WO2012127094A1

WO2012127094A1 - Isolated cells from the blood of the coronary sinus, and uses thereof

Info

Publication number: WO2012127094A1
Application number: PCT/ES2012/070194
Authority: WO
Inventors: Eduardo DE TERESA GALVAN; Noela RODRIGUEZ LOSADA
Original assignee: Servicio Andaluz De Salud; Universidad De Málaga; Fundación Instituto Mediterraneo Para El Avance De La Biotecnología Y La Investigación Sanitaria (Imabis)
Priority date: 2011-03-23
Filing date: 2012-03-23
Publication date: 2012-09-27

Abstract

The invention falls in the field of cellular biology and medicine, and relates to a cell and/or cell population that has a new cellular phenotype and carries out a contractile process without non-physiological external inductors, in a sub-optimal non-specific culture medium, in such a way that they can contract even in non-favourable media. The invention also relates to the appropriate methodology for isolating, cultivating, expanding and characterising said cell population with characteristics of embryonic cells such as nanog, Oct3/4, and cMyc, to the pharmaceutical compositions comprising said cell populations, and to the use of said compositions in the regeneration of tissues, especially for the regeneration of damaged cardiac tissue, such as the myocardium.

Description

Isolated blood cells from the coronary sinus and uses.

FIELD OF THE INVENTION

The present invention is within the field of cell biology and medicine, and refers to a cell and / or cell population that presents a new cell phenotype, and that performs a contractile process without external, non-physiological inductors, in a suboptimal non-specific culture medium, so that they can be contracted even in non-favoring media. It also refers to the appropriate methodology for isolating, culturing, expanding and characterizing said cell population, with characteristics of embryonic cells such as nanog, Oct3 / 4, and cMyc, to pharmaceutical compositions comprising said cell populations, and to the use of said compositions in tissue regeneration, and particularly for the regeneration of damaged heart tissue, such as myocardium.

STATE OF THE TECHNIQUE

Cardiovascular diseases (CVD) are the leading cause of mortality in developed countries. WHO estimates that 17.5 million people died from this cause in 2005, representing 30% of all deaths recorded in the world; 7.6 million of those deaths were due to coronary heart disease.

Ischemic heart disease (Ischemic heart disease -IHD-) and heart failure (heart failure -HF-) are two of the three leading causes of mortality in women and men. Both share the same pathophysiological substrate: the loss of cardiomyocytes due to necrosis and / or apoptosis. The death of cardiomyocytes results in an impediment to the ability of the myocardium to pump blood, which results in a decrease in the left ventricular ejection fraction (left ventricular ejection fraction - LVEF-). In fact, a residual LVEF after an acute myocardial infarction (AMI) is a strong predictor of one-year survival (Jimenez-Quevedo et al., 2008. Rev. Esp. Cardiol. 61, 635-639). It was thought that once the cardiomyocyte had died, the heart was unable to replace it. Until recently, this was attributed to the terminal nature of differentiation of the heart, considering it a postmitotic organ. However, this concept has changed recently, and it is accepted that the heart has regenerative capacity, thinking that this capacity is limited, and probably unable to regenerate / replace the heart to a significant extent (Nadal-Ginard et al., 2003. Circ .Res. 92,139-150; Barile et ai, 2007. Nat. Clin. Pract. Cardiovasc. Med. 4 Suppl 1, S9-S14)

Medical treatment has significantly improved patient survival, and is based primarily on pharmacological and surgical therapies. These are aimed at preventing the progression of the disease, discharging the heart, or reversing the consequences of the disease, but they do not solve the underlying problem, that is, the functional loss of myocardium. The only treatment that involves replacing the tissue is heart transplantation, but this approach is limited by donor availability. There is evidence that stem cells of various origins can differentiate into cardiomyocytes, blood vessels or connective tissue, and are therefore potentially capable of regenerating myocardial tissue (Ellison et al., 2007. Nat. Clin. Pract. Cardiovasc. Med .4 Suppl 1, S52-9). In general, research groups working in cardiovascular regenerative therapy use either peripheral circulation progenitor cells (Leone 2009, Eur Heart J. 2009; 30: 890-899), heart-resident cells (Beltrami et al., 2001. N. Engl. J. Med. 344, 1750-1757; Beltrami et al., 2003. Cell 1 14, 763-776; Torella et al., 2007 Cell Mol. Life Sci. 64, 661-673) or cells resident in the bone marrow (Psaltis et al., 2008. Stem Cells 26, 2201-222) or mesenchymal derivatives such as fat (Gwak et al., 2009. Cell Biochem. Funct. 27, 148-154). In recent years, new strategies have been developed aimed at stimulating the regeneration of the heart muscle or its replacement by cells with myogenic capacity (Kessler and Byrne, 1999. Annu Rev Physiol 61, 219-42). Stem cells have been studied intensively as a source of cardiomyocytes. Cardiomyocytes are generated from a cell precursor that divides and gives rise to groups of cells of the same type. During fetal life, these cells begin to differentiate and contractile myofibrils appear in their cytoplasm. These contractile fetal cardiomyocytes still retain the ability to divide despite being in a differentiated state and, in the case of humans, this ability is maintained until 3-4 months of postnatal life (Gupta et al., 1991 Biochem Biophys. Res Commun. 1991 Feb 14; 174 (3): 1 196-203). In general, it is assumed that within a few months of birth we already have the maximum number of cardiac myocytes that we can have, and that from this moment the cells that are lost will no longer be able to be replaced, which leads to a progressive decrease in their number until death. This is the growth model that has been considered valid for cardiac muscle cells and for central nervous system neurons. On the contrary, the rest of the body's cells retain the ability to divide after reaching a differentiated steady state.

However, there are results showing that the heart is an organ in continuous regeneration that increases the production of new muscle cells in response to different physiological and pathological stimuli (Olivetti et al., 1996, J Mol Cell Cardiol. Sep; 28 (9) : 2005-16). This regenerative capacity offers new opportunities for the treatment of heart failure, such as the use of embryonic stem cells as a source of cardiomyocytes to repair the damaged myocardium and improve cardiac function (Emanueli et al., 2005. Thromb. Haemost. 94, 738 -749). The potential benefit of stem cell transplantation in animal models has been investigated, using bone marrow cells (Fukuda. & Fujita, 2005. Kidney Int. 68, 1940-1943; Orlic et al., 2001. Nature 410, 701-705), cardiac stem cells (Beltrami et al., 2003. Cell 1 14, 763-776), embryonic stem cells (ES) (Behfar et al., 2002. FASEB J. 1 6, 1 558 -1566; Menard et al., 2005. Lancet 366, 1005-1012), and fetal cardiomyocytes (Rubart et al., 2003. Circ. Res. 92, 1217-1224), injected at the site of cardiac damage.

Recently, populations of stem cells in the heart have been identified (Leri et al., 2005. Physiol Rev. 85, 1373-1416), including cells expressing the c-Kit stem cell receptor (Beltrami et al., 2003. Cell 1 14, 763-776), Sca-1 (Oh et al., 2003. Proc. Nati. Acad. Sci. US A 100, 12313-12318), or the islet-1 transcription factor (Laugwitz et al. , 2005. Nature 433, 647-653) on the cell surface, side population cells (SP) (Pfister et al., 2005. Circ. Res. 97, 52-61), and cells capable of growing in cardio-spheres (Messina et al., 2005. Circ. Res. 95, 91 1-921).

However, despite the observation of the existence of cells capable of subdividing and becoming cardiomyocytes, the proliferation / regeneration rate is so low that it is not sufficient to regenerate dead ischemic tissue, and tissue replacement is inevitable. Functional death due to non-functional tissue such as non-contractile fibroblasts. In addition, in the case of stem cells of embryonic origin, there is a serious histocompatibility problem, since it is evident that no two embryos are the same, and less those that you want to use for therapy, which will be those discarded for fertilization, of what arises another great ethical-moral problem.

It is necessary, therefore, to develop a method of isolation of adult cell populations, which possess the appropriate characteristics for tissue regeneration, and particularly, that are capable of secreting growth factors and cytokines that are capable of protecting the myocardium from ischemic damage, inhibiting the inflammatory response that follows the death of myocardial cells, and acting as cardiac resident stem cells, progenitor and precursor cells, contributing to the regeneration of damaged contractile cells, and microvascularization.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1. Image of the culture of peripheral blood adherent cells: 1 a) 15-day culture; 1 b) 30-day culture of adherent peripheral cells; 1 c) 60-day culture of adherent peripheral cells. Figure 1 d) non-adherent floating cell culture from the coronary sinus in patients with acute myocardial infarction with a culture time of 15 days; 1 e) 30-day culture of floating cells; 1 f) 60-day culture of floating cells.

Fig. 2. Detailed image of floating cells from the coronary sinus that form a cluster of 60 days of cell culture (2a, 4x). In fig. 2c) the asymmetric division is observed originating small cells (40 x magnifications) and larger cells (2b, 20 x magnifications).

Fig. 3. Image of a cell cluster with a size of 1 00 μιτι whose morphological structure has been preserved with the 4% Paraformaldehyde fixative. In this figure you can see the spheroidal aspect of the cells that make up the structure.

Fig. 4. Image of a cell whose cell size is between 10-20 μιτι, prefixed with paraformaldehyde and from a cluster where spheroidal morphology is shown.

Fig. 5. Image of three cells smaller than 10 μιτι. preset with paraformaldehyde and from a cell cluster. Fig. 6. Shows cell cultures in vivo. Fig. 6a, shows a cell culture at an early stage of the culture after having subdivided the cells. In the first 7 days of cultivation, clusters formed by a number of cells between 17-20. Fig. 6b, a culture is observed between 7-15 days formed by 80-200 cells, whose cluster size has a diameter of 100 μιτι. Fig. 6c 15-day cluster culture whose size has increased to 200 μιτι. Fig6.d. Cell cluster culture between 15-30 days of culture whose size is between 500 μιτι.

Fig 7. Table showing the doubling time of cultured cells in 3 independent experiments. The table shows the results of the records obtained between 0 and 48 hours of cultivation and 120h - 144 hours of cultivation. The table shows the parameters such as the growth rate = the number of doubles that occur per unit of time, the equation generated by the software used: http://www.doublinq-time.com/compute.php.

Fig. 8. Graph of the growth curve in relation to the number of cells and days of culture (0, 1, 3, 5, 7 days of culture quantified). The experiments were performed using three conditions 1: counting cultured cells with pre-incubation medium and de novo medium, in relation 1 vol: 1 vol.2: quantified cells but cultured in completely replaced medium. 3: quantified and cultured cells in pre-incubation medium and de novo 2n medium perform 1 vol: 2 vol. The data represent the average of 3 independent experiments in duplicate in cell cultures with a 5-month incubation maintenance period.

Fig. 9. Graph of the growth curve of the number of cells quantified over 30 days taking as cut-off points: at 0 days, 5, 10, 1, 5 and 30 days respectively. The graph shows the mean ± standard deviation of 3 independent experiments in cell cultures after 5 months of maintenance. Fig. 10. In vivo culture image of cluster-forming cells, where the cells have been tripisinized and subcultured, the photographic records are shown 24, 48 and 3 days after subculture. Fig. 11. Image of cells of the culture in vivo, at 6, 7 and 15 days post subculture (consecutive figure to Figure 10). Fig. 12. Image of the structural morphology of the arrangement and shape of the dular nuclei arranged within the cluster. They are shown in blue Dapi nuclear marker of a cluster prefixed with paraformaldehyde. The images were obtained in confocal microscope LEICA, mod. SP5 II and analyzed in the Leica LAS AF software (60x magnification).

Fig. 13. Image of a prefixed cluster with cellular paraformaldehyde where the formation of a central cavity is shown in detail. The images were obtained in confocal microscope LEICA, mod. SP5 II and analyzed in the Leica LAS AF software (60x magnification).

Fig. 14. Image of the layout of a previously fixed cell cluster with paraformaldehyde and marker with a) Phalloidin staining f-actin filaments and b) Dapi as a nuclear marker. Fig. 15. Image of the phalloidin made in pre-set cells with paraformaldehyde. The figure shows the cytoskeleton marked with pahlloidin in green, visualizing the detail of the cavity or pore found in the cell clusters. Cells from 3-month cell cultures in culture

Fig. 16. Image of the phalloidin made in pre-set cells with paraformaldehyde where the structure and morphology of the cellular cytoskeleton are detailed. Cells from 3-month cell cultures in culture. Fig. 17. Detail image of the cell nuclei marked with Dapi, as a nucelar marker, where the nuclei are observed structurally without anomalies due to cellular necrosis. Cells from cultures 3-month cell culture. Images obtained with Nikon model TE2000-U fluorescence microscope and images were made with the Nikon DS5MC model camera. Fig. 18. Image of the superposition of the nuclear tide (DAPI) with the cytoskeletal tide (Phalloidin). The images were made with the Nikon microscope model TE2000-U and with a cold camera model Nikon DS5MC.

