MXPA00009398A - Cardiac-derived stem cells - Google Patents

Abstract

The invention provides cardiac-derived pluripotent stem cells, which on proliferation and differentiation can produce a variety of cell types including cardiocytes, fibroblasts, smooth muscle cells, skeletal muscle cells, keratinocytes, osteoblasts and chondrocytes. The cells can be used in methods of treating patients suffering from necrotic heart tissue. The stem cells proliferate and differentiate to produce cardiocytes replacing the necrotic tissue. The cells can also be used to screen compounds for activity in promoting proliferation and/or differentiation of cardiac-derived stem cells.

Description

NON-DIFFERENTIATED CELLS DERIVED, CARDIAC TECHNICAL FIELD The invention lies in the technical fields of cell biology, the discovery of drugs and medicines.

BACKGROUND OF THE INVENTION In various tissues, the terminal state of cell differentiation is incompatible with cell division. For example, in the skin cells of the outermost layer, the cell nucleus disintegrates, in red blood cells, there is no nucleus, and in muscle cells, myofibrils obstruct mitosis and cytokinesis. The ability of these cells and tissues to develop, regenerate and repair depends on the existence of differentiated cells that, after division, form additional differentiated progenitor cells. Cells not terminally differentiated, can be divided without limit, and give rise to a progeny, which can continue dividing or can be differentiated. The cells . undifferentiated can be totipotent, pluripotent or unipotent. Differentiated totipotent cells (eg, embryonic differentiated cells) can give rise to each cell type in the adult organism. Undifferentiated pluripotent cells can give rise to more than one differentiated cell type. A unipotent undifferentiated cell can give rise to a single type of differentiated cell. The undifferentiated cells are generally characterized by being of small size, low granularity, a low nuclear cytoplasmic ratio and without expression of osteopontin, collagens and alkaline phosphatase. The existence of undifferentiated cells is well documented for the epidermis, the intestinal and hematopoietic epithelial systems. Undifferentiated hemotopoietic cells are present in the circulation as well as in the bone marrow. Circulating hemotoietic undifferentiated cells can colonize organs such as the spleen. It is thought that undifferentiated cells for bone, cartilage, fat cells and three types of mullets (smooth, skeletal, and cardiocytic) are a common mesenchymal undifferentiated cell precursor, but no undifferentiated mesenchymal cell or tissue-specific progenitors more concomitantly have been characterized (Owen et al., Ciba Fdn. Symp. 136, 42-46, 1988); Owen et al., J. Cell Sci 87, 731-738 (1987)). Zohar, Blood 90, 34-71-3481 (1997) reports attempts to identify progenitors of osteogenic cells by isolating cells from the fetal rat periostere and selecting the differential expression of markers, protein content and cell cycle position. Huss et al.m PNAS 92, 748-752 (1995) reports the identification of a canine bone marrow culture stromal cell precursor. It was reported that precursors differentiate in the presence of growth factor and present mature differentiation markers. Robbins et al., Trends Cardiovasc. Med 2, 44-50 (1992) reports that mouse embryonic non-differentiated cells can differentiate into embryo bodies that simulate some aspects of cardiogenesis.

DEFINITIONS An isolated cell is a cell that has been purified at least partially from other cell types with which it is naturally associated. Frequently an isolated cell exists in a population of cells of at least 25, 50, 75, 90, 95 or 99% which are the isolated cell type. Sometimes an isolated cell gives rise to a cell line in which all cells are essentially identical except for spontaneous mutations that may arise in the propagation of the cell line. Sometimes isolated cells undergo spontaneous differentiation to generate a mixed population of the mature cell type. A set of differentiation markers means one or more phenotypic properties that can be identified and are specific to a particular cell type. The differentiation markers are exhibited transiently in several cell lineage stages. Undifferentiated pluripotent cells that can be regenerated without compromising a lineage express a set of differentiation markers that can be lost when they commit to a reduced cell lineage. The precursor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress to the cell lineage towards maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cellular products and receptors. The adhesion capacity of the cells in culture is a marker of the progress of an undifferentiated state to a differentiated one. Adherent cells form a layer on a plastic substrate. A cell lineage refers to a differentiated cell type and the ancestors of the cell types from which the differentiated cell type was derived. For example, cardiocytes and myoblasts are two types of cells in the same lineage.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the invention provides cardiac derived, pluripotent, non-adherent, isolated, non-differentiated human cells. After proliferation and differentiation of such cells, progenitor cells are produced which comprise a cell type selected from the group consisting of an undifferentiated, cardiac derived, adherent, fibroblast, smooth muscle cells, a skeletal muscle cell, an cardiocito, a keratinocyte, an osteoblast and a chondrocyte. Some of such undifferentiated cells generate all types of cells selected from the group. The non-differentiated, non-adherent cardiac cells of the invention can be produced by propagating a population of cardiac tissue-derived cells in a liquid medium on a substrate; and discarding the cells of the population that adhere to the substrate and leaving a suspension of undifferentiated derived, cardiac, nonadherent cells. The invention further provides a non-differentiated, cardiac, pluripotent, non-adherent, non-isolated cell, which upon proliferation and differentiation produces progenitor cells comprising cardiocytes and either chondrocytes or keratinocytes. Some such undifferentiated cells upon proliferation and differentiation produce progenitor cells further comprising at least one cell type selected from the group consisting of an undifferentiated, derived, cardiac, adherent cell, a fibroblast, a smooth muscle cell and a Skeletal muscle cells. Such undifferentiated cells can be undifferentiated human or mouse cells, for example. Optionally, mouse undifferentiated cells can be obtained from a mouse deficient in p53. The invention further provides a non-differentiated, cardiac, human, adherent, isolated cell, which proliferates and differentiates to produce progenitor cells comprising a cell type selected from the group consisting of a fibroblast, a smooth muscle cell, cell of skeletal muscle, a cardiocyte, a chondrocyte, a keratinocyte and an osteoblast. The invention further provides an isolated, cardiac derived, non-differentiated cell, which proliferates and differentiates to produce progenitor cells comprising a cardiocyte and any of a chondrocyte or keratinocyte. In another aspect, the invention provides a method for preparing a non-differentiated, derived, cardiac, non-adherent, isolated cell. Such a method involves centrifuging a suspension of cells from the cardiac tissue of a subject on a density gradient; isolate a band of cells comprising monocytes; propagating the cells until the adherent cardiocytes have dried or been discarded leaving the cells in suspension; and culturing the cells in suspension until a population of nonadherent cardiac cells is detectable. The invention also provides methods for preparing derived, cardiac, non-differentiated, adherent cells. Such methods start with a non-differentiated, cardiac, non-adherent derived cell as described above. The undifferentiated cell is propagated until adherent progenitor cells appear. An adherent cell lacking markers of a cell selected from the group consisting of myoblasts, smooth muscle cells, skeletal muscle cells, cardiocytes, osteoblasts, keratinocytes and chondroblasts, which upon proliferation and differentiation produce progenitor cells comprising at least a cell type of the group that is then identified. This invention also provides methods for preparing a cardiocyte. Some such methods involve providing an undifferentiated, derived, cardiac, non-adherent cell, as described above; propagate the cell under conditions in which the cell proliferates and differentiates to produce progenitor cells that comprise adherent cells; and identify an adherent cell with markers of differentiation characteristic of a cardiocyte. Alternatively, such methods may begin with an undifferentiated, derived, cardiac, non-adherent cell, as described above. The cell spreads under conditions in which the cell proliferates and differentiates to produce adherent progenitor cells. An adherent cell is then identified with markers of differentiation characteristic of a cardiocyte. In another aspect the invention provides an isolated population of cells comprising smooth muscle cells, skeletal muscle cells, cardiocytes, fibroblasts, keratinocytes, osteoblasts and chondrocytes. In another aspect the invention provides methods for treating a patient suffering from necrotic cardiac tissue. Such methods involve administering to the patient an effective dose of undifferentiated or adherent cells as described above, whereby the non-adherent cells proliferate and differentiate to produce cardiocytes, which replace the necrotic tissue. In some such methods, non-differentiated non-adherent cells are administered directly to the patient's heart. In other methods, non-differentiated non-adherent cells are administered intravenously. In some methods, fibroblast growth factor is also administered to the patient to stimulate the proliferation and / or differentiation of the non-adherent cells. In some methods, the undifferentiated cell factor is administered to the patient to stimulate the differentiation of nonadherent cells to cardiocytes. In some methods, the patient has a congestive heart defect. In some methods, undifferentiated cells are obtained from the patient's blood, and are propagated in vi tro before being administered to the patient. In another aspect, the invention provides methods for screening potential agents for the activity to promote the proliferation and / or differentiation of cardiac derived, non-differentiated cells. Such methods involve propagating non-differentiated, cardiac, non-adherent or adherent cells in the presence of a potential agent, and verifying a change in the differentiation status of progenitor cells relative to non-differentiated, non-adherent or adherent cardiac derived cells. In "some methods, the change in the state of differentiation is the adhesion of the progenitor cells, in some cases, the change in the state of differentiation is verified by detecting the appearance of cardiocytes." In some methods, the appearance of undifferentiated cells is verified, derived, cardiac, adherent.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the differentiation pathways of undifferentiated cells derived from cardiac tissue to various types of differentiated cells.

