by techniques of fresh or cryopreserved apheresis, CD34-fresh or cryopreserved fraction obtained from peripheral blood or its mobilized blood products, "buffy coats" and any combination thereof.

6. Mesenchymal stem cell according to any one of claims 1 to 5, wherein the peripheral blood comes from a subject to whom a stimulation factor has been administered.

7. Mesenchymal stem cell according to claim 6, wherein the stimulation factor is selected from the group consisting of granulocyte colony growth factor (G-CSF), colony growth factor granulomacrophages (GM-CSF), a CXCR4 receptor antagonist, a catecholamine, and combinations thereof.

8. An isolated cell population comprising mesenchymal stem cells from peripheral blood or its blood products according to any one of claims 1 to 7.

9. A composition of mesenchymal stem cells from peripheral blood, or their blood products, in which at least 50% of mesenchymal stem cells from peripheral blood, or from their blood products, comprising said composition are stem cells mesenchymal expressing IL13RA2 according to any one of claims 1 to 7.

A pharmaceutical composition comprising a mesenchymal stem cell according to any one of claims 1 to 7, a cell population according to claim 8, or a mesenchymal stem cell composition according to claim 9, and a pharmaceutically acceptable carrier.

In vitro method for the identification and / or isolation of a mesenchymal stem cell from peripheral blood or its blood products comprising detecting the expression of the alpha-2 receptor of interleukin 13 (IL13RA2) in cells of a peripheral blood sample or from its blood products and, if desired, isolate said cells expressing IL13RA2.

12. The method of claim 11, wherein the peripheral blood used is selected from the group consisting of fresh or cryopreserved peripheral blood, fresh or cryopreserved mobilized peripheral blood, mobilized and unmobilized peripheral blood obtained by fresh or cryopreserved apheresis techniques, fresh or cryopreserved CD34 fraction obtained from peripheral blood or its mobilized blood products, "buffy coats", and any combination thereof.

13. A method according to any of claims 11 or 12, wherein said peripheral blood is derived from a subject to which a stimulation factor has been administered.

14. The method of claim 13, wherein said stimulation factor is selected from the group consisting of granulocyte colony growth factor (G-CSF), granulomachophageal colony growth factor (GM-CSF), an antagonist of the CXCR4 receiver, a cateco lamina, and their combinations.

15. Method according to any of claims 11 to 14, wherein said peripheral or blood-derived blood is of human origin.

16. An isolated mesenchymal stem cell obtainable by a method according to any of claims 11 to 15.

17. An isolated cell population comprising mesenchymal stem cells according to claim 16.

18. A composition of mesenchymal stem cells from peripheral blood, or from its blood products, in which at least 50% of mesenchymal stem cells from peripheral blood, or from their blood products, comprising said composition are stem cells mesenchymal expressing IL13RA2 according to claim 16.

19. A pharmaceutical composition comprising a mesenchymal stem cell according to claim 16, a cell population according to claim 17 or a mesenchymal stem cell composition according to claim 18, and a pharmaceutically acceptable carrier.

20. Use of the interleukin 13 alpha-2 receptor (IL13RA2) as a marker of a mesenchymal stem cell from peripheral blood or its blood products.

21. Use according to claim 20, for the identification and / or isolation in vitro of a mesenchymal stem cell from peripheral blood, or a blood product thereof, of a subject.

22. Use of a reagent capable of detecting the alpha-2 receptor of interleukin 13 (IL 13RA2) for the identification and / or isolation of a mesenchymal stem cell from peripheral blood or its blood products.

23. Use of a mesenchymal stem cell according to any one of claims 1 to 7, or 16, or a cell population according to claim 8 or 17, or a stem cell composition according to claim 9 or 18, or a composition pharmaceutical according to claim 10 or 19, in the preparation of a medicament for the treatment of an autoimmune disease.

24. Use of a mesenchymal stem cell according to any one of claims 1 to 7, or 16, or of a cell population according to claim 8 or 17, or of a stem cell composition according to claim 9 or 18, or of a composition pharmaceutical according to claim 10 or 19, in the preparation of a medicament for the treatment of an inflammatory disease.

25. Use of a mesenchymal stem cell according to any one of claims 1 to 7, or 16, or a cell population according to claim 8 or 17, or a stem cell composition according to claim 9 or 18, or a composition pharmaceutical according to claim 10 or 19, in the preparation of a medicament to induce transplant tolerance.

26. Use of a mesenchymal stem cell according to any one of claims 1 to 7, or 16, or of a cell population according to claim 8 or 17, or of a stem cell composition according to claim 9 or 18, or of a composition pharmaceutical according to claim 10 or 19, in the preparation of a medicament for tissue repair and regeneration.

27. Use of a mesenchymal stem cell according to any one of claims 1 to 7, or 16, or a cell population according to claim 8 or 17, or a stem cell composition according to claim 9 or 18, or a composition pharmaceutical according to claim 10 or 19, as a transport system or vehicle of a biologically active compound to a site of interest.