Fig. 19. Marking with Calcein AM and detailed in Fig. 19-1 the Image in 20x magnification, of the blue marking: DAPI as a nuclear and green marker: Calcein AM (green-Calcein AM) showing the cell viability and active metabolism, in cultures of 2 months incubation. Fig19-2: Image in 4x increases in cell culture of 2 months incubation. Fig. 19-3 Images of cultures of 5 months of incubation whose red marking is Calcein AM. Observing in b) figure of microcopy in clear field 10x increases, b-1) shows the red Calcein AM (10x increases). Figure 19-3c) shows a cluster (20x magnification) of 5 months of incubation. Fig. 19-4 a) shows in a clear field a cell cluster and in detail is shown in normal light a-1) a 40x magnification detail of the central region of the cluster. fig. a-2) The metabolically active central region is observed in red (40x magnification). The images were taken with the Nikon microscope model TE2000-U and with a cold chamber model Nikon DS5MC.

Figure 20. Image of a cell transfected with p-eGFP-C1 using Fugene 6 Transfection Reagent (Roche) in vivo, Fig 20.a) light field microscopy with phase contrast. Fig. 20. B) GFP fluorescent green signal. . The images were obtained with Nikon inverted microscope model TE2000-U and with cold camera model Nikon DS5MC. Fig. 21 .- Immunofluorescence image of a clone of cells, in vivo after four days in clonal growth in green the p-eGFP-C1 dizziness is shown (Fig 21 c) and in bright field the cells are observed in vivo. The images were obtained with a Nikon inverted microscope model TE2000-U and with a cold chamber model Nikon DS5MC.

Fig. 22.- Immunofluorescence image of Bromodeoxyuridine BrdU in Red and in Blue the Dapi nuclear marker. Fig 22 a) 10 x DAPI cluster catkins; Fig. 22 b) Mix 10x of nuclear Dapi Blue and BrdU in red showing positive cell proliferation .; Fig 22 c) 20 x nuclear Dapi increases. Fig 22 d) Mixture of Dapi blue and BrdU red positive for cell proliferation (20 x magnification). The images were made with the Nikon microscope model TE2000-U and with a cold camera model Nikon DS5MC.

Fig. 23.- Immunofluorescence image of a phalloidin green marking showing f-actin filaments in green. The 40 x magnification image was obtained using the Nikon model TE2000-U inverted microscope and with a Nikon DS5MC cold camera.

Fig. 24. Immunofluorescence image showing the green nuclear macaje in blue. 40x magnification, inverted microscope model Nikon model TE2000-U and with cold camera model Nikon DS5MC.

Fig 25. Image of immunofluorescence marking in 40 x magnification of DAPI marking (Fig 25a), Nanog (Fig 25b). OCT3 / 4 (Fig 25c) and mixing (merge) (Fig. 25d), the Nikon model TE2000-U microscope was used and with a Nikon DS5MC cold room model.

Fig 26. Image in 20 x magnification of DAPI marking (Fig 26a), Nanog (Fig 26b). OCT3 / 4 (Fig 26c) and mixing (merge) (Fig. 26d), the Nikon model TE2000-U microscope was used and with a Nikon DS5MC cold room model. Fig 27. Image of DAPI marking (Fig 27a), Nanog (Fig 27b). OCT3 / 4 (Fig 27c) and mixing (merge) (Fig. 27d) the LEICA confocal microscope was used, mod. SP5 II and the images were made with the Leica LAS AF software. (20x increases).

Fig 28. Image of DAPI tide (Fig 28a), Nanog (Fig 28b). OCT3 / 4 (Fig 28c) and mixing (merge) (Fig. 28d) the Nikon microscope model TE2000-U was used and with a cold chamber model Nikon DS5MC

Fig 29. Immunoflurescence image with cells fixed in 4% paraformaldehyde In green, the positive marking for Ssea-4, in red for Ssea-3, and in blue the nuclear marker dapi.the confocal microscope LEICA, mod. SP5 II and the images were made with the Leica LAS AF software. (20x increases).

Fig 30. Image of Immunoflurescence with cells fixed in 4% paraformaldehyde In green the positive marking for Ssea-4 is observed and in blue the dapi nuclear marker. The images were obtained with the LEICA confocal microscope, mod. SP5 II and the images were made with the Leica LAS AF software. (20x increases).

Fig. 31. C-Kit (4a) and DAPI (4b) marking the Nikon model TE2000-U fluorescence microscope was used and the images were made with the Nikon model DS5MC camera. (20x increases). Fig. 32. Figure showing the flow cytometry performed on the Cyan Dako-cytomation cytometer (Summit software v3.4) using membrane markers and transcription factors: Fig. 32a) Oct3 / 4-FITC; Fig. 32b) NanogPE; Fig 32. c) CD15-PerCP; Fig 32.d) cKit-PeCY7; Fig 32.e) CD105-APC; Fig 32.f) CD90-APC; Fig 32.g) KDR-APC; Fig 32.h) CXCR4-PE; Fig 32.i) CD34- FITC. Fig. 33. RT-PCR Image of the expression of genes involved in pluripotence: Nanog, Oct4; SSEA1; SOX-2, Sox-7; Brachyury; Runx-1; Slc; GATA-1; c-Myc; genes in differentiation towards cardiomyocyte: GATA-4; Isl1 +; Mef2c; Nkx2.5; Tnl; genes involved in neurogenesis and endoderm NeruroDI, Foxa2 / HNF3 and other genes involved in the differentiation of endothelial or smooth muscle tissue such as CXCR4, KDR and Sma. The gene coding for b-actin has been used as a constitutive or houskeeping gene.

Fig. 34. qRT-PCR. Graph of the increase in the relative expression (ddCt) of the genes obtained from the quantification through qRT-PCR comparing the gene expression of cluster-forming cells of the coronary sinus and peripheral blood cells. The data has been relativized using the peripheral blood sample as a calibrator. The houskeeping gene used for data normalization has been b-actin.

Fig. 35. Angiogenesis assay (BD matrigel-angiogenesis assay) where adherent endothelial cells from peripheral blood (35a, 35b, 35c, 35d) and floating cells from the coronary sinus (35e, 35f, 35g, 35h are compared ). Fig. 35a) shows adherent morphology of adherent cultured peripheral blood cells; used in the test fig. 35b) shows the peripheral blood cells in the green Calcein AM (Sigma Aldrich) green matrigel-angiogenesis system a positive cell viability marker Figure 35d) shows the peripheral blood after 5 days of culture. Fig. 35g) shows the floating cells of the coronary sinus in the matrigel / angiogenesis system (BD bioscience) labeled with green Calcein AM (Sigma Aldrich) after 5 days of culture. Figure 35d) shows the formation of vascular structures after 15 days of culture, typical of endothelial cells, of peripheral blood, however, in comparison with peripheral blood cells (fig. 35d), fig. 35h shows that after 15 days under induced conditions of angiogenesis, coronary sinus cells maintain their cluster structure without observing a cellular compromise Apparently forming a vascular structure as occurs in peripheral blood cells.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have developed the appropriate methodology for isolating and culturing cells with embryonic cell characteristics by expressing specific pluripotent transcription factors of embryonic cells, such as Nanog or Oct3 / 4 and in certain populations besides others such as SSEA-1 or cMyc (Belmonte et al., 2009. Nat Rev Genet. 10: 878-883. 9; Kalmar 2009. PLoS Biol. 7: e1000149). These cells have been obtained from mature adult tissue, not embryonic tissue, and are located in the blood that drains from the heart from the coronary sinus, and not in the cardiac niche as the cardiac resident progenitor cells described to date (Urbanek 2006. Nati Acad Sci US A. 103: 9226-9231). Although, cells of embryonic origin in mouse heart tissue have been described as those that mark for Isl1 +, Sca1 + (Laugwit et al., Nature. 2005; 433: 647-653, Liang et al., 2010. Int J Cardiol. 2010; 138: 40-49) has not been reproducible in human cardiac tissue. They are isolated non-adherent cultured cells forming clusters, from the coronary sinus.

According to current criteria, an embryonic cell must possess the clonogenic ability to divide or clone up to 8 times or subcultures. The authors of the present invention have been able to subculture the cells up to 15 times, being able to amplify up to 56 times more in three days.

Based on these experiments, the inventive aspects that are explained in detail below have been developed.

BLOOD ISOLATED ADULT CELLS FROM THE BREAST

CORONARY OF A MAMMAL A first aspect of the invention relates to an isolated adult cell that is positive for the Nanog and Oct3 / 4 markers, and negative for the CD34 markers, those of the adult endothelial cell CD31, CD146, and that of the adult blood cell CD45, and which does not show the expression of the transcription factor of adult smooth muscle cell Sma. Morphologically, the cells of the invention are not attached but floating and forming clusters or cell clusters, which propagate by jumping off a "mother cluster". These clusters are very morphologically similar to those described by other authors for embryonic bodies or cardiac or cardiac progenitor cells, also showing an asymmetric division of the cells, so that a (large) stem cell and small, smaller cells are generated, surrounding them. In a preferred embodiment of this first aspect of the invention, the isolated cells are further characterized by being able to undergo synchronous contraction in culture with an adrenergic factor, preferably with Norepinephrine. Therefore, preferably the cells of the invention are non-adherent cluster-forming cells, originating from the coronary sinus, which show positive immunoreaction for flow cytometric tests: Nanog +, Oct3 / 4 +, and which however, no immunoreaction is observed for The cytometry markers CD34 -, CD45-, CD31 -, CD146- and Sma are characterized in that they are capable of experiencing synchronous contraction in culture with an adrenergic factor, preferably with norepinephrine. In the context of the present invention, adrenergic factor is understood as any substance that exerts effects similar or identical to those of epinephrine (adrenaline).

Adult cells isolated from the first aspect of the invention are also positive for the Sox2 marker. Thus, in a preferred embodiment, the adult isolated cell, that is, non-adherent cultured culturing cells of clusters from the coronary sinus, also show the natural positive expression of Sox2.

Adult cells isolated from the first aspect of the invention are also positive for the CD90 and CD105 markers. Thus, in a preferred embodiment, the adult isolated cell, that is, the non-adherent cultured cluster-forming cells from the coronary sinus, also show the natural positive expression of CD90 and CD105. In another preferred embodiment, the adult cell isolated from the first aspect of the invention also naturally expresses the transcription factors IsM, NeuroDI and Foxa2.

In another more preferred embodiment, the cell of the invention also shows positive immunoreaction to the markers CD1 17 (c-kit), Sox7 and Runxl. In another even more preferred embodiment, the cell of the first aspect of the invention also shows positive immunoreaction for CXCR4 and CD15 (SSEA1), and naturally expresses the transcription factors SSEA4 and cMyc. In an even more preferred embodiment of the invention, the cell of the first aspect of the invention further expresses the transcription factors CD133 (Tip 1), KDR, GATA4, Mef2c, Tnl and NKx2.5.

A second aspect of the invention relates to an isolated adult cell that is positive (shows positive immunoreaction for flow cytometry markers) for markers: Nanog, Oct3 / 4, CD15 (SSEA1), CD133 (Tip 1), and negative for CD34 markers, those for adult endothelial cell CD31, CD146, and adult blood cell CD45. As was the case in the first aspect of the invention, the cells are not attached but floating and forming clusters or cell clusters, which propagate by jumping off a "mother cluster." These clusters are morphologically very similar to those described by other authors for embryonic bodies or cardiac or cardiac progenitor cells, also showing an asymmetric division of the cells, so that a stem cell (large) and small cells of smaller size are generated, surrounding them. Therefore, preferably the cells of the second aspect of the invention are non-adherent cells. Cluster-forming non-adherent cultured cells from the coronary sinus of the second aspect of the invention thus show positive immunoreaction for flow cytometric tests: Nanog +, Oct3 / 4 +, CD15 (SSEA1) +, CD133 +, however no immunoreaction is observed for cytometry markers CD34 -, CD45-, CD31 -, CD146-

Adult cells isolated from the second aspect of the invention also naturally express the transcription factors: SSEA4, Sox2, Sox7, Brachyury, Runxl, Slc, cMyc not being observed gene expression of the genes that transcribe for smooth muscle tissue: Sma. Thus, in a preferred embodiment, the adult isolated cell, that is, non-adherent cultured cluster-forming cells from the coronary sinus, show the natural positive expression of the transcription factors: Nanog, Oct3 / 4, CD15 (SSEA1) +, CD133 +, SSEA4, Sox2, Sox7, Brachyury, Runxl, Slc, cMyc and does not show the expression of the transcription factor of smooth adult muscle cell Sma nor immunoreaction is observed for the cytometry markers CD34 -, CD45-, CD31 -, CD146-.