Figure 2 shows a density gradient of cells obtained by digesting cardiac tissue. The myocyte band is the third from above.

DETAILED DESCRIPTION I. General Aspects The invention provides at least two classes of differentiated, cardiac, pluripotent cells. One class of cells is non-adherent and the other is adherent. Adherent cells are progenitors partially differentiated from non-adherent cells. Both classes of undifferentiated cells can be propagated and differentiated into a variety of differentiated cell types. The undifferentiated cells, cardiac, can be isolated from humans and other vertebrate animals. The cells are isolated from a fraction of a suspension of derived, cardiac cells, which have typically been discarded as remnants by previous workers in the field. Typically, a band of myocyte cells is isolated from a density gradient, and the cells are propagated in a culture medium on a substrate. Previous workers have retained the cells by adhering them to the surface and discarding the culture medium in what is erroneously believed to contain only residues. The inventors of the present have maintained the culture medium and discarded the adherent cells. After propagation of the culture medium a population of non-adherent cells in suspension becomes evident. The cells have several applications in therapy and drug discovery. In therapeutic applications, the cells are administered to patients suffering from cardiac defects, such as necrotic tissue resulting from myocardial infarction. The undifferentiated cells administered colonize the heart of a patient and give rise to myocardial progenitor cells that replace the necrotic tissue and / or supplement the preexisting cardiac tissue. In drug discovery applications, undifferentiated cells are used to select compounds for their activity in promoting or inhibiting the differentiation of undifferentiated cells into cardiocytes or other mature differentiated cell types. Compounds that promote the differentiation of undifferentiated cells or cardiocytes can be used for the therapy of patients with cardiac defects, optionally in conjunction with the undifferentiated cells of the invention. Other compounds find uses in other therapeutic applications in which the promotion or inhibition of differentiation of undifferentiated cells is desirable. Another use for the undifferentiated cells of the present invention is in the selection of compounds that expand the population of undifferentiated cells in culture. Some of these compounds also induce differentiation and others do not. Other therapeutic uses for the cells of the present invention is to provide a method for selecting compounds that promote the mobilization of derived undifferentiated, cardiac cells from the heart to the circulatory system. In vivo assays to assess cardiac neogenesis include neonatal and mature animals with the cells of the present invention. The cardiac function of the animals is measured as the cardiac rhythm, blood pressure, VL pressure reduction (tdp / dt) a cardiac value to determine the left ventricular function. Post-mortem methods to assess cardiac improvement include: increase in cardiac weight, core / cytoplasmic volume, staining of cardiac cytological sections to determine the levels of proliferation of cellular nuclear antigen (PCNA) against cytoplasmic actin (Quainic et al., Circula Res. 75: 1050-1063, 1994 and Reiss et al., Proc. Na ti. Acad. Sci. 93: 8630-8635, 1996). The undifferentiated mesenchymal cells derived, cardiac, of the present invention can be used in the treatment of disorders associated with cardiac diseases, i.e., myocardial infarction, coronary artery disease, congestive heart failure, hypertrophic cardiomyopathy, myocarditis, congenital heart defects and dilated cardiomyopathy. The cells of the present invention are useful for improving cardiac function, either by induction of myocytic cardiac neogenesis and / or hyperplasia, by induction of coronary collateral formation, or by induction of remodeling of the necrotic myocardial area. The cells of the present invention are also useful for promoting angiogenesis and healing wounds after angioplasty or endaterectomy, for developing coronary collateral circulation, for revascularization in the eye, for complications related to poor circulation such as diabetic foot ulcers. , for apoplexy, after coronary perfusion using pharmacological methods and other indications where angiogenesis is of benefit. An ischemic event is the interruption of blood flow to an organ, which results in necrosis or infarction of the non-perfused region. Ischemia, reperfusion is the interruption of blood flow to an organ, such as the heart or brain, and the subsequent (often abrupt) restoration of blood flow. Although restoration of blood flow is essential to preserve tissue functions, it is known that reperfusion itself can be harmful. Indeed, there is evidence that reperfusion of an ischemic area includes endothelium-dependent vasorelaxation that affects vasospasm, and compromised cardiac coronary vasodilation, which is not observed in an ischemic event without reperfusion (Cuevas et al., Growth Factors 15: 39-40, 1997). Both ischemia and reperfusion are important contributors to tissue necrosis, such as myocardial infarction or stroke. The cells of the present invention have therapeutic value to reduce tissue damage caused by ischemia or ischemia-reperfusion events, particularly in heart or brain. Other therapeutic uses for the present invention include the induction of neogenesis and / or skeletal muscle hyperplasia, cartilage regeneration, bone formation, tendon regeneration, neural neogenesis, neogenesis in the pancreas and kidney, and / or for the treatment of systemic and pulmonary hypertension. The different functions of specialized cells are achieved by stimulating the differentiation of mesenchymal, derived, cardiac (MSC) undifferentiated cells into the appropriate cell pathway, for example, in lineage cells of osteoblasts, chondrocytes, skeletal monocytes or fibroblasts. The pathway of differentiation is determined by exposure to a growth factor that promotes the formation of a particular differentiated cell. Various combinations of growth factor and differentiated cell types different from those known in the art are described below. The coronary collateral development induced by cardiac derived MSCs was measured in rabbits, in dogs or pigs using the models of chronic coronary occlusion (Landau et al., Arner, Heart J. 29: 924-931, 1995).; Sellke et al., Surgery 120 (2): 182-188, 1996 and Lazarous et al., 1996, ibid.). Cardiac derived MSCs that induced benefits for the treatment of stroke were tested in vivo in rats using bilateral carotid artery occlusion and measuring histological changes, as well as maze functioning (Gage et al., Neurobiol.Aging 9: 645-655, 1988). Derivative, cardiac MSCs that induced efficacy in hypertension were tested in vivo using spontaneously hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl. 1: S114-116, 1995) .

II. Cells of the Invention 1. Derived, Cardiac, Non-differentiated Cells, Pluripotent, Non-Adherent These cells are characterized by being highly refractory to light, have a small size (typically less than 10 microns and often less than 5 microns), spherical shape and low growth. The cells are further characterized by their ability to proliferate (ie, self-renew) in a medium containing a carbon source, a nitrogen source, insulin and transferrin. In addition to the factor of undifferentiated cells (commercially available from Amgen), acidic or basic fibroblast growth factor, zFGF-5, factor 1 inhibitor of leukemia (commercially available) or the increase in serum concentration that stimulates the spread. The cells are also characterized by their ability to differentiate into various cell types. Cell types include cells in several differentiation states more advanced than non-differentiated, derived, cardiac, nonadherent cells. These cell types include undifferentiated, derived, cardiac, adherent cells (see below), fibroblasts, myoblasts, smooth muscle cells, skeletal muscle cells, cardiocytes, chondrodoblasts, keratinocytes, and chondrocytes. Probably, adipoblast, adipocytes, osteoblasts and osteocytes are also present in the progenitor cells. Tendocytes may also be present. The relationship of undifferentiated, derived, cardiac, non-adherent cells to differentiated progenitor cell lines and types is shown in Figure 1. Undifferentiated, derived, cardiac cells can be stimulated to differentiate by propagation in media with FBS. The definition can be stimulated by treatment with growth factors, such as the undifferentiated cell growth factor, and by contact inhibition. Higher concentrations of horse serum, although they stimulate proliferation, have a tendency to inhibit differentiation. The type of growth factor used to induce differentiation can divert the differentiation towards a selected lineage. Retinoic acid, TGF-β, bone macrophogenic proteins (BPM), ascorbic acid, and β-glycerophosphate lead to the production of osteoblasts. Indomethacin, IBMX (3-isobutyl-l-methylxanthine), insulin, and triiodohirocin (T3) lead to the production of adipocytes. The aFGF, bFGF, vitamin D3, TNF-ß and retinoic acid lead to the production of myocytes, zFGF-5 leads to the expansion of progenitors of cardiocytes, although it may also be more effective later in the pathway of adherent cells. cardiocytes (2) Non-differentiated, derived, cardiac, pluripotent, adherent cells Undifferentiated, derived, cardiac, pluripotent, adherent cells result from the proliferation and partial differentiation of undifferentiated, nonadherent cells described in the previous section. The cells are characterized by the amorphous form and the lack of markers of differentiation tested to date. The markers of differentiation tested include the expression of alkaline phosphatase, which is a marker for pericytes, osteoblast precursors and chondrocyte precursors, and a-actin expression. The cells can be induced to proliferate and / or differentiate into the same cell types as non-differentiated non-adherent cells. The rate of proliferation and the lineage at which differentiation can be induced and can be controlled by the supplementation of the media with growth factors as described above.

III. Production of Non-Differentiated, Derived, Non-Adherent Cardiac Cells. The initial material is a heart or cardiac biopsy of a human or animal subject. The subject can be embryonic beyond the mesodermal, neonate, infant or adult stage. If immortalized cells are desired, the initial material can be obtained from the heart of a transgenic animal that is deficient in one or both copies of a tumor suppressor gene. Examples of tumor suppressor genes include p53, p21, and the retinoblastoma gene. In transgenic mice with a homozygous mutation in p53, they are commercially available from Taconica Farms or Jackson Labs. Methods of processing whole tissue to prepare a cell suspension are described, for example, by Methods In Molecular Biology: Animal Cell Cul ture , 5, (Pollard et al., Eds. Humana Press, NJ, 1990), incorporated herein by reference). Typically, cardiac tissue is digested with collagenase, trypsin, other proteases and / or DNase to release the cells. Polinger, Exp. Cell Res. 63: 78-82 (1970); Owens et al., J. Na ti. Cancer Insti t. , 53: 261-269, 1974; Milo et al., In Vi tro 16: 20-30, 1980; Lasfargues, "Human Breast Tumors", in Kruse et al. (eds) Tissue Cul ture Methods and Applications (Academic Press, NY, 1973), Paul, Cell and Tissue Cul ture, Churchill Livingston, Edinburgh, 1975). The intact cells are then separated from the debris by low speed centrifugation. The remains are collected in the supernatant. The cell pellet is resuspended and the cells are fractionated. The fractionation can be carried out on a density gradient such as Ficoll or sucrose gradient. The cells form four bands as shown in Figure 2, the myocyte band is the third from above. The myocyte band is removed and the cells are cultured in a complete medium. Such a medium contains the least one source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals, and whey. The medium can also be supplemented with growth factors. The cells are allowed to grow at about 37 ° C in a 95% atmosphere of 02 and 5% C02 on a plastic substrate. Initially, most cells are adherent monocytes. However, 1 the majority of adherent monocytes (eg, at least 75 or 90%) die within 5-30 days and small non-adherent cells appear in suspension at increasing concentrations. The time period is the shorter end of the previous range for cells deficient in p53 and the longer end of the range for normal cells. The small nonadherent cells are undifferentiated, cardiac, pluripotent derived cells.