In another preferred embodiment of the second aspect of the invention, the adult cell isolated from the second aspect of the invention also naturally expresses the transcription factor NeuroDI and Foxa2.

In another more preferred embodiment of the second aspect of the invention, the cell of the invention also shows positive immunoreaction the CD90, CD105, CXCR4 and KDR markers. In another even more preferred embodiment, the cell of the second aspect of the invention also shows positive immunoreaction for the CD1 17 marker (c-kit), and naturally expresses the IsM transcription factor. In an even more preferred embodiment of the invention, the cell of the second aspect of the invention also expresses the transcription factors GATA4, Mef2c, Tnl and NKx2.5.

These cells, both those of the first and those of the second aspect of the invention, although they are adult cells, that is, they have not been obtained from an embryo but from post-natal and adult tissue, naturally express a series of biochemical markers at the level of mRNA and protein, many of which have previously been associated with pluripotency capacity, and have even shown the ability to confer totipotency when transfected into somatic adult fibroblasts, such as Oct4, Sox2 and c-Myc (Takahashi et al., Cell 2007, 131 pg. 1-12). The cells also express Nanog, which is one of the best studied genes that contribute to the multipotent state. Therefore, cells of any aspect of the present invention, despite being present in post-natal and adult tissue, possess many of the biochemical characteristics that are classically associated with embryonic stem cells. That is, the presence of transcription factors in a cell indicates that cells have the potential to differentiate into other cell types, when grown in a specific culture medium of those other cell types, that is, they are adult stem cells.

The term "adult stem cell" means that the stem cell is isolated from a tissue or organ of an animal in a state of growth after the embryonic state. Preferably, the stem cells of the invention have been isolated in a post-natal state. Preferably they have been isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents and adults.

Isolated adult cells of the invention are also characterized by not naturally expressing a series of markers (CD34, CD31, CD146, CD45), mainly associated with cell differentiation.

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.

The cells of the invention are positive for certain phenotypic markers and negative for others. "Positive" means that the cell expresses the marker. To consider that the marker is expressed, it must be present at a "detectable level". In this report, "detectable level" means that the marker can be detected by one of the standard methodologies, such as PCR, blotting or FACS. A gene is considered to be expressed by a cell of the invention if it can be reasonably detected after 20 cycles, preferably 25 cycles, and more preferably 30 cycles of PCR, which corresponds to a level of expression in the cell of at least 100 copies per cell. It is considered that a marker is not expressed by a cell of the invention, if the expression cannot be detected at a level of about 10-20 copies per cell. Among these positive / negative levels, the cell may be weakly positive for a given marker. The term "natural expression", or "naturally expressed" means that the cells have not been manipulated by recombinant technology, in any way. This is, for example, that the cells have not been artificially induced to express these markers or modulate the expression. of these markers by the introduction into the cells of exogenous material, such as the introduction of heterologous promoters, or other sequences operatively linked to any of the endogenous genes, or by the introduction of exogenous genes.

More preferably, the adult cell isolated from any aspect of the invention is obtained from blood from the coronary sinus of a subject, preferably a mammal, or from its blood products. In another more preferred embodiment, the mammal has a heart condition. In another embodiment Preferred, the heart condition is an ischemic heart disease (Ischemic heart disease -IHD-) or a heart failure. In another even more preferred embodiment, the heart condition is acute coronary syndrome, and more preferably, acute coronary syndrome with ST segment elevation (AMI).

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. The term mammal, as understood herein, refers to any organism of the Eukaryota super kingdom, Metazoa kingdom, Phylum Chordata, Mammalia class. Thus, blood can be obtained from the coronary sinus of mouse, rat, pig and human. In another even more preferred embodiment, the mammal is the human being.

In the current literature, only embryonic cells or derivatives of embryonic lines, such as mouse Isl1 +, Sca1 + cells, are able to beat spontaneously. However, at present, no cell species of adult tissue, which has the ability to beat, without inductions is described. In any case, for a cell to experience contraction or beat, embryonic cell cultures or iPS with 5-Azitidine are needed (Nelson et al., 2009. Circulation 1 20: 408-416, Deng 2010. EMBO Rep. 1 1: 161-165) for four or five weeks of treatment. However, in mesenchymal progenitor cells, only myosin filaments or cardiac troponin I filaments have been observed under fluorescence microscopy, but not the cellular beat (Toma et al., 2002. Circulation 105: 93-8; Peran et al., 2010. Cytotherapy). Another mechanism of differentiation is through co-culture with rat fetal cardiomyocytes, being criticized by various authors for considering it a fusion and not a differentiation, (Quaini et al. 2002. N EngI J Med. 346: 5-15.) . Recently, it has been achieved through low frequency waves to stimulate contraction in cardiac progenitor cells (Gaetani et al., 2009. Cardiovascular research. 82:41 1-20). However, the authors of The present invention has proven that cells of any aspect of the invention were capable of contracting synchronously (every 3 seconds) after 12 hours in culture with Noerepinephrine, with a long refractory period, probably due to the lack of ATP and Calcium in the medium, which are two molecules directly involved in the contraction and not used, since they wanted to see if spontaneous contraction arose. Thus, in another preferred embodiment, the cell of any aspect of the invention also has the ability to contract synchronously in a medium with norepinephrine or any other adrenergic factor.

If desired, the cell of any aspect 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 its basic properties (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences encoding proteins of interest such as, for example, proteins with therapeutic properties. Therefore, in another preferred embodiment, the cell of any aspect of the invention has been genetically modified.

As stated above, the progeny of a single clonal cell can be expanded by numerous passes, without apparently suffering any chromosomal abnormality, or the loss of its growth and differentiation properties.

Therefore, if desired, cells of any aspect of the invention can be clonally expanded using a suitable method for cloning cell populations. For example, a proliferated population of 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 (for example, 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. Another aspect of the invention relates to an isolated cell population, hereinafter cell population of the invention, comprising at least one adult cell of any aspect of the invention. In a preferred embodiment the cell population of the invention comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of adult cells of the invention.

The cell population of the invention is positive for a given marker if at least 20% of the cells in the population show a detectable expression of the marker, preferably 70%, 80%, 90%, 95%, and much more. preferably, 98%. Sometimes, 99% or 100% of the cells of the invention show a detectable expression of the marker. As in the case of cells, expression can be detected, for example but not limited, by PCR techniques, using FACS (fluorescence activated cell sorting), or by immunohistochemistry using specific antibodies.

The term "isolated" indicates that the cell or cell population of the invention to which it refers, are not in their natural environment. That is, the cell or cell population has been separated from its surrounding tissue. Particularly it means that said cell or the cell population is substantially free (free) of other cells normally present in the blood, or in a blood product thereof, of a subject from which the blood has been extracted for the isolation of said cell or cell population In general, a cell derived from blood, or from a blood product thereof, is essentially free of other cells normally present in said blood or blood product thereof when separated from at least 60%, preferably at least 80%, preferably of 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 or blood products thereof.

It also refers to cells or cell populations that have been isolated from the organism in which they originate. The term also includes cells that have been isolated from one organism and re-introduced into the same organism, or another.

In another preferred embodiment, the cell population of any aspect of the invention is obtainable by a method comprising:

to. obtain a blood sample from the coronary sinus of a mammal,

b. isolate the blood sample cells from step (a),

Preferably, the method further comprises

C. purify the isolated cells of (b)

More preferably, it also comprises:

d. culturing the purified cells of step (c), until floating cells are generated,

and. subculture the floating cells of step (d),

Even more preferably, it also comprises:

F. keep them in cultivation until their confluence and subculture them again.

Preferably, the blood sample from the coronary sinus step (a) is diluted, in an approximate ratio of 1: 6 in a 5% PBS solution of FBS and 1% of antibiotics. In another preferred embodiment, the diluted sample from step (a) is centrifuged with Ficoll. In another preferred embodiment, after centrifugation, the mononucleated cells are washed with 5% FBS PBS and 1% antibiotic by centrifugation. In another preferred embodiment, after washing, the cells are re-suspended in FBS and EBM-2G. In another preferred embodiment, the re-suspended cells are seeded in wells with 1 ml of EBM-2G medium. More preferably, the 7th day of incubation the supernatant phase where the floating cells are taken up and incubated in a new culture dish with a medium comprising DMEM and HamF12 in proportion 4: 6, FBS, antibiotic, hydrocortisone, EFG, FGF-B, IGF-1, glutamine, 1M Hepes and ascorbic acid, in fibronectin-coated plates. More preferably, on the 12th day the cells are subdivided into plates treated with fibronectin. Even more preferably, at least 8 subcultures of the cells are made in plates treated with fibronectin.

In this report, Nanog means a transcription factor of pluripotency (Kalmar et al., 2009. PLoS Biol. Jul; 7 (7): e1000149. Epub 2009 Jul 7; Pan & Thomson 2007. Cell Res. Jan; 17 ( 1): 42-9). It is also called hNanog; homeobox protein NANOG; homeobox transcription factor Nanog. The human gene is found on chromosome 12 (12p13.31), and its sequence is collected in GeneBank NP_079141 .2 NM_024865.2 (mRNA)

In this report, Oct3 / 4 means a transcription factor of pluripotency (Kim et al., 2009. Cell. Feb 6; 136 (3): 41 1-9), also referred to as POU5F1, OTTHUMP00000221 150; OTTHUMP00000221 151; POU domain, class 5, transcription factor 1; POU-type homeodomain-containing DNA-binding protein; octamer-binding protein 3; octamer-binding protein 4; octamer-binding transcription factor 3; octamer-binding transcription factor-3. The human gene is found on chromosome 6 (6p21 .31), and its sequence is found in GenBank NP_976034.4 and NM_024865.2 (mRNA) This report refers to CD15 (Pruszak et al., 2009. Stem Cells. Dec; 27 (12): 2928-40; Kucia et al., 2006. Leukemia. May; 20 (5): 857-69) also denominated as FUT4, ELFT, FCT3A, FUC-TIV, FUTIV, LeX, SSEA-1, ELAM ligand fucosyltransferase; ELAM-1 ligand fucosyltransferase; Lewis X; alpha- (1, 3) -fucosyltransferase; fucT-IV; Fucosyltransferase IV; galactoside 3-L-fucosyltransferase; stage-specific embryonic antigen 1. The human gene is found on chromosome 1 1 (1 1 q21), and its sequence is found in GenBank NP_002024.1 and NM_002033.3 (mRNA). In this report it is understood by CD133 also called Tip 1, MSTP061, AC133, PROM1, CORD12, MCDR2, PROML1, RP41, STGD4, ΟΤΤΉΙΙΜΡ00000217744; OTTHUMP00000217745; ΟΤΤΉΙΙΜΡ00000217746; AC133 antigen; hProminin; hematopoietic stem cell antigen; prominin-1; prominin-like 1; prominin-like protein 1. It is an early progenitor cell marker (Rufaihah et al., 2010. Regen Med. Mar; 5 (2): 231-44). The human gene is found on chromosome 1 1 (1 1 q21), and its sequence is found in GenBank NP_002024.1 and NM_002033.3 (mRNA)

In this report, CD34 is understood as a hematopoietic and progenitor cell marker (Aiuti et al., 1997. J.Exp.Med. 185, 1 1 1 -120). It is also called RP1 1-328D5.2, CD34 antigen; hematopoietic progenitor cell antigen CD34. The human gene is found on chromosome 1 (1 q32), and its sequence is found in GenBank NP_001020280.1 and NC_000001 .10 (mRNA).

In this report, CD31 is understood as an endothelial cell marker (Pfister et al., 2005. Circ.Res. 97, 52-61). Also called PECAM1, FLJ34100, FLJ58394, PECAM-1, CD31 antigen; CD31 / EndoCAM; GPIIA ';PECA1; PECAM-1, CD31 / EndoCAM; adhesion molecule; endoCAM; platelet endothelial cell adhesion molecule. The human gene is found on chromosome 17 (17q23), and its sequence is found in GenBank NP_000433.3 and NM_000442.3 (mRNA). In this report, CD146 is understood as a differentiated endothelial cell marker (Kebir et al., 2010. Circ Res. Jul 9; 107 (1): 66-75). Also called MCAM (melanoma cell adhesion molecule), Gicerin; S-endo 1 endothelial-associated antigen; cell surface glycoprotein MUC18; cell surface glycoprotein P1 H12; melanoma adhesion molecule; melanoma-associated antigen A32; melanoma-associated antigen MUC18. The human gene is found on chromosome 1 1 (1 1 q23.3), and its sequence is found in GenBank NP_006491 .2 and NM_006500.2 (mRNA).