IV. Markers of Differentiation of Different Types of Cells The progeny of differentiated cells of undifferentiated cells is recognized in part due to the presence of differentiation markers. There are three types of muscle cells, cardiocytes (ie cardiac muscle cells), striated muscle and smooth muscle cells. The three cell types are derived from a common precursor, called myoblast. The expression of myosin cardiac isozyme and the specific cardiac pattern of creatine kinase isozyme expression when identified together on the same cell or on a clonal population of cells are markers for cardiac muscle cells. Cardiocytes can also be recognized by their bifurcated appearance and their ability to form junctions in holes by light microscopy. Such cells can be recognized by the formation of an electrical potential through confluent cells and by detecting the transfer of signals through the cells. The muscle a-actin mRNA, and the smooth muscle cell actin are markers of myocyte differentiation. The expression of the myosin isozyme and a muscle-specific pattern of isozyme creatine kinase expression when identified in a cell or clonal population are markers of skeletal muscle cells. Osteoblastic cells secrete the material from the bone matrix. The expression of ALP, osteocalcin, the expression of AMPc induced by PTH, and the capacity of bone mineralization, identified together in a cell or polyclonal population of cells are markers of differentiation for osteoblasts. Chondrocytes secrete type II cartilage. The aggrecan and the Type IIB collagen identified in a cell or clonal population of cells, the stain with Alcian Blue, which detects the production of chondroitin sulfate, are markers for the chondrocytes. Keratinocytes secrete keratin and can be recognized using commercially available stains. Adipocytes produce lipids and can be recognized by Oily Red staining 0.

V. Factors of Growth 1 Basic FGF (also known as FGF-2) is mitogenic in vivo for endothelial cells, vascular smooth muscle cells, fibroblasts, and generally for mesodermal or neuroectodermal cells, including cardiac and skeletal myocytes (Gospodarowics et al., J. Cell. Biol. 70: 395-405, 1976; Gospodarowica 'et al., J. Cell. Biol. 89: 568-578, 1981 and Kardami, J. Mol. Cell, Biochem, 92, 124-134, .1990). Non-proliferative activities associated with acidic and / or basic FGF include: increased endothelial release of tissue plasminogen activator, stimulation of extracellular matrix synthesis, chemotaxis for endothelial cells, induced expression of fetal contractile genes in cardiomyocytes (Parker et al. al., J. Clin. Invest. 85, 507-514, 1990), and improved pituitary hormone responses (Baird et al., J. Cellular Physiol., 5, 101-106, 1987).

ZFGF-5 is a type of fibroblast growth factor that is expressed at high levels in fetal cardiac tissue and human adult. This growth factor is also expressed at lower levels in lung tissues, skeletal muscle, fetal smooth muscle such as in the small intestine, colon and trachea. The high level of expression of zFGF-5 in fetal and adult heart and its effects on cells derived from cardiac tissue suggests that zFGF-5 has a particular potency in the stimulation of proliferation and / or differentiation of cells, no differences derived, cardiac , to cardiocytes. The isolation of zFGF-5 and its properties are described in greater detail in commonly owned PCT US97 / 18635, filed October 16, 1997 (incorporated herein by reference). The nucleotide sequence 'which codes for zFGF-5 is described in SEQ ID NO. 1, and its deduced amino acid sequence is described in SEQ ID NO. 2. The sequence of zFGF-5 shows some sequence similarity to FGF-8.

SAW . Immortalization of Cells Cells that can be continuously cultured and do not die after a limited number of cell generations are called immortalized cells. A cell that survives only 20 to 80 reproductions of its population is considered finite (Freshney, Cul ture of Animal Cells (Wiley-Liss, NY, 1994), incorporated herein by reference), and a cell that survives more than 80, of preferably at least 100, cell generations is considered immortalized. Some but not all immortalized cells are tumorigenic. As noted above, the cells can be immortalized using a transgenic animal deficient in a tumor suppressor gene as a cardiac tissue donor. Cells from other sources can be immortalized by other means. These means include transformation and expression by a gene whose product plays a role in cellular senescence, or overexpression or mutation of one or more oncogenes that impede the action of senescence genes. Expression of the genes that results in positive signals for cell proliferation include the SV40 large T antigen (Linder et al., Exp. Cell Res. 191, 1-7 (1990), _ large polyoma antigen T ( Ogris et al., Oncogene 8, 1277-1283, 1993), ElA adenovirus (Braithwaite et al., J. Virol. 45, 192-199, 1983), myc oncogene (Khoobyarian et al., Virus Res. 30, 113 -128, 1993), and the E87 gene of papilloma virus type 16 (McDougall, Curr. Top, Microbiol, Immunol., 186: 101-119, 1994).

II. Therapeutic Regimens Heart disease is the leading cause of death in the United States, contributing up to 30% of all deaths. More than 5 million people are diagnosed with heart disease in the United States. Coronary disease may be due to congestive heart failure, hypertrophic cardiomyopathy, cardioneopathy, viral infection or myocardial infarction (MI). Myocardial infarction accounts for 750.00 hospital admissions per year in the United States. In IM patients, ischemia stimulates the growth of fibroblasts and promotes the development of larger than normal amounts of fibrous tissue that replaces necrotic muscle tissue. Risk factors for MI include diabetes mellitus, hypertension, trunk obesity, smoking, high levels of low density lipoprotein plasma or genetic predisposition. Although apparently some proliferation of cardiocytes occurs with the normal aging of humans and animals (Olivetti et al., J. Am. Coil, Cardiol., 24 (1), 140-9 (1994) and Anversa et al., Circ. Res. 67, 871-885, 1990), this is inadequate to repair damages due to coronary diseases. In this way, the necrosis that occurs in MI and other coronary diseases is essentially irreversible. Such conditions are treated by the demodulation of undifferentiated, derived, cardiac cells, as described above. Adherent or non-adherent cells can be used. The cells can be administered intravenously, intracoronary, or intraventricularly. A catheter can be used for the last two routes of administration, which are more usual for adherent cells. The cells are administered in a therapeutically effective dose. Such a dose is sufficient to generate significant numbers of new cardiac cells in the heart, and / or replace at least partially the necrotic cardiac tissue, and / or produce a clinically significant change in cardiac function. A clinically significant improvement in cardiac function can be determined by measuring the left ventricular ejection fraction, before, and after the administration of the cells, and determining at least an increase of 5%, preferably 10% or more, in the fraction and total ejection. Standard procedures are available to determine the ejection fraction, measured by the beats by blood ejected. Lights may vary from approximately 100-107, 1000-106 or 104-105 cells. The cells can be administered as pharmaceutical compositions, which also include, depending on the desired formulation, pharmaceutically acceptable, typically sterile, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. . The diluent is selected so that it does not affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered physiological saline, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. The administration of undifferentiated cells can be preceded, accompanied or followed by the administration of growth factors that stimulate the proliferation and / or differentiation of undifferentiated cells into cardiocytes. Growth factors can be administered intravenously or intraventricularly. Growth factors are administered in a dose sufficient to cooperate with the undifferentiated cells administered in the generation of significant numbers of new cardiac cells in the heart, and / or replace at least partially the necrotic cardiac tissue, and / or produce a change clinically significant in cardiac function. Suitable growth factors include zFGF-5 and growth factor of undifferentiated cells.

In some methods, the undifferentiated, derived, cardiac cells are administered in combination with anti-inflammatory agents that counteract, reverse or partially alleviate the inflammation associated with coronary disease. Suitable anti-inflammatory agents that include antibodies to Mac-1, and E and P selectins (See Springer, Na ture 346, 425-433 (1990) Osborn, Cell 62, 3 (1990); Hynes, Cell 69, 11 (1992)). The undifferentiated cells administered, cardiac, can also be administered with diuretics, ACE inhibitors and β-adrenergic blockers. In some methods, the patient receiving the undifferentiated cells and the donor from which the cells were obtained are of similar HLA to reduce allotypic rejections. In other methods, the cells are administered under the cover of an immunosuppressive regimen to reduce the risk of rejection. The immunosuppressive agents that can be used include cyclosporine, corticosteroids and OKT3. In other methods, immune responses are avoided by obtaining undifferentiated cells from the patient to be treated. Undifferentiated cells can be obtained by biopsy of cardiac tissue, and expanded in vi tro before readministration. Alternatively, given the provision of the present, derived, cardiac, isolated undifferentiated cells, the differentiation markers for those cells can be identified, and the cells can be isolated from the blood of the patient to be treated.

VIII. Use of Derived, Cardiac, Non-differentiated Cells in the Selection of Drugs The undifferentiated, derived, cardiac cells described above can be used to test compounds to determine their activity in the promotion or inhibition of cell proliferation and / or differentiation. . In general, a compound is contacted with a population of undifferentiated, derived, cardiac cells, optionally, in the presence of other agents that are known to promote or inhibit the metabolic pathway or phenotype of interest, and the phenotypic and metabolic changes are verified in comparison with a control in which the compound being treated is absent. The treated s.er compounds include known or suspected growth factors and analogs thereof, libraries of previously known or natural compounds that have proliferation promotion or differentiation activity and combined libraries of compounds. Combined libraries of the compounds can be constructed by the synthetic library encoded (ESL) method described in Affymax, WO 95/12608, Affimax, WO 93/06121, Columbia University, WO94 / 08051, Pharmacopeia, WO 95/35503, and Manuscripts, WO 95/30642 (each of which is incorporated by reference for all purposes). Peptide libraries can also be generated by phage display methods. See, for example, Devlin, WO 91/18980. Compounds that cause undifferentiated, derived (cardiac) cells to proliferate and / or differentiate into cardiocytes are useful as therapeutic agents under the same conditions that undifferentiated, derived, cardiac cells are useful.Such compounds can be administered alone to stimulate the proliferation and differentiation of undifferentiated, derived, cardiac, endogenous cells, or they can be administered in conjunction with undifferentiated, derived, cardiac, exogenous cells Such compounds are selected for their proliferating activity by contacting them with undifferentiated, derived cells , cardiac, in growth media, and verifying an increase in cell count, or the incorporation of 3H-thymidine Compounds are selected to promote differentiation to cardiocytes by verifying the cells with the morphological appearance and differentiation markers characteristic of cardiocytes as noted previously, similarly, the compounds can be verified to determine their activity in the promotion of the differentiation of undifferentiated cells, derived, cardiac, to other types of differentiated cells, such as smooth muscle cells, skeletal muscle cells, osteoblasts and chondrocytes. The activity is detected by detecting the morphological appearance and markers of differentiation characteristic of one of these cell types. Compounds with activity in promoting the differentiation of one of the above cell types are useful in the treatment of patients with degenerative diseases of bones, muscles or cartilage. Compounds that inhibit the differentiation of undifferentiated cells from certain cell types such as adipocytes may also be useful in some circumstances. For example, compounds that inhibit the differentiation of undifferentiated cells into adipocytes can be used to treat obesity. Such compounds are identified by contacting a compound under test with undifferentiated, derived, cardiac cells, under conditions that in other circumstances would lead to the differentiation of undifferentiated cells to a certain type of cell, and verifying a decrease in the frequency or degree of conversion to the cell type in relation to a control in which the compound was omitted. The compounds identified as therapeutic agents by such screening with the cell lines of the invention are formulated for therapeutic use as pharmaceutical compositions. The compositions may also include, depending on the desired formulation, pharmaceutically acceptable, non-toxic carriers or diluents, as described above.