In this report, CD45 is understood as a hematopoietic and lymphocyte marker (Timmermans et al., 2007. Arterioscler. Thromb. Vasc. Biol. 27, 1572-1579; Reyes et ai, 2002 .. J. Clin. Invest. 1 09 , 337-346). Also called PTPRC (protein tyrosine phosphatase, receptor type, C), RP1 1-553K8.4, B220, CD45, CD45R, GP180, LCA, LY5, T200, CD45 antigen; L-CA; OTTHUMP00000033816; OTTHUMP00000038575; T200 glycoprotein; T200 leukocyte common antigen; glycoprotein; leukocyte common antigen; leukocyte-common antigen; protein tyrosine phosphatase, receptor type, c polypeptide; receptor-type tyrosine-protein phosphatase C. The human gene is located on chromosome 1 (1 q31 -q32), and its sequence is found in GenBank NP_563580.1 and NM_080923.2 (mRNA).

In this report, SSEA-4 is understood as the cellular marker originally described as: Stage-specific embryonic antigen-4. It is a marker of the surface of embryonic cells, only expressing itself in embryonic stages and specifically in the morula state in division 2-8 and stages of preimplantation of the embryo (Draper et al., 2002, J Anat. Mar (Pt 3): 249-58; Rao et al., 2007. Methods Mol Biol. 407: 51-61). Also called MSGb5 (Monosialosyl globopentaosylceramide), SIAT4B; ST3GALII; Gal-NAc6S; ST3GalA.2; ST3GAL2. (Saito et al., 2003. J Biol Chem. Jul 18; 278 (29): 26474-9). In the human gene it is found on chromosome 16 (16q22.1), and its sequence is found in GenBank NM_006927.3 mRNA).

In this report, Sox2 is understood as a transcription factor of pluripotency (Kashyap et al., 2009. Stem Cells Dev. Sep; 18 (7): 1093-108). Also called SRY (sex determining region Y) -box 2, NOP3, MCOPS3, MGC2413, SRY-related HMG-box gene 2; transcription factor SOX-2; transcription factor SOX2. The human gene is found on chromosome 3 (3q26.3-q27), and its sequence is found in GenBank NP_003097.1 and NM_003106.2 (mRNA).

In this report, Sox7 is understood as a transcription factor involved in embryonic development and cell fate. Also called SRY (sex determining region Y) -box 7, MGC10895, SOX7 transcription factor; transcription factor SOX-7. The human gene is found on chromosome 8, and its sequence is found in GenBank NP_1 13627.1 and NM_031439.2 (mRNA).

In this report, Brachyury is understood as a transcription factor of the T-box family that regulates the development of the early mesoderm controls such important processes as the determination of the left and right axis of the body. (Wardle & Papaioannou, 2008. Curr Opin Genet Dev. 2008 Oct; 18 (5): 418-25. Epub 2008 Sep 7). It is also called RP1 1-459F1 .1, MGC104817, TFT, OTTHUMP00000017588; T brachyury homolog; brachyury protein; protein T. In the human gene it is found on chromosome 6 (6q27), and its sequence is found in GenBank NP_033335.1 and NM_009309.2 (mRNA)

In this report, Runxl is understood as a transcription factor belonging to the Box Run genes that is related to development processes (Coffman 2003. Cell Biol Int. 27 (4): 315-24). Also called AML1, AML1 -EVI-1, AMLCR1, CBFA2, EVI-1, PEBP2aB, ML1 -EVI-1 fusion protein; CBF-alpha-2; OTTHUMP00000108697; OTTHUMP00000108700; OTTHUMP00000108702; PEA2-alpha B; PEBP2-alpha B; SL3-3 enhancer factor 1 alpha B subunit; SL3 / AKV core-binding factor alpha B subunit; acute myeloid leukemia 1 protein; amll oncogene; core-binding factor subunit alpha-2; core-binding factor, runt domain, alpha subunit 2; oncogene AML-1; polyomavirus enhancer-binding protein 2 alpha B subunit. The human gene is found on chromosome 21 (21 q22.3), and its sequence is found in GenBank NP_001 1 16079.1 and NM_001 122607.1 (mRNA).

In this report, Slc is understood as one of the cluster genes of the CC cytokine family, involved in inflammatory and immunoregulatory processes. Also called CCL21 (chemokine (C-C motif) ligand 21), RP1 1-195F19.22-001, 6Ckine, CKb9, ECL, MGC34555, SCYA21, SLC, TCA4, C-C motif chemokine 21; Efficient Chemoattractant for Lymphocytes; OTTHUMP00000021303; beta chemokine exodus-2; beta-chemokine exodus-2; exodus-2; secondary lymphoid tissue chemokine; secondary lymphoid-tissue chemokine; small inducible cytokine subfamily A (Cys-Cys), member 21; small-inducible cytokine A21. The human gene is found on chromosome 9 (9p13), and its sequence is found in GenBank NP_002980.1 and NM_002989.2 (mRNA)

In this report cMyc is understood as a regulatory transcription factor of differentiation towards cardiomyocyte (Conacci-Sorrell et al., 2010. Cell. Aug 6; 142 (3): 480-93; So et al., 2010. Int J Cardiol Sep 24; Nelson et al., 2009. Clin. Transí Sci. Apr; 2 (2): 1 18-26). Also called MRTL, bHLHe39, c-Myc. The human gene is found on chromosome 8 (8q24.21) and its sequence is found in GenBank NP_002458.2 and NM_002467.4 (mRNA).

In this report Sma is understood as a smooth muscle cell marker (Coco et al., 2009. Am J Surg Pathol. Dec; 33 (12): 1795-80). Also called ACTG2, ACT, ACTA3, ACTE, ACTL3, ACTSG, OTTHUMP00000202509; actin, gamma-enteric smooth muscle; actin-like protein; alpha-actin 3; alpha-actin-3; gamma-2-actin; smooth muscle gamma actin; smooth muscle gamma-actin. In The human gene is found on chromosome 2 (2p13.1). Its sequence is found in GenBank NP_001606.1 and NM_001615.3 (mRNA).

NeuroDI is understood herein as a transcription factor involved in the development of the endoderm as well as neurogenesis. (Lee et al., 1995. Science May 12; 268 (5212): 836-44). It is also called neurogenic differentiation 1, BETA2; BHF-1; MODY6; NEUROD1; bHLHa3. The human gene is found on chromosome 2 (2q32) and its sequence is found in GenBank NP_002491 .2 and NM_002500.2 (mRNA).

In this report, FOXA2 or HNF-3-beta2 -3 nuclear hepatic factor 3 beta 2 is understood as a transcription factor involved in the development of the endoderm, as well as its derived tissues, lung, pancreas, and liver and neurogenesis. (Lee CS, Sund NJ, Behr R, Herrera PL, Kaestner KH. Foxa2 is required for the differentiation of pancreatic alpha-cells. Dev Biol. 2005 Feb 15; 278 (2): 484-95.) It is also called RP23- 207P16.2, HNF3-beta, HNF3beta, Hnf-3b, Hnf3b, Tcf-3b, Tcf3b. The human gene is found on chromosome 20 and its sequence is found in GenBank NC_000020.10 NT_01 1387.8 (mRNA).

In this report, CD90 is understood as a marker of mesenchymal cell and of the cardiac derived stem cells (Ricke et al., 2010. Cell Mol Life Sci. Jun 18; Davis et al., 2009. PLoS One. Sep 25; 4 ( 9): e7195; Smith et al., 2007. Circulation. 1 15, 896-908. Also called THY1 (Thy-1 cell surface antigen), FLJ33325, CDw90; Thy-1 T-cell antigen; thy-1 antigen ; thy-1 membrane glycoprotein.The human gene is found on chromosome 1 1 (1 1 q23.3) and its sequence is found in GenBank NP_006279.2 and NM_006288.3 (mRNA) CD105 or also called endoglin 1, a marker of mesenchymal and mesenchymal bone marrow cells and of the cardiac derived stem cells (Bayes-Genis et al., 2009. Ann N YAcad Sci. Sep; 1 176: 1 18-23; Smith et ai, 2007. Circulation. 1 15, 896-908). It is also called RP1 1-228B15.2, CD105, END, FLJ41744, HHT1, ORW, ORW1. The human gene is found on chromosome 1 1 (1 1 q23.3) and its sequence is found in GenBank NP_001 108225.1 and NM_001 1 14753.1 (mRNA) for isoform 1, and NP_000109.1 and NM_0001 18.2 (mRNA) for isoform 2.

CXCR4 (chemokine (CXC motif) receptor 4), a marker of stem cells and receptor of the chemokine SDF-1 (Penn 2009. Circ.Res. 104, 1 133-1 135; Wojakowski et al., 2006. Eur.Heart J., 27, 283-289). The human gene is found on chromosome 9 and its sequence is collected in GenBank NP_000109.1 and NM_0001 18.2 (mRNA)

In this specification, KDR (kinase insert domain receptor) is understood as a type I II tyrosine kinase receptor (Yang et al., 2008. Nature May 22; 453 (7194): 524-8). Also called CD309, FLK1, VEGFR, VEGFR2, fetal liver kinase 1; fetal liver kinase-1; protein-tyrosine kinase receptor Flk-1; soluble VEGFR2; tyrosine kinase growth factor receptor; vascular endothelial growth factor receptor 2. The human gene is located on chromosome 4 (4q1 1 -q12) and its sequence is collected in GenBank NP_002244.1 and NM_002253.2 (mRNA)

In this report it is understood CD1 17 (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog), a marker of cardiac stem cells, cardiospheres, resident cardiac stem cells (Zamba et al., 2010. Circulation. May 1 1; 121 (18): 1992-2000 Epub 2010 Apr 26; Xaymardan et al., 2009. Stem Cells. Aug; 27 (8): 191 1-20; Miyamoto et al., 2010. Stem Cells Dev. Jan; 19 ( 1): 105-16. PubMed PMID: 19580375; Barile et al., 2007. Prog Cardiovasc Dis. 2007 Jul-Aug; 50 (1): 31-48; Tallini et al., 2009. Proc. Nati. Acad. Sci. USA, 106, 1808-1813). Also called C-Kit, CD1 17, PBT, SCFR, mast / stem cell growth receptor factor; proto-oncogene c-Kit; proto-oncogene tyrosine-protein kinase Kit; soluble KIT variant 1; tyrosine-protein kinase Kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene-like protein. The human gene is found in the chromosome 4 (4q1 1 -q12) and its sequence is found in GenBank NP_001087241 .1 and NM_001093772.1 (mRNA)

In this report Isl1 is understood as a homeobox transcription factor involved in the differentiation towards the cells that make up the heart (Bu et al., 2009 Nature. Jul 2; 460 (7251): 1 13-7; Laugwitz et al. , 2005. Nature. 433, 647-653; Moretti et al., 2007. Cell Mol. Life Sci. 64, 674-682). Also called ISL1 transcription factor, LIM / homeodomain; OTTHUMP00000221834; insulin gene enhancer protein ISL-1; islet-1. The human gene is found on chromosome 5 (5q1 1 .1), and its sequence is found in GenBank NP_002193.2 and NM_002202.2 (mRNA)

In this report, GATA4 (GATA binding protein 4) is understood as a transcription factor that regulates the expression of the genes involved in the development of the heart. (Durocher et al., 1997. EMBO J. 16: 5687-5696). Also called MGC126629, GATA-binding factor 4; GATA-binding protein 4; transcription factor GATA-4. The human gene is found on chromosome 8 (8p23.1 -p22) and its sequence is found in GenBank NP_002043.2 and NM_002052.3 (mRNA)

Mef2c (myocyte enhancer factor 2C) is understood herein as a transcription factor involved in myogenesis (McDermott et al., 1993. Molec. Cell. Biol. 13: 2564-2577). It is also called C5DELq14.3; DEL5q14.3. The human gene is found on chromosome 13 (13 45.0 cM) and its sequence is found in GenBank NP_079558.1 and NM_025282.3 (mRNA).

In this report NKx2.5 means a cardiomyocyte lineage marker (Prall et ai, 2007. Cell. Mar9; 128 (5): 947-59). Also called CHNG5, CSX, CSX1, FLJ52202, FLJ97166, FLJ97195, FLJ97197, FLJ99536, NKX2.5, NKX2E, NKX4-1, cardiac-specific homeobox 1; homeobox protein CSX; homeobox protein NK-2 homolog E; homeobox protein Nkx-2.5; Tinman Paralog The human gene is found on chromosome 5 (5q34) and its sequence is found in GenBank NP_001 159648.1 and NM_001 166176.1 (mRNA).