EXAMPLES Materials a. Serum Free Media for Myocytes Mixture of Dulbecco's Modified Eagle's Medium / ham Nutrient F12 (1: 1, Gibco Laboratories) Insulin (JRH 5C2059), 1 μg / ml Transferrin (JRH 6K2222) 5 μg / ml Selenium (Aldrich Chem Co 20010/7), 1 nM Thyroxine (Sigma T-0397), 1 nM Ascorbic Acid (Sigma A-4034), 25 μg / ml LiCl (Sigma L-0505), 1 nM b. Culture Medium (MC) Mixture of Dulbecco's Modified Eagle's Medium / ham Nutrient F12 (1: 1, Gibco Laboratories), containing 15% HIA FBS c. Media for Myocyte Isolation Mixture of Dulbecco's Modified Eagle's Medium / ham Nutrient F12 (1: 1; Gibco Laboratories) DF 20, containing 20% of HIA FBS DF 10, containing 10% of HIA FBS DF 5, Containing 5% HIA FBS HIA FBS (HyClone lot # AEL4614) d. Enzymes for Cardiac Tissue Digestion Collagenase (Worthington LS004176), 2% solution DNase (Worthington LS002007), 0.5% solution Enzymes were dissolved in OBS (Gibco Laboratories) with / 1% Glucose and. Purification with Percoll Percoll (Pharmacia # 17-0891-01) Additional cushions (g / L for lx; og / lOOml for lOx), pH 7 6.8 NaCl 1.0 Dextrose 1.5 NaH2P04 0.4 KCl 0.1 MgSO4 4.76 HEPES 0.02 Phenol Red - Sigma All the reagents were passed through a 0.22 μM filter before being used. A standard solution was produced using 9 parts (45 ml) of Percoll, plus 1 part (5 ml) of lOx Ads without Phenol Red. The steps of the gradient were made as follows. f. Human Fibronectin Plates Standard solution is 1 mg / ml (1000 μg / ml) Concentration of the coating 1 μg / cm2 Standard solution diluted to 10 μg / ml (100X: 200 μl in ml of SFM) Cultivate tissue culture dishes of 3 ml / 60 mm Incubate at room temperature for 1 hour. Vacuum the remaining material. Rinse the plates carefully with dH20 to prevent detachment of the outer surface. The plates are ready to use, store at 4 ° C 2. Isolation of Non-Differentiated, Derivative, Cardiac, Non-Adherent Cells from Mice Deficient in p53 In this example, a code was used to designate the months. Thus, A, B and C represent successive months starting with A. In A / 19, the hearts of 10 female P53 - / - mice (approximately 2 months old) were digested as follows: 1) Hearts were cut with scissors in a minimum volume of PBS (-). 2) PBS (-) + 1% glucose was added for a final volume of 18 ml. 3) 1 ml of DNase and 1 ml of collagenase were added. 4) The suspension was stirred in a 50 ml Falcon tube on a rotary shaker at 37 ° C for 30 minutes. 5) This first supernatant was discarded since it contains most of the remains of RBC and fibrots. 6) Steps 2-5 were repeated until the hearts were completely digested. 7) After a stirring period of 30 minutes of each (except for the first digestion, which was discarded) the supernatant was removed and added to 20 ml of DF20. The supernatant with DF20 was centrifuged at 1650 rpm, 4 ° C for 10 minutes. The resulting pellet was stored and resuspended in 10 ml of DF10 and kept on ice. 8) After completing the digestion of the hearts, the pellets were combined (5 pellets per 50 ml of Falcon tube) and centrifuged at 1650 rpm, 4 ° C for 10 minutes. 9) The final resulting pellet was resuspended in 1082 gm / ml Percoll (12 ml). 10) A Percoll gradient was prepared in a 50 ml Falcon tube as follows: 12 ml of 1050 gm / ml of Percoll was loaded, then 12 ml of 1.060 gm / ml of bottom Percoll were loaded, followed by the cell suspension at 1082 gm / ml Percoll, loaded on the bottom. The 50 ml Falcon tube was centrifuged in a Beckman CS-6R centrifuge for 30 minutes at 3000 rpm, TA. 11) The resulting myocyte fraction in the band between the 1082 gm / ml layers of lower Percoll and 1.06 gm / ml of medium Percoll was removed and an equal volume of DF10 was added. 12) The myocyte fraction was centrifuged at 1650 rpm for 10 minutes at RT. 13) The resulting pellet contained 3.75 x 106 cells, which were also cultured in 5 wells in 5 ml of DFS. 14) At A / 20 media were changed to culture media (MC) in 6-well tissue culture plates, which were replaced again with fresh CM on A / 22 and A / 25. Versatile adherent cardiocytes were evident from A / 20 to A / 25, although in decreasing numbers. Additionally, very small suspension cells were present in the wells. 15) At A / 26 those cells in suspension were divided 1: 3 into MC, MC plus 1% horse serum (SC), MC plus 10% SC (HyClone, Logan, Utah). 16) AB / 01 cells in suspension in MC plus 1% horse serum (SC) and MC plus 10% SC were divided 1:10 into T25 bottles (0.5 ml of cell suspension plus 4.5 ml of fresh MC with 1 % SC or 10% SC). A B / 15 cells in suspension in MC plus 1% SC were divided 1: 1. The flasks were not manipulated again until C / 19. The flasks were incubated at 37 ° C in 02 at 95% and C02 at 5%. At C / 19, cells maintained on MC plus 10% SC were available by light refraction and extrusion of tripin blue. Only the cells in MC were not available. Cells in MC and 1% SC became adherent cells and are described in more detail below. The cell density of the suspension was determined, using a hemacytometer, and was 6 x 106 / ml. Also, at C / 19, the cells that grew in MC plus 10% SC were centrifuged at 1500 rpm for 10 minutes and resuspended at 0.5 x 106 / ml in serum free media (MLS); MLS plus 1% SC, MLS plus 10% SC, MC, MC plus 1% SC, or MC plus 10% SC. At C / 26 the cells in suspension in MC plus 10% of SC were at 8.5 x 105 / ml, while cells in suspension in MLS plus 10% SC were 8.75 x 10 5 / ml. Half of these cells in suspension (both MLS and MC cells) were resuspended in MC plus 10% SC or MLS plus 10% SC, and were cultured on methylcellulose in the presence of several cytokines and growth factors. The cells in suspension did not form colonies on methylcellulose, even after 12 days suggesting that the cells could be primitive undifferentiated cells. At this time, culturing the cells in suspension in 10% SC did not appear to have significant additional impact on the cells, so that the cells were passed on MC only and MLS only. The zFGF-5 (150 pg / mL) increased the proliferation of the cells 2.5-5.0 times, and the undifferentiated cell factor (SCF 10 ng / ml) increased the proliferation approximately 1.0-2.0 times determined by absorption of tritiated thymi in short impulses. One month of treatment of the cells in suspension with 10 ng / ml SCF in MC resulted in the appearance of an adherent layer of mixed lineage cells designated as cells derived from SCF / MC. In contrast, treatment of cells in suspension with SCF in MLS did not result in adherent cell lineages, implying that some other growth factor present in the 15% FBS found in MC is essential, in addition to SCF. The addition of zFGF-5 (150 lg / ml) to bottles with SCF in MC prevents the induction of adherent cell lines suggesting that zFGF-5 blocked the differentiating activity of SCF. By 'simple microscopy, the morphology of cells derived from SCF / MC suggested lineages which include cardiocytes, chondrocytes, fibroblasts, smooth muscle monocytes, and skeletal muscle myoblasts. Cells similar to cardiocytes had the appearance of cigarettes or floating wood. Cells similar to chondrocytes had the appearance of nests or cobblestone streets. The fibroblasts were large and long. The cells with a smooth muscle monocyte phenotype were amorphous or star-shaped, the skeletal muscle cells had the shape of a feather. The myoblasts were intermediate between the fibroblasts and skeletal muscle cells. Other lineages are probably present, and can be characterized using immunohistochemistry, immunofluorescence and FACS analysis. 3. Non-differentiated cells, cardiac, adherent to C / 19, cells maintained in MC plus 1% of SC were differentiated into adherent cells of mixed morphology. Those cells were trypsimized, pelleted, and then divided 1: 6 into MLS, MLS plus 1% SC, MLS plus 10% SC, MC, MC plus 1% SC or MC plus 10% SC. At C / 26, the adherent cells expanded in PM only since the different media or SC concentration had no noticeable effect on the morphology of the adherent cell. By simple microscopy the adherent cells appear to be a mixed population that includes pericytes, fibroblasts, cardiocytes, smooth muscle myocytes, skeletal muscle myocytes. The adherent cells were subcloned on C / 08. From the subcloning, 11 clones were obtained and expanded on 95% 02/5% C02 culture media at 37 ° C. The cells were characterized by the following criteria. (1) Morphology by simple microscopy. (2) Staining with alkaline phosphatase (identifies pericytes, osteoblastic precursors and chondrocytic precursors and mature chondrocytes and osteoblasts) (3) Absorption of acetylated LDL (marker of endothelial cells) (4) Ac MF-20 (anti-pectoral myosin mouse adult chicken, which cross-reacts with mouse myosin and detects myocin from adult cardiac and skeletal myocytes) (5) M0636 (mouse human muscle actin Dako # 5/56 recognizes alpha myocyte actin from cardiac muscle, skeletal and smooth muscle and gamma actin from smooth muscle myocytes). (6) mitogenic response to zFGF-5, acid FGF and basic FGF. This "test proves the proliferation capacity." Cells were grown in 96-well plates, in MC and grown to confluence and then switched to serum-free media for 24 hours after which growth factor was added and Thymidine was titrated (7) Production of zFGF-5 mRNA identified by Northern immunoblotting which is expressed in high concentration by cells of cardiac lineage. (8) Differentiation induced by contact inhibition or step in the MLS to identify proliferation and / or differentiation capacity. (9) Differentiation induced by ascorbic acid / β-glycerophosphate (AA / β-GP) to identify osteoblasts. (a) Mixed population spread in MS + 1% SC before isolation of clones Cells in this population showed different morphological characteristics to those of pericytes, fibroblasts, cardiocytes, smooth muscle myocytes, skeletal muscle myocytes, and other cells. Approximately 20-25% of the cells were positive for alkaline phosphatase, indicating cells of osteoblastic or chondrocytic lineage. All cells were negative to the absorption of acetylated LALD identifying the absence of endothelial cells. All cells were negative at the binding of MF-20 indicating an absence of differential skeletal cells terminally. All cells were positive for the binding of M0636 indicating the presence of precursors of cardiocytes, myocytes, skeletal muscle cells and smooth muscle cells. The cells showed a mitogenic response to z FgF-5 (cFGF), acid FGF (aFGF) and basic FGF. BFGF was more potent in the induction of a mitogenic response. The response was increased in a manner dependent on the concentration of one pg / ml to 1 μg / ml. After treatment with serum-free media, the cells showed moderate growth and differentiation in cells with an appearance similar to that of fried eggs. This appearance suggests early stage myocytes. In a treatment with ascorbic acid / ß-Gp, the cells differentiated into chondrocyte-like cells on day 6 in their morphology. No mineralization occurred even after day 25 indicating the absence of osteoblastic lineage cells.

Clone 1D9: Clone 1D9 was isolated from only one of the mixed population described above in 3a. Some cells propagated from 1D9 showed morphology of cardiocytes, and other cells showed a different morphology. A few cells stained slightly positive for alkaline phosphatase. The cells did not absorb acetylated LDL. MF-20 did not bind to the cells. M0636 bound to the cells indicating that some cells were smooth muscle, skeletal muscle and / or cardiac lineage. The zFGF-5, FGF acid and basic FGF all induced a mitogenic response and the bFGF was the most potent. The serum free media induced slow growth but no obvious morphological changes. The contact-dependent differentiation produced chondrocytes, cardiocytes, and other types of cells recognized by their morphology. Treatment with ascorbic acid / β-GP resulted in the appearance of chondrocyte-like cells on day 6. No mineralization occurred even after day 25. These results indicate that clone 1D9 gives rise to differentiated cells of different lineages.

Clone 2E7: The expanded cells of 2E7 have the morphology of myoblasts (precursors of skeletal monocytes / fibroblasts / adipocytes). The cells were negative for alkaline phosphatase staining, acetylated LDL absorption and MF-20 binding. The cells were positive at the binding of M0636 indicating the presence of precursors of smooth muscle, skeletal cells and / or cardiocytes. Cells proliferated in response to zFGF-5, acidic FGF and basic FGF, with basic FGF being the most potent. After treatment with serum-free media, the cells proliferated and changed from morphology to an appearance of large polygonal cells, which may be early-stage monocytes. The cells were negative for the binding of MF-20. After treatment with ascorbic acid // ß-GP, the cells differentiated into "chain-like" formations on day 11. No mineralization was observed even after day 25.

Clone 1611: Clone 1G11 was also isolated as a single cell from the mixed population of the adherent p53 deficient cells described above. The expanded cells of 1G11 had the morphology of skeletal myocytes, smooth muscle myocytes, cardiocytes, and other cell types. Approximately 20-25% of the cells stained positive for alkaline phosphatase. The cells showed the mitogenic response zFGF-5, acidic FGF and basic FGF, with bFGF being the most potent and bFGF and aFGF equally active. The adhesion of serum-free media caused a low growth rate and the appearance of star-shaped cells, which are precursors of myocytes. The cells did not bind to MF-20. Treatment with ascorbic acid / β-GP did not result in mineralization even after day 25.

Clone 2B6: This clone showed the morphology of skeletal myocytes, smooth muscle myocytes, cardiocytes, and other cell types. The cells were negative for alkaline phosphatase staining and acetylated LDL absorption of MF-20. The cells showed the mitogenic response to bFGF and aFGF, but not to zFGF-5. MLS-induced differentiation, and slower growth rate, produced thicker, rod-shaped cells. Treatment with ascorbic acid / ß-GP induced resulted in cells that were grouped in "reverse chain" formations on day 11. No mineralization occurred even after day 25.

Clone 2A7: This clone showed the morphology of the myoblasts (precursors of skeletal myocytes / fibroblasts / adipocytes). The cells were negative for alkaline phosphatase staining, acetylated LDL staining, MF-20 binding. The cells were slightly positive for the binding of M0636. The cells showed the mitogenic response to zFGF-5, acidic FGF and basic FGF. The aFGF and the bFGF were more potent, the three FGFs were equally active. The MLS did not cause changes in the growth rate or morphology. Treatment with ascorbic acid / β-GP resulted in mineralization even after day 25.

Clone 2G7: This clone had the morphology of multinucleated cardiocytes, and other cell types. Some cells were positive for alkaline phosphatase staining. The cells were negative for the absorption of acetylated LDL and binding of MF-20. The cells were positive for the binding of M0636. The cells showed a mitogenic response to zFGF-5, acid FGF and basic FGF being the most potent, with aFGF being the most potent, and bFGF being equally active. The MLS induced a moderate growth rate, and without different morphological changes. The cells were negative for the binding of MF-20. Treatment with ascorbic acid / β-GP resulted in mineralization even after day 25.

Clone 2F11: This clone. Had the morphology of myoblasts (precursors of skeletal myocytes / fibroblasts / adipocytes). 5% of the cells were stained with alkaline phosphatase. The cells were negative for the absorption of Acetylated LDL, MF-20 binding, and M0636 binding. The MLS induced a moderate growth rate resulting in cells in starry form. Differentiation induced by ascorbic acid / ß-GP resulted in cells that gathered in a "chain-like" formation on day 11. No mineralization occurred even after day 25.

Clone 2A6: Those cells had a stellar appearance characteristic of myoblasts. The cells were negative to staining with alkaline phosphatase, negative for acetylated LDL, negative for the binding of MF-20 negative for the binding of M0636. The cells showed a mitogenic response to zFGF-5, acidic FGF and basic FGF, with bFGF being the most potent, and all three FGF being equally active. Treatment with MLS produced a moderate growth and appearance of some larger star-shaped cells. The treatment with ascorbic acid / β-GP induced differentiation with cells that gathered in formations "similar to a chain" on the 11th (many formations were evident). No mineralization occurred even after day 25.

Clone 2G12: These cells showed the morphology of cardiocytes, skeletal myocytes, smooth muscle myocytes, and others. The cells were negative for alkaline phosphatase staining, acetylated LDL absorption, MP-20 binding, M0636 binding. The cells showed a mitogenic response to zFGF-5, acidic FGF and basic FGF. BFGF and cFGF were equally potent and active from 10 g / ml to 1 μm / ml. Stimulation with FGF regenerated the progenitor population of the original suspension cells and yielded zFGF-5 RNA. The zFGF-5 150 pg / ml (1 day) produced a clear stimulation in adult cardiocytes judging by their morphology. The treatment for 5 days resulted in the formation of a hay stack structure, chains or passages. Treatment with MLS resulted in strong growth, loss of multinucleation, and appearance of large polygonal cells. The contact-dependent differentiation produced cardiocytes, chondrocytes and other cell types. The treatment with ascorbic acid / ß-GP resulted in the differentiation of larger cells on day 6. No mineralization occurred even after day 25.