In this report, Tnl is understood as a cardiomyocyte lineage marker (Homo sapiens troponin I type 3 (cardiac) (Parmacek et al., 2004 Nov-Dec; 47 (3): 159-76; Robertson et al., J Mol Cell Cardiol Dec; 49 (6): 1031-41 Epub 2010 Aug 27) Also called TNNI3, CMD1 FF, CMD2A, CMH7, MGC1 16817, RCM1, TNNC1, cTnl. The human gene is found on chromosome 19 ( 19q13.4) and its sequence is included in GenBank NP_000354.4 and NM_ NM_000363.4 (mRNA).

Another aspect of the invention relates to a composition of adult cells of any aspect of the invention, hereafter referred to as a composition of the invention, comprising an isolated cell or an isolated population of the invention. Preferably, the cells of the adult cell composition of the invention have at least 50%, at least 60%, preferably 70%, more preferably 80%, even more preferably, 90%, and, Even more preferably, 95% of adult cells are adult cells of the invention.

Said adult cell composition of the invention may contain a medium in which the cells of the invention are found; said medium must be compatible with said cells. For example, but not limited to, isotonic solutions, optionally supplemented with serum; cell culture media or, alternatively, a solid, semi-solid, gelatinous or viscous support medium.

PHARMACEUTICAL COMPOSITION OF THE INVENTION

Adult cells of any aspect of the invention, the cell population of the invention, as well as the composition of the invention, may be part of a pharmaceutical composition for administration to a subject. Therefore, another aspect of the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, which comprises an adult cell isolated from any aspect of the invention or a cell population of the invention. In a preferred embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier. In another preferred embodiment, the pharmaceutical composition of the invention further comprises another active ingredient.

The term "pharmaceutically acceptable vehicle" refers to a vehicle that must be approved by a federal government regulatory agency or a state government 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" refers to a diluent, adjuvant, excipient or carrier with which the cells or the cell population of the invention or of said composition comprising stem cells of the invention obtainable according to the method of the invention should be administered; obviously, said vehicle must be compatible with said cells. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (eg, 0.9% sterile saline NaCI, 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 collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polynithine, hydrogels, agarose, silicone dextran sulfate. 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 cells 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 cells, which comprise said pharmaceutical composition, and / or auxiliary substances or Pharmaceutically acceptable substances, such as buffering agents, surfactants, cosolvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators. The stability of the cells 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, and so on. 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).

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.

USES OF THE INVENTION CELLS

Another aspect of the invention relates to the use of a cell or a population of cells of the invention, or of a pharmaceutical composition of any of aspects of the invention, in the preparation of a medicament. Alternatively, it refers to a cell or a population of cells of any aspect of the invention for use as a medicament. Another aspect of the invention relates to the use of a cell or a population of cells of any aspect of the invention, or of a pharmaceutical composition of the invention, in the preparation of a medicament for tissue repair and regeneration. Alternatively, it refers to a cell or a population of cells of any aspect of the invention for use in the manufacture of a medicament for tissue repair and regeneration. In a preferred embodiment of this aspect of the invention, the tissue is the myocardium.

Another aspect of the invention relates to the use of a cell or a population of cells of any aspect of the invention, or of a pharmaceutical composition of the invention, in the preparation of a medicament for the treatment of cardiovascular diseases. Alternatively, it refers to a cell or a population of cells of any aspect of the invention for use in the preparation of a medicament for the treatment of cardiovascular diseases.

In a preferred embodiment of this aspect of the invention, cardiovascular disease is selected from the group comprising: myocardial infarction, cardiac hypertrophy and cardiac arrhythmia, or any combination thereof. In another even more preferred embodiment, cardiovascular disease is cardiac ischemia.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, the disease is a cardiovascular disease, and more preferably it is a heart attack. The pharmaceutical composition of the invention will contain a prophylactic or therapeutically effective amount of the cells of the invention or of the cell population 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 cells of the invention contained in the pharmaceutical composition that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, due to the characteristics of the cells and the desired therapeutic effect sought. In general, the therapeutically effective amount of cells of the invention to be administered will depend, among other factors, on the subject's own characteristics, the severity of the disease, the manner of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the person skilled in the art, who should adjust this dose depending on the factors described above. As an illustrative and non-limiting example, the pharmaceutical composition of the invention can be administered as a single dose, containing approximately between 1 x 10 ⁵ and 10x10 ⁶ cells of the invention per kilo of body weight of the recipient, and more preferably between 5x10 ⁵ and 5x10 ⁶ cells of the invention per kilo of body weight of the recipient, in a more preferred embodiment even said pharmaceutical composition contains about 1 x10 ⁶ and 2x10 ⁶ cells of the invention per kilo of body weight of recipient, depending on the factors described previously. The dose of cells of the invention can be repeated, depending on the condition and evolution of the patient, 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 to the individual. Examples Illustrative, non-limiting, 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 appropriate pharmaceutically acceptable carrier. , for administration to a subject parenterally, eg, a human being, preferably intravenously, intraperitoneally, subcutaneously, etc., although other alternative routes of administration are also possible. Administration of the pharmaceutical composition of the invention to the individual will be carried out by conventional means. For example, said pharmaceutical composition can be administered to said individual 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 serial inflation and deflation of distal and proximal balloons located within the patient's vasculature, 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 that any of these components can be mixed in the same composition and other active ingredients. The isolated adult cell of the invention, or the cell population of the invention, may be of autologous, allogeneic or xenogenic origin. Preferably, they are of autologous origin. The administration can also be carried out with the help of a "scaffold" or bioactive scaffolding, that is, of a support structure that serves to transport cells and / or growth or differentiation factors, and which usually consist of a structure quite rigid, covered or impregnated with a drug that helps cells establish themselves and build a tissue that is the same as what you want to replace. Different scaffoid are known in the state of the art. As previously stated, if desired, the adult cell 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 they are important for their basic properties (proliferation, migration, transdifferentiation, etc.).

Another aspect of the invention relates to its use in a screening or drug selection method comprising:

a) Contacting a cell or a population of adult cells isolated from any aspect of the invention with a substance to be tested,

b) Evaluate the effect of said substance on the cell phenotype or cell population of any aspect of the invention. METHOD OF OBTAINING CELLS OF THE INVENTION

The inventors have also developed a method of isolation and identification of cells and cell populations of adult cells isolated from any aspect of the invention. Thus, in another aspect, the invention relates to a method for the isolation of cells from any aspect of the invention, from blood from the coronary sinus of a mammal, or from its blood products, hereinafter "method of invention ", comprising:

to. obtain a blood sample from the coronary sinus of a mammal,

b. isolate the blood sample cells from step (a), Preferably, it also includes:

C. purify the isolated cells of (b),

and. subculture the floating cells of step (d) in an enriched medium,

and even more preferably, it comprises:

F. keep them in cultivation until their confluence and subculture them again.

Preferably, the coronary sinus blood sample step (a) is diluted, in an approximate ratio of 1: 6 in a 5% PBS solution of FBS and 1% of antibiotics. In another preferred embodiment, the diluted sample from step (a) is centrifuged with Ficoll. In another preferred embodiment, after centrifugation, the mononucleated cells are washed with 5% FBS PBS and 1% antibiotic by centrifugation. In another preferred embodiment, after washing, the cells are re-suspended in FBS and EBM-2G. In another preferred embodiment, the re-suspended cells are seeded in wells with 1 ml of EBM-2G medium. More preferably, the 7th day of incubation the supernatant phase where the floating cells are taken up and incubated in a new culture dish with a medium comprising DMEM and HamF12 in proportion 4: 6, FBS, antibiotic, hydrocortisone, EFG, FGF-B, IGF-1, glutamine, 1M Hepes and ascorbic acid, in fibronectin-coated plates. More preferably, on the 12th day the cells are subdivided into plates treated with fibronectin. Even more preferably, at least 8 subcultures of the cells are made in plates treated with fibronectin.

Subsequently, adult cells isolated from any aspect of the invention, or the cell population of the invention, can be identified by any method known in the state of the art. In the present invention, "identification" means the process that allows a cell to be recognized. isolated adult of the invention present in a sample and distinguish it from the rest of cells present in said sample.

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

Once the blood sample, preferably from the coronary sinus of a mammal, has been obtained, it should be treated to isolate the mononuclear cells. Any of the methods in the state of the art for isolating mononuclear cells from 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.

For the identification of adult cells isolated from any aspect of the invention, the markers described above can be used. These markers are proteins, and in some of them 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. The detection of the marker of the invention and the subsequent isolation of the cells expressing said marker can be carried out by any method that allows cell separation based on a given phenotypic or genotypic characteristic.

In the identification and / or isolation tests, the cell or 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.

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 monoclonal antibodies 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.

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.

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 they are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors.

Materials and methods

Example 1.- IN VITRO MORPHOLOGICAL STUDY OF THE PRIMARY CULTURE OF CELLS FROM THE CORONARY SINE OF PATIENTS WITH ACUTE INFARTO OF MYOCARDIUM.

In vitro studies of floating cell culture were obtained by extracting blood from the coronary sinus through catheterization of n = 9 patients with acute coronary syndrome and ST elevation. Peripheral blood was obtained through venous puncture of the same patients. Heparin (BD-Vacutainer) was used as an anticoagulant for cell isolation. Mononuclear cells were isolated by gradient with Ficoll (Sigma-Aldrich) at 800g, 15 minutes RT. The cells were washed with 1% PBS of FBS. The culture was previously performed in EBM-2G medium supplemented with FGF, EGF, IGF, glucagon, amphotericin D, penicillin-streptomycin, 20% FBS (Cambrex, Lonza) for 7 days the cells were incubated at 37 ° C in an atmosphere wet at 5% CO2. Floating cells that were seeded in HamF12: 10% DMEM FBS, 0.01% penicillin-streptomycin were obtained.

Example 2.- STUDY OF THE CELLULAR SIZE OF THE COLONING FORMER CELLS.

Photographs were taken in vivo to analyze the size and external morphology of the clusters and adherent cells (Fig7-a-7-d). A Nikon TE2000-U microscope was used and with a Nikon DS5MC cold camera Likewise, photographs were taken with cells and clusters previously fixed with 4% paraformaldehyde in order to preserve their morphology (Fig. 3-4). With Normanski microscopy in Nikon microscope model Eclipse E800. Example 3- STUDY OF PROLIFERATIVE CAPACITY.

Cluster forming cells were collected, trypsinized and subcultured for 5 months. The studies were carried out in triplicate at 14 culture passes. These studies were performed by sowing a total of 5,105 cells per cm2 and counting was performed using a Neubauer camera and the Nikon microscope model TE2000-U. The four fields in each quadrant were counted, the corresponding 16 squares were counted, of the total counts the arithmetic mean was made. For the total count, the cells were considered to be resuspended in 1 ml of cell medium and 10μΙ of cell suspension was used for counting.

To check the proliferative capacity over 30 days, in cultures of long incubation period (5 months and more than 14 passes) a total cell number was sown: 5,105 per cm2 at time zero t = 0, subsequently they were collected at t = 1: 24 hours, t = 2: 48 hours, t = 3: 3 days, t = 4: 6 days, t = 5: 7 days, t = 15 days, t = 8 30 days from culture. It was done in triplicate. Photographic monitoring was performed in vivo, using the Nikon microscope model TE2000-U and with a cold camera model Nikon DS5MC. The cell count in Neubauer chamber gave rise to a growth curve (where the average data of the count and the deviation of the average of each count were shown) and to the study of doubling-time.

For the study of the time it takes for a cell to divide in half, the following free software obtained from the following web address http://mathworld.wolfram.com/LeastSquaresFittinqExponential.html used by the following bibliographical references was used: Roth V. 2006 <http://www.doubling-time.com/compute.php>

Weisstein, Eric W. "Least Squares Fitting— Exponential." From MathWorld— A

Wolfram Web

To study the rate of cell-mediated cell proliferation, cells were seeded at a concentration per well of: 5,105 per cm2 by performing a cell count on days 0, 1, 3, 5 and 7. The means used to resuspend the cells they varied as follows: an experiment was sown where the culture medium consisted of medium "where the cells were pre-incubated previously filtered (0.22μηη) and floating medium (Hamf12: DMEM 20% FBS) The volumes used were 1 vol : 1 vol for pre-culture medium and replacement medium (floating medium).

The second experiment was to sow the same number of cells per cm2 using entirely renewed floting medium. And the third experiment was the sowing of the same number of cells but replacing a volume 1 vol: 3vol. All experiments were performed at the same time. They were counted on the same day and counted at 0, 1, 3, 5 and 7 days of cultivation.

Example 4- MORPHOLOGICAL STUDY OF COLONIES FORMING CELLS USING PHALLOIDIN AS A CYTO SKELETON MARKER.

The clusters were collected using a 3ml pasteur pipette and deposited in a conical neck falcon tube. The clusters were washed twice with a buffered saline phosphate buffer, pH7.4. The clusters were introduced in a paraformaldehyde solution for 10 minutes at room temperature. They were washed twice after staining with Phalloidin with PBS. The clusters were incubated for 30 minutes at room temperature in a humidified chamber. They were washed again twice with PBS solution and nuclear staining was performed with Dapi (Sigma-Aldrich) 1: 200 The images were made with the Nikon microscope model TE2000-U and with a cold camera model Nikon DS5MC.

Example 5- STUDY OF CELLULAR VIABILITY IN LONG-TERM CROPS (OVER 5 MONTHS) THROUGH CALCEINA AM

Cell cultures were used with cluster-forming cells that were trypsinized (Trypsin / Edtta 1 x) for 1 minute in a cell incubator (37 ° C, 5% CO2) and resuspended at a concentration of: 5,105 per cm2. The cell viability was monitored for 7 days, in cultures of 1 month of incubation as in cultures of 3 months of incubation or 5 months of incubation. The photographic recording was carried out in a Nikon fluorescence microscope model TE2000-U and with a cold camera model Nikon DS5MC.

Example 6- STUDY OF CLONOGENESIS USING BRDU

Previously trypsinized clusters were taken and individualized cells were seeded in a 96-well multipoint (in 2000 cel / well) in floating medium: pre-incubation medium (1 vol: 1 vol) for 4 days the cells were treated with BrdU (10μΜ) subsequently collected with a 3 ml pasteur pipette, gently centrifuged and mixed with untreated BrdU cells, in medium (1 vol: 1 vol, in floating medium: preincubation medium) in order to observe if mixed aggregates were formed or maintained the clonogenic capacity. After 15 days of incubation the immunofluorescence was performed. Clusters were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS for 10 minutes 3 times and incubated with 4M HCI for 5 minutes afterwards permeabilization was performed with HCI, subsequently blocked with BSA (Sigma Aldrich) and PBS for 20 minutes, and washed with PBS 3 times, for 2 hours the 1 ^or antibody (Becton and Dickinson, BD Ref: 555627) was incubated at a concentration of 1: 75, washed with PBS for 10 minutes in 3 successive washes each. It was incubated for 1 hour with the secondary polyclonal antibody TRIC (1: 200) and Dapi as a nuclear marker (concentration 1: 200). Immunofluorescence was mounted with medium montain gel and was visualized in Nikon inverted microscope model TE2000-U and with cold chamber model Nikon DS5MC

Example 7- STUDY OF CLONOGENESIS USING GFP (p-eGFP-C1)

To observe clonal growth by means of plasmid e-GFP-C1 expressing the green fluorescent protein GFP. The cell clusters were disintegrated with Trispine / Edta (1 x, Sigma-Aldrich) for 1 minute and subsequently the cells were washed with floating medium (DMEM: HamF12 20% FBS). And they were kept in a 15ml falcon in the cell incubator (37 ° C, 5% CO2 in a humid atmosphere) until they were transfected. Plasmid p-eGFP-C1 (Clontech, Spain) previously pre-incubated for 30 minutes with Fugene 6 Transfection Reagent (Roche) with a DNA: Fugene ratio of 3: 2 was used. The cells were incubated with the Fugene: p-eGFP-C1 for 1 hour in an incubator to promote plasmid transfection and subsequently seeded at a rate of 5,105 per cm2, 50% of the cells seeded by each well being positively transfected and the other 50% untransfected cells. The images with e-GFP-C1 were obtained in real time in vivo after 4 days in incubation, with Nikon inverted microscope model TE2000-U and with cold camera model Nikon DS5MC. The proportion of cells transfected with the GFP plasmid is visualized as the formation of cell clones.

Example 8- Visualization of the ASYMMETRIC DISSION

In order to assess cell size, cells and cell clusters with paraformaldehyde were fixed. Cultures of 14 passes were used, which were collected and fixed with 4% paraformaldehyde for 10 minutes and washed with PBS for 10 minutes in three independent incubations. Subsequently, 50mM ammonium chloride was used as a neutralizer and the washings were repeated. The cells fixed and resuspended in PBS were visualized and photographed on the Nikon model TE2000-U and with a cold chamber model Nikon DS5MC. To be able to check the structural integrity and visualize the polar bodies products of the asymmetric division of the typically stem cells, a fixation with 4% Paraformaldehyde was made subsequently the cells were stained with phalloidin at a concentration 5 μΙ: 200μΙ PBS subsequently stained with Dapi for nuclear marking (1: 200). The images were obtained in the inverted microscope model Nikon model TE2000-U and with cold camera model Nikon DS5MC. Example 9- PHENOTYPICAL STUDY OF THE CLUSTERS OF FLOATING CELLS FROM THE CORONARY BREAST USING THE CELL BIOLOGY TECHNIQUE: IMMUNOFLUORESCENCE (Oct3 / 4, Nanog, Ssea-1, Ssea4, Tra81 -1, cKit).

The cells from coronary sinus blood of patients with infarction, cluster-forming medium cultured (DMEM: HamF12, 10% FBS), were collected in 15 ml falcon tube, fixed with 4% paraformaldehyde (Sigma Aldrich) for 20 minutes Then, 3 successive washes were performed with PBS, centrifuging the cells at 400 g at room temperature for 10 minutes. Cells were stained with Faloidine (Sigma Aldrich) as a cytoplasmic marker (1: 50) for 35 minutes of incubation and 15 minutes with DAPI (1: 200) as a nuclear marker. They were subsequently washed and the immunofluorescence was mounted with Montain Medium Gel by incubating them 30 minutes in the dark before being visualized (Fig. 2). The c-Kit marker (BD bioscience) was also used as a cardiac resident cell marker, incubating at a 1: 10 concentration for 1 hour (Fig. 3). For the mapping of Oct3 / 4 and Nanog the manufacturing protocols of the commercial house (BD bioscience) were followed and after fixation with 4% paraformaldehyde the cells were washed and fixed with Methanol (- 20 ° C) for 5 minutes, subsequently they were centrifuged and the supernatant was aspirated, subsequently incubated 10 minutes with Triton x-100 (0.01%) and washed with PBS. The cells were subsequently blocked for 1 hour at room temperature with 1% BSA and Tween20 (0.05%). Be performed the washing and incubation for 1 hour of Oct4 conjugated with FITC (1: 10) in PBS subsequently washing (3 washes with 10-minute incubations of 10 minutes in PBS 0.05% Tween20) and incubation with Nanog conjugated with PE for 1 hour (1: 10) and the last wash was performed (3 washes with 3 10-minute incubations in 0.05% PBS Tween20) and the immunofluorescence was mounted with a Montain Medium (TissueTeK) gel solution for 30 minutes remained incubating in darkness (Fig. 4). The images were obtained in confocal microscope LEICA, mod. SP5 II and analyzed in the Leica LAS AF software.

Example 10.- PHENOTYPICAL STUDY OF THE CLUSTERS OF FLOATING CELLS FROM THE CORONARY SENUS USING THE FLOW CYTOMETRY TECHNIQUE

The cells were labeled for 30 minutes at room temperature with the following conjugated antibodies: CD15-PerCeP, OCT3 / 4-FITC, Nanog-APC (BDbioscience), CD105-APC, CD34-FITC, CD133-PE, cKit-PeCy7, CD90 - APC, CD146-FITC, CD31-FITC, CD45-FITC, KDR-APC, CXCR4-PE (R&D Systems), negative control isotypes were used for each corresponding immunoglobulin IgG-PerCep, IgG-FITC, IgG-APC, IgG -PE (R & Dsystems). The manufacturing advice of the BD biosystems commercial house was followed for the immobilization of Oct-4 and Nanog, so the cells were fixed in paraformaldehyde for 4 minutes, then 5 minutes of incubation with 0.1% triton-x100, blocked with BSA, 0.5% PBS for 1 hour. The flow cytometry results obtained with the Dako-cytomation Cyan cytometer (Summit software v3.4).

Example 11.- PHENOTYPICAL STUDY OF THE FLOATING CELL CLUSTERS FROM THE CORONARY BREAST USING THE MOLECULAR BIOLOGY TECHNIQUE: RT-PCR end-point and qRT-PCR in real time.

Total RNA was extracted from human floating cells from the coronary sinus using the RNeasy total RNA kit (Quiagen). The quantity and purity of RNA samples were made in the Nanodrop (Thermo Scientific D-1000). Only samples with a 260/280 nm ratio of 2.0 were accepted for analysis. 2 g of total RNA sample were used to perform the témplate (cDNA) using the reverse transcriptase enzyme MMLV (Promega, Madison, USA). 200 ng of cDNA were used for semiquantitative PCR detection with specific human gene primers detailed in the table.

The endogenous b-actin gene was used as an endogenous control for the normalization of gene expression levels. The sequences of the primers were determined through the GenBank sequences, and published in previous studies (Kennedy et al., 2007. Blood. Apr 1; 109 (7): 2679-87; Tomescot et al., 2007. Stem Cells. Sep; 25 (9): 2200-5. Epub 2007 May 31; Gaetani et al., 2009. Cardiovascular research 82:41 1-20).

The temperature conditions of the end-point PCR cycle began with a first cycle of denaturation of 95 ° for 2 minutes and 25 cycles of consecutive stages of denaturation at 94 ° C for 30 seconds, primers mating was performed at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes and finally 72 ° C for 7 minutes. These reactions were carried out in Thermocycler (Applied Biosystems). It was used as a physical support for the separation of the different DNA fragments by electrophoresis, a 2% agarose gel stained with Ethidium Bromide. The ChemiDoc ™ XRS gel developer (Bio-Rad Laboratories) was used to visualize the PCR products. The software used for analysis was version 460.

For the study of quantitative expression of genes in floating cells extracted from venous blood from the coronary sinus compared to cells extracted from peripheral venous blood, cultures treated with the same medium and the same incubation time (30 days) were used. Quantitative real-time PCR was performed on the 7500 Fast (Applied Biosystems) equipment. For the qRT-PCR reaction a concentration of 50 ng cDNA and a final reaction volume of 20 μΙ. SYBR Green (Roche, FastStart Universal SYBR Green Master (ROX) Applied Science, Germany) was used for the detection of PCR products the following genes were used: b-actin, Nanog, Oct4, GATA-1, Brachyury, SOX -1, SSEA1, Runxl, cMyc, Mef2c, Nkx2.5, IsM +, CXCR4 and KDR, NeuroD, Foxa2 / NHF-3.

For the amplification the following program was used: denaturation at 55 ° C for 20 seconds and a second denaturation at 95 ° C for 10 minutes, 40 cycles subdivided into two stages were used: first stage at 95 ° C for 15 seconds and the second stage at 55 ° C for 1 minute. The conditions of the melting curve were at 95 ° C for 15 seconds; at 60 ° for 1 minute; at 95 ° C for 30 seconds and at 60 ° C for 1 5 seconds. The software used for the analysis was the 7500 fast version SDS 2.0.5.

Data were analyzed according to the relative ddCT expression analysis method, previously described by Pfaffl (Pfaffl et al., 2002. Nucleic Acids Res. May 1; 30 (9): e36) Gene expression data of the floating cells from the coronary sinus are normalized according to the peripheral blood in Log10 scale.