Clone 2D6: The cells had a stellar or haystack appearance when they were subconfluents. suggesting myoblasts (precursors of skeletal myocytes / fibroblasts / adipocytes). The cells were negative for the absorption of acetylated LDL, negative for alkaline phosphatase staining, negative for the binding of MF-20 and positive for the binding of M0636. Treatment with MLS produced rapid growth, and loss of the appearance of a hay stack or stellar. Treatment with ascorbic acid / β-GP resulted in the appearance of chondrocyte-like cells on day 6. No mineralization occurred even after day 25.

Clone 2H6: The cells showed the morphology of cardiocytes and other cells. The cells were negative for alkaline phosphatase staining, acetylated LDL absorption, binding of MF-20 and positive for M0636 binding. The cells showed the mitogenic response to zFGF-5, acidic FGF and basic FGF. BFGF was the most potent, and all three FGFs were equally active. The treatment with MLS produced a reduced growth rate, reduced adherence to plastic, and the appearance of large polygonal cells. Treatment with ascorbic acid / ß-GM resulted in cells that gathered in "chain-like" formations on day 11. No mineralization occurred even after day 25.

. Isolation of undifferentiated cardiac derived cells from neonatal mice 1) Neonatal mice of 1-4 days of age were sacrificed by cervical dislocation and placed in a 70% ETOH bath. When 10-15 neonates had been collected, they were placed on their back and the sternotomy was performed in the midline. Pressure was applied to the chest cavity with forceps, the heart was removed and the ventricle was easily removed. The ventricles were placed in ice-cold PBS (50 ml Falcon tube, with PBS on ice), with approximately 100 ventricles in each tube. 2) When all the hearts had been collected, it was rinsed in PBS several times. The heart tissue was then cut with scissors in a minimum volume of PBS, and rinsed again in PBS several times. (It was rinsed until all the red blood cells and debris were removed). From step 3) the PBS is supplemented with 1% glucose. 3) a) To begin the dissociation, the hearts were taken to 18 ml of PBS. b) 1 ml of each standard Dnasa / Collagenase solution was added. 4) Was stirred on a rotary shaker at 37 ° C for minutes (DIGESTION 1). 5) The supernatant of 1. Was discarded. The digestion which mainly contained fibroblasts, red blood cells and debris. 6) a) To begin the dissociation, the hearts were taken to 18 ml of PBS. b) 1 ml of each DNase / Collagenase standard solution was removed. 7) Shake on a rotary shaker at 37 ° C for 20 minutes. 8) Transfer the supernatant (20 ml) from the tube to a 50 ml Falcon tube containing 20 ml of DF 20. Mix carefully (turn the tube upside down). 9) Centrifuge the tubes at 1650 rpm, 4 ° C for 10 minutes. 10) Keep the sedimented cells on the bottom, and discard the sobrensadante (~ 40 ml). 11) Add 10 ml of DF10 to the sediment, mix well upwards and downwards (using the sterile pipette), keeping the cell suspensions on ice. Repeat steps 6) -11) until all tissue is digested. 12) When the dissociation is complete, combine all steps 11). 13) Sediment the cells (centrifuge - the tubes at 1650 rpms at 4 ° C, for 10 minutes, discard the supernatant). 14) Resuspend the monocytes in 1.082 g / ml of Percoll (12 ml of Percoll / 100 nonaonates). 15) Prepare the Percoll gradient using a 10 ml pipette to load from the bottom 12 ml of each of 1050 g / ml, 1060 g / ml, then 1082 g / ml (containing cells) of Percoll into so many tubes of Falcon 50 ml as necessary. (1 Percoll gradient tube / 100 neonates). 16) Centrifuge for 30 minutes at 3000 rpms, room temperature. A Beckman CS-6R centrifuge is suitable. 18) After the centrifugation step in a Percoll gradient, 4 bands appear (see Figure 2). 19) Aspirate the upper band, collect (first layer of fibroblasts, if you wish to keep, transfer in a 50 ml Falcon tube, add the same amount of DF10 as the volume of cell suspension collected.) 20 Mix well but carefully, and centrifuge the cells at 1650 rpm, 10 minutes, at room temperature. 21) Discard the supernatant. Add 5 ml DF / "100 neonates". 22) Incubate the myocytes on human fibronectin (HFn) "for 1 hour at 37 ° C. The remaining fibroblasts were taken to the coated plate for 1 hour 23) Collect the non-adherent cells, wash the disc several times with DF5 for make sure all nonadherent cells are collected 24) Centrifuge the cells 10 minutes at 1650 rpm, remove the supernatant, and resuspend the cells in DF5. 6. Bone Marrow MSC Expansion Trials were performed to measure the frequency of fibroblast colonies that form units of non-adherent, low-density monkey cells isolated from the bone marrow. This test is indicative of the frequency of non-differentiated mesenchymal cells. Half of a 96-well microtiter plate was inoculated with cells at a density of 10,000 cells / well and the other half of the plate was inoculated with cells at a density of 1,000 cells / well. The culture medium is aMEM (GIBCO-BRI, Gaithersburg, MD) 2% bovine serum albumin, 10 μg / ml insulin, 200 μg / ml transferrin, antibiotic and 50 μm mercaptoethanol. The cells were incubated at 37 ° C in 5% C02 for 14 days and then stained with toluidine blue to improve the visibility of the cells and examined microscopically. The positive wells had at least 50 cells that exhibit a "stromal" morphology, ie large, scattered cells. The positive control is a medium containing 20% fetal bovine serum. The results showed that zFGF5 at a concentration of 100 ng / ml increased the frequency of CFU-F to levels equivalent to the positive control of 20% FBS. 7. Identification of cardiac derived MCSs for zFGF5 Identification of a putative mesenchymal non-differentiated cells as a target (ie, having a receptor) for zFGF was done using a FITC-labeled protein and cardiac tissue of neonatal mouse or fetal sheep ( third trimester) . The zFGF5, purified as described above, was dialyzed in 0.1 M sodium bicarbonate pH 9.0. Fluorescein isothiocyanate was dissolved (FITC; Molecular Probes, Eugene, OR) at 1 mg / ml in the same buffer without exposure to strong light. The mixture was prepared with a content of 1 mg of FITC / 1 mg of zFGF5, and reacted for 1-2 hours in the dark at room temperature. The reaction was stopped by adding 1 M glycerin to a final concentration of 0.1 M, and then reacting for 1 hour at room temperature. The mixture was then dialyzed against 0.1 M sodium bicarbonate to make a 1: 500-1: 100 dilution for 3 hours. The dialysis solution was changed and the process was repeated for 3-18 hours to remove the labeled FITC. The cardiac mouse ventricles of neonatal mice or fetal sheep were isolated, cut, and repeatedly washed in