Table 1 .- Genes

Oct3 / 4 FW: SEQ ID NO: 1 RW: SEQ ID NO: 2

NANOG FW: SEQ ID NO: 3 RW: SEQ ID NO: 4

B-ACTIN FW: SEQ ID NO: 5 RW: SEQ ID NO: 6

GATA1 FW: SEQ ID NO: 7 RW: SEQ ID NO: 8

KDR FW: SEQ ID NO: 9 RW: SEQ ID NO: 10

NeuroDI FW: SEQ ID NO: 1 1 RW: SEQ ID NO: 12

RUNX1 FW: SEQ ID NO: 13 RW: SEQ ID NO: 14

SCL FW: SEQ ID NO: 15 RW: SEQ ID NO: 16

SOX7 FW: SEQ ID NO: 17 RW: SEQ ID NO: 18

BRACHYURI FW: SEQ ID NO: 19 RW: SEQ ID NO: 20

TNI FW: SEQ ID NO: 21 RW: SEQ ID NO: 22

SMA FW: SEQ ID NO: 23 RW: SEQ ID NO: 24

SSEA-1 FW: SEQ ID NO: 25 RW: SEQ ID NO: 26 CXCR4 FW: SEQ ID NO: 27 RW SEQ ID NO: 28

SOX2 FW: SEQ ID NO: 29 RW SEQ ID NO: 30

C. Myc FW: SEQ ID NO: 31 RW SEQ ID NO: 32

CAT 4 FW: SEQ ID NO: 33 RW SEQ ID NO: 34

MEF2C FW: SEQ ID NO: 35 RW SEQ ID NO: 36

ISL1 + FW: SEQ ID NO: 37 RW SEQ ID NO: 38

* The oligonucleotides have been previously published (Kennedy et al Blood. 2007 Apr 1; 109 (7): 2679-87; Tomescot A et al Stem Cells. 2007 Sep; 25 (9): 2200-5. Epub 2007 May 31; Gaetani et al Cardiovascular research. 2009; 82: 41 1-20)

Sequences SEQ ID NO: 39 and 40 represent primers fw and rw respectively of GATA4-h.

Sequences SEQ ID NO: 41 and 42 represent primers fw and rw respectively of C-KIT

Sequences SEQ ID NO: 43 and 44 represent primers fw and rw respectively of FOXA2 / HNF-3 The sequences used in the present invention have been previously described in the literature as depicted below:

- SMA, Nkx2.5, Tnl from Gaetani R, Ledda M, Barile L et al. Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields. Cardiovasc Res 2009; 82: 41 1-20.

- FOXA2, GATA-1, KDR, NeuroD, RUNX1, SOX7, SCLb, b-actin:

Kennedy M, D'Souza SL, Lynch-Kattman M, Schwantz S, Keller G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 2007; 109: 2679-87.

- Nanog, c-KIT from Kalmar T, Lim C, Hayward P, Munoz-Descalzo S, Nichols J, Garcia-Ojalvo J, Martínez Arias A. Regulated fluctuations in nanog expression mediated cell fate decisions in embryonic stem cells. PLoS Biol 2009; 7 .: Adamo L, Naveiras O, Wenzel PL et al. Biomechanical forces promote embryonic haematopoiesis. Nature 2009; 459: 1 131-5

- Mef2c, Isl1 +, Brachyury, GATA4: Tomescot A, Leschik J, Bellamy V et al. Differentiation in vivo of cardiac committed human embryonic stem cells in postmyocardial infarcted rats. Stem Cells 2007; 25: 2200-5.

- cMyc, SSEA-1: Conacci-Sorrell M, Ngouenet C, Eisenman RN. Myc- nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation. Cell 2010 Aug 6; 142 (3): 480-93.

- SSEa-4: Kashyap V, Rezende NC, Scotland KB, Shaffer SM, Persson JL, Gudas LJ, MonganNP. Regulation of stem cell pluripotency and differentiation involves a mutualn regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cells Dev. 2009 Sep; 18 (7): 1093-108

- Oct3 / 4: Kim JB, Sebastiano V, Wu G, Araúzo-Bravo MJ, Sasse P, Gentile L, Ko K, Ruau D, Ehrich M, van den Boom D, Meyer J, Hübner K, Bernemann C, Ortmeier C , Zenke M, Fleischmann BK, Zaehres H, Schóler HR. Oct4-induced pluripotency in adult neural stem cells. Cell 2009 Feb 6; 136 (3): 41 1-9.

Example 12- FUNCTIONAL STUDY OF THE FLOATING CELL CLUSTERS FROM THE CORONARY BREAST: CONTRACTILITY STUDY

Cluster-forming floating cells were collected in 15ml falcon tube and centrifuged at 250g for 10 minutes. The supernatant was aspirated and 1 ml of trypsin was added to 1 X trypsin (Sigma Aldrich) by incubating them for 3 minutes in incubation (37 ° C, 5% CO2 and humid atmosphere). Subsequently, 500,000 cells were quantified and seeded in each well (multiwell of 24). GelTrex-DMEM-HamF12 medium was seeded in a 1: 29 ratio, whereby the texture of the gelatinous matrix was low) and 5% FBS (without supplementation). The cells remained 24 hours in incubation, before applying the treatment of NE, to promote cell adaptation to the new culture medium. After that time, they were treated with Norepinephrine (100μΜ) and after 12 hours, it was observed with phase contrast microscopy if the cells had the ability to contract using the Nikon model TE2000-U microscope and the images were made with the camera Nikon model DS5MC.

Example 13- FUNCTIONAL STUDY OF THE CLUSTERS OF FLOATING CELLS FROM THE CORONARY SINE: STUDY OF ANGIOGENESIS.

The floating cells of the coronary sinus and the peripheral blood cells of 30 days of culture, were collected and trypsinized with 1 X trypsin, seeded at a concentration of 50,000 cells per well in the Matrix system Angiogenesys Systems (BD bioscience) previously stained with Calceina AM (1 hour in darkness). 5% humid atmosphere of C02, 37 ° C was incubated for 15 days. Continuous monitoring of vessel formation was performed using the Nikon model TE2000-U microscope, the photographs were taken with the Nikon model DS5MC model camera. Obtaining results of 5 days and 15 days respectively.

Example 14- RESULTS OF THE IN VITRO STUDY, OF THE PRIMARY CULTURE OF CELLS FROM THE CORONARY SINE OF PATIENTS WITH ACUTE INFARTO OF MYOCARDIUM.

Through the primary culture of two different blood sources: cells from peripheral blood and blood from the coronary sinus of patients with acute myocardial infarction, we verify a different cellular behavior.

While it is true that cells from circulating blood adhered and formed tubule-like structures (Figure 1 a-1 c) as typically described in the literature, coronary sinus cells clustered and did not differentiate no other tissue (Fig. 1 d-1f), Both cells progenitors discovered in resident cardiac tissue by Messina et al. (Messina et al., 2004 Circ Res. 95: 91 1-921), such as human or mouse embryonic progenitor cells (Davis et al., 2010. J Mol Cell Cardiol. Aug; 49 (2): 312-21), such as neuronal progenitor cells (Reynolds et al., 1992. Science. Mar27; 255 (5052): 1707-10) have the common characteristic of forming cell clusters similar to those described in this studio. Asymmetric cell division, resulting in larger cells and smaller cells (Snippert et al., 201 1. EMBO Rep. Feb; 12 (2): 1 13-22) is an inherent characteristic of progenitor cells which share the cells of the invention (Fig. 2).

Example 15- RESULTS OF THE STUDY OF THE CELLULAR SIZE OF THE FORMER CELLS OF COLONIES.

The cells multiply giving rise to asymmetric division, with cells of approximate size between 1 1 and 20 μιτι (figure 4) and smaller cells that do not exceed 6 μιτι (figure 5). In the study of asymmetric division using phalloidin (Figure 23-24) as a cytoskeletal marker, the presence of the development of a secondary body is observed in nuclear tide (Figure 24)

The images obtained with inverted microscopy show a variable cluster size between 50μηη and 500μηη in diameter. In the initial stages of cell culture, (Fig 6-a) cluster is observed in vivo, with a number between 17 and 20 cells per cluster, which gradually increase in size (40 cells per cluster, Fig 6b) later (80 cells per cluster, I observed in Fig. 3 in samples fixed with paraformaldehyde with a diameter of 100 μιτι), doubling their size reaching a diameter of 200 μιτι (fig 6c). This cellular increase reaches over 500 μιτι in diameter in cultures with 60 days of incubation. Example 16- RESULTS OF THE STUDY OF THE IN VITRO CELLULAR PROLIFERATION OF CELLS FROM THE CORONARY SINE OF PATIENTS WITH ACUTE INFO OF MYOCARDIUM

As a result of the proliferation study, it was observed how cluster-forming cells have as particular feature the secretion of proliferation stimulating factors. Therefore, the replacement of the medium, total or de novo, causes a change in the initial characteristics of the culture medium that retard the proliferative process. The colony-forming clusters take 72 hours to reach exponential proliferative activity (fig 10-1 1).

From time 0 to 48 hours (Figure 10) of culture the cells have a Doubling Time Average equal to 57 hours (Figure 7). However, from 48 hours to 72 hours and later, the average dubbing time is approximately 18 hours. Between 120 and 144 hours (figure 1 1) the doubling time is approximately 19 hours. (figure 7). This phenomenon is enhanced when crops whose percentage of replacement of the medium is not total but partial. However, in those crops whose replacement of the culture medium is total, the growth rate and the growth rate are reduced as shown in Figure 8.

The analysis over 30 days of three cell samples from different patients, shows in graph 9, the average of the counts made at each point of the study, show a Doubling Time = 29.31 hours. Figure 8 shows how, for the exponential amplification of the clusters, cell culture is required in a ratio of 5,105 cells per cm2 and with a fraction of cell medium where they have been previously generated or cultured. Figure 8 also shows the culture with a stationary proliferation window, whose growth rate is lower, caused by a total replacement of the culture medium by means of de novo "floating" culture. Example 17- RESULTS OF THE MORPHOLOGICAL STUDY OF COLONY FORMER CELLS USING PHALLOIDIN AS A CYTO SKELETON MARKER. Through the use of Phalloidin and DAPI nuclear marker it has been found that cell clusters have a particular morphology since they have a central cavity, until now it has not been observed by cardiosphere-forming cells, nor mesenchymal stem cells, nor hematopoietic stem cells ( Figure 12 and 13).

The results on the preservation of the structure using phalloidin, in cultures for a long time have allowed to observe how the time of permanence in culture does not produce a disorganization of the cytoskeletal structure as it happens in necrosis or apoptotic processes, nor are structural anomalies of the DNA such as apoptotic bodies, condensed heterochromatin, etc. On the contrary, in these cluster of 3 months of cultivation they remain morphologically and structurally well organized (Figure 15, 16 and 17 in detail). Phalloidin marking allows the cytoskeletal distribution of cells to be observed (Figure 15, 16), observing a central nucleus (Figure 17) and a circular cyskeleton (Figure 18-merge) that occupies less than 20% of the cell surface. Both nuclear tides (Figure 12,13 and 14-b) and phalloidin tide figure 14-a) show the presence of the orifice present in the cell cluster. In figure 15, it is observed in detail how this pore is configured, not described in the literature of cardiac stem cells or cells derived from cardiac stem cells. In Figure 18 and 23 it can be seen how the cell nucleus is monoclonal and occupies more than 80% of the cell surface. The cytoskeleton is organized, and has different densities of accumulation of f-actin marking, assuming a higher cytoskeletal density for certain areas clearly more intensely marked, and others less marked with green phalloidin as observed in figure 23. It can be seen in both figure 16 and 23 that the cytoplasm is circumcentric and the spherical nucleus. Cluster-forming cells are moriologically spheroidal, with a central nucleus, as seen in Figure 17-18 and 23-24.

Example 18- STUDY OF CELLULAR VIABILITY IN LONG-TERM CROPS (OVER 5 MONTHS) BY CALCEINA AM

The marking with calcein AM (green or red, Invitrogen) allows to know the viability of the cell, and if it is metabolically active. The results show that in initial cultures as in cluster-forming cells (figure 19) they remain active and viable (Figure 19-2 cultures of 2 months of incubation).

In cultures of 5 months of incubation, it is observed how the cluster structure remains positively marked with Calcein Red AM. Similarly, it is observed that the center of clusters larger than δΟΌμιτι also have metabolic activity and viability as shown in Figure 19-4.

Example 19- RESULTS OF THE CLONOGENESIS STUDY USING GFP (p-eGFP-C1)

The results with positive GFP transfection show GFP positive cells (Fig 20-b and Fig 21 d) and clone proliferative cells (Figure 21). By incorporating cells transfected with GFP vs not transfected with GFP, it can be seen how those cells that incorporated the vector form clones and the cells that did not incorporate the vector are not labeled and do not form the clones.

Example 20- RESULTS STUDY OF CLONOGENESIS USING BRDU

This is another confirmatory test that the clusters come from clonal structures. To rule out the formation of adhesion aggregates, both cells treated with untreated BrdU were cultured in the same conditions and in the same wells. The result is that shown in Figure 22, a clonal growth, with an increase in proliferative capacity inside the cluster, which is where the highest BrdU signal is observed (Fig byd).

Example 21 - RESULTS OF THE PHENOTYPICAL STUDY OF THE CLUSTERS OF FLOATING CELLS FROM THE CORONARY SINE BY IMMUNOFLUORESCENCE. The results of confocal microscopy show how cluster-forming cells form a compact structure formed by cells whose nucleus (DAPI nuclear marker: fig. 22 and 23) occupies a large part of the cytoplasmic region of the cell. Positive colocalization is demonstrated (Figure 25d) of the pluripotent markers: Nanog (25b) and Oct 3/4 (25c). So we observe that these non-adherent cells, found in venous blood that has previously irrigated in the heart, are cells that are capable of expressing both pluripotent markers typical of embryonic stem cells. We found the expression of cells in early stages of culture, individualized (figure 25 a-d) as in a cluster of <20Όμηη in diameter, made with Nikon fluorescent inverted microscope.

However, when we perform in a larger cluster> 200 μιτι, we observe in confocal microscopy how the distribution of pluripotent markers is not random, on the contrary it seems to house an organized structure (figure 26 ad) of pluripotent markers, showing a significant internal signal within the cell cluster. Therefore, it presupposes that the interior of the cell cluster that coincides with the highest BrdU uptake (fig. 22b and d) are the most pluripotent cells. In fig. 32 cKit positive marking is observed by sharing this marker with resident cells in cardiac tissue. Example 22- RESULTS OF THE PHENOTYPICAL STUDY OF THE CLUSTERS OF FLOATING CELLS FROM THE CORONARY SENUS USING THE FLOW CYTOMETRY TECHNIQUE. (Fig. 33 a- 33 i).

Cultured non-adherent cells from the coronary sinus capable of forming cell clusters have a majority expression of the Nanog + pluripotent markers, Oct3 / 4, as shown in Figure 33 a and b. Likewise, a majority percentage (<80%) of the immunoreactive cells is observed for the CD90 + CD105 + mesenchymal markers as shown in Figures 32e and 32f. However, in a percentage of 20% of cluster-forming cells express cKit +, as a cardiac stem cell marker (Fig32d) and as well as CD133, CXCR4 +, CD15 (SSEA1) +. KDR + expression is less than 5% of cells. Negative immunoreaction has been observed for the CD34-, CD45-, CD31- and CD146- markers, so it is shown that this cell type does not share the specific markers of adult or adult progenitor hematopoietic cell, nor of adult endothelial cell. Example 23- RESULTS OF THE STUDY OF GENE EXPRESSION AND RELATIVE QUANTIFICATION OF THE CLUSTERS OF FLOATING CELLS FROM THE BREAST.

End Point PCR results show how floating cultured cells from the coronary sinus express the coding genes for transcription factors and pluripotent homeoboxes: Nanog, Oct3 / 4, SSEA-1, Sox-2, Sox-7, Brachyury, Runxl , Slc, cMyc (Fig. 34). Current literature describes that the activity of embryonic transcription factors cMyc, Klf-4, SSEA1, Otc3 / 4, Nanog facilitate the acquisition of the pluripotent ability to transform mouse fibroblast cells into pluripotent cells that are capable of acquiring the phenotype Cardiac (Martínez-Fernandez et al., 2009. Circ Res. 105: 648-656; Nelson et al., 2009, Circulation. 120: 408-416). Studies in circulating non-resident cells peripherals observe the presence of circulating cells that have the characteristic of expressing transcription factors such as Oct3 / 4, Nanog and SSEA1 such as VSEL cells, which are increased after an acute coronary syndrome. In this study, a cell type has been found in venous circulation of the coronary sinus that shares the characteristics of resident cardiac-forming cells and with the ability to express transcription factors involved in the maintenance of pluripotency (Brachyury, Runxl, cMyc, SSEA1, SSEA4, SSEA3, Oct3 / 4, Nanog, SOX2, SOX7, GATA-1) typical of human embryonic cells and in the development of embryonic cells towards cardiac tissue such as Nkx2.4, cMef, Isl 1 +, GATA4, Tnl. The results of PCR-End Pont show expression of genes involved in the cardiac lineage: GATA4, lsl-1, Mef2c, Nkx2.5. In the study of the pluripotent and indifferentiation characteristics of the cluster-forming floating cells of the coronary sinus we find the expression of markers characteristic of neurospheres such as NeuroDI (NeurDI). Advocating for the pluripotential capacity of cells isolated from the coronary sinus, expression of the NeuroDI gene was observed. Expression of the transcripts for the chemokine 4 CXCR4 / CXCL12 receptor and the VEGF type 2 receptor called KDR or VEGFR2 was found. However, no expression of the transcript encoding the SMA-alpha was observed, so it follows that it is not smooth muscle cells. Like cardiospheres, and neurospheres are cells that remain floating clusters in the crop and do not remain in adherence. But unlike the cells that form cardiospheres, these cells have not been extracted from a cardiac explant but from the blood that has drained the heart and remains residual in the coronary sinus.

The results of the real-time quantitative PCR (fig. 35) show the results in logarithmic scale. It is observed when the expression levels of the floating cells of the coronary sinus are compared with the peripheral blood cells, the floating cells have an expression genes involved in development and with pluripotent characteristics between 103 and 10-106 times greater than the cells of peripherally blood. It has also been proven that Floating coronary sinus cells have an expression between 103 and 102 times greater of genes involved in cardiomyocyte differentiation than peripheral blood cells. There are no significant changes in the expression of CXCR4 but nevertheless, if an increase in the expression in peripheral blood cells of the KDR receptor (VEGFR2) associated with angiogenesis processes is observed.

Example 24- RESULTS OF THE FUNCTIONAL STUDY OF THE FLOATING CELL CLUSTERS FROM THE CORONARY SINE: CONTRACTILITY STUDY.

The capacity of contraction and generate heartbeat of the floating cells of the coronary sinus was verified synchronously (every 3 seconds) with a large refractory period, probably due to the lack in the middle of the two fundamental substrates for the contractile machinery: ATP and Calcio . Since the study has not enriched the environment with these two factors. To date, experiments on the capacity of latency or contraction require co-culture with fetal rat cardiomyocytes, or by adding azitidine (5-AZA) to the medium for 4 or 5 weeks, and after that period of time, it can be observed to fluorescence microscopy of myosin or troponin filaments (Zhang et al., 2009. Interact Cardiovasc Thorac Surg. Dec; 9 (6): 943-6). Other researchers have recently published a study showing that the use of low frequency waves stimulates the contractility of cardiac progenitor cells. In the current literature, only embryonic cells or derivatives of embryonic lines, such as mouse Isl1 +, Sca1 + cells, are able to beat spontaneously (Liang et al., 2010. Int J Cardiol. Jan 7; 138 (1) : 40-9). However, at present, no cell species of adult tissue, which has the ability to beat, without pharmacological inductions is described. Norepinephrine is a contractility stimulator, and acts on the receptors of NE stimulating the impulse. Although the cells of the invention are capable in 12 hours of developing the right machinery for contraction, it is because they probably possess the right machinery for contractility and appropriate NE receptors to trigger the stimulus. The results of gene expression confirm the presence of the genes involved in the cardiac lineage (Mef2c, GATA4, Nkx2.5) and contractile as they are TNI (cardiac) that express the proteins involved in the contractile machinery.

Example 25- FUNCTIONAL STUDY OF THE FLOATING CELL CLUSTERS FROM THE CORONARY SINE: ANGIOGENESIS STUDY.

The angiogenesis test (Figure 36) is an assay that is composed of a physical support formed by a gelatinous matrix in an essential medium supplemented with inductors for angiogenesis. The test results show that both peripheral blood cells (36b) and coronary sinus blood cells (36f) are positive for the vital marking of calcein AM. Therefore, the cells seeded in the supplemented gelatinous matrix are metabolically active and functional in this inducing medium for angiogenesis. However, as shown in Figures 9 c and 9d, cells from peripheral blood are capable of forming vascular structures such as those previously described by the literature on adult and progenitor endothelial cells (EPC). However, cells from the coronary sinus that form clusters (fig. 36 e) and that are metabolically active and viable (fig. 36f) but unable to form vascular structures (fig. 36g and 36h). These data corroborate what was previously described in our study of gene expression, where the data obtained by qPCR (fig 34) show a detriment of the expression of KDR (VEGF receptor), a fundamental receptor in angiogenesis, in cells from the coronary sinus. .

Claims

An isolated adult cell that is positive for the markers Nanog and Oct3/4, and negative for the markers CD34, the adult endothelial cell markers CD31, CD146, and the adult blood cell marker CD45, and that does not show expression of the transcription factor adult smooth muscle cell Sma, characterized because it is capable of undergoing synchronous contraction in culture with an adrenergic factor.

The isolated adult cell according to the previous claim, which is also positive for the CD90 and CD105 markers.

The isolated adult cell according to any of claims 1-2, which also naturally expresses the transcription factors IsM, NeuroDI and Foxa2.

The isolated adult cell according to any of claims 1-3, which is also positive for the CD1 17 (c-kit) markers, and naturally expresses the transcription factors Sox2, Sox7 and Runxl.

An isolated adult cell according to any of claims 1-4, which is also positive for the markers CXCR4, CD15 (SSEA1), and which also naturally expresses the transcription factors SSEA4 and cMyc.

An isolated adult cell according to any of claims 1 -5, which is also positive for the markers CD133 (Propina 1), KDR, GATA4, Mef2c, Tnl and NKx2.5.

An isolated adult cell according to any of claims 1-6, wherein the adrenergic factor is Norepinephrine.

An isolated adult cell according to any of claims 1-7, wherein the cell is characterized by having a circumcentric cytoplasm, by be mononucleated and because said nucleus occupies more than 70% of the total cell surface.

9. An isolated adult cell according to any of claims 1-8, wherein the cell is characterized by having the clonogenic capacity to subdivide or clone itself at least 8 times without suffering any chromosomal abnormality or the loss of its differentiation properties.

10. An isolated adult cell according to any of claims 1-9, wherein the cell is characterized by having an asymmetric division.

eleven . An isolated adult cell according to any of claims 1-10, which has been genetically modified.

12. An isolated cell population comprising adult cells according to any of claims 1-1 1.

13. An isolated cell population according to the preceding claim, comprising at least 80% of cells according to any of claims 1-1 1.

14. An isolated adult cell according to any of claims 1-11, or a cell population according to any of claims 12-13, obtainable by a method comprising: a. obtain a blood sample from the coronary sinus of a mammal, b. isolate the cells from the blood sample from step (a),

c. purify the cells isolated from (b),

15. An isolated adult cell according to any of claims 1-12, or a cell population according to any of claims 13-14, obtainable by the method according to claim 15, further comprising:

d. culture the purified cells from step (c), until floating cells are generated, and. subculture the floating cells from step (d),

16. The isolated cell of any of claims 14 or 15, wherein the mammal has heart disease.

17. The isolated cell of the preceding claim wherein the heart disease is acute coronary syndrome.

18. The isolated cell of any of claims 16 or 17, wherein the mammal is a human.

19. A composition comprising an isolated adult cell or an isolated population of adult cells according to any of claims 1-18.

20. A pharmaceutical composition comprising an isolated adult cell or an isolated population of adult cells according to any of claims 1-18, or a composition according to claim 19.

twenty-one . The pharmaceutical composition according to the preceding claim, further comprising a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to any of claims 20-21, which also comprises another active ingredient.

23. Use of an isolated adult cell or an isolated cell population according to any of claims 1-18, or a composition according to any of claims 19-22, in the preparation of a medicine.

24. Use of an isolated adult cell or an isolated cell population according to any of claims 1-18, or a composition according to any of claims 19-22, in the preparation of a medicine for tissue repair and regeneration.

25. Use of an isolated adult cell or an isolated cell population according to any of claims 1-18, or a composition according to any of claims 19-22, in the preparation of a medicine for the treatment of cardiovascular diseases.

26. Use of an isolated adult cell, an isolated cell population or a composition according to the preceding claim, wherein the cardiovascular disease is selected from the group comprising: myocardial infarction, cardiac ischemia, cardiac hypertrophy and cardiac arrhythmia, or any combination thereof. .

27. Use of a cell or an isolated population of adult cells according to claim 26, wherein the cardiovascular disease is cardiac ischemia.

28. A method of drug selection that includes:

to. Contact a cell or an isolated population of adult cells according to any of claims 1-18 with a substance to be tested, and

b. evaluate the effect of said substance on the phenotype of the cell or isolated population of adult cells.

29. A method of obtaining a cell or an isolated population of adult cells according to any of claims 1 -18, comprising: a. Obtain a blood sample from the coronary sinus of a mammal, b. isolate the cells from the blood sample from step (a),

c. purify the cells isolated from (b),

d. culture the purified cells from step (c), until floating cells are generated,

and. subculture the floating cells from step (d).

30. The method of obtaining a cell or an isolated population of adult cells according to the preceding claim, which also comprises: f. Keep the floating cells from step (d) in culture until confluence and subculture them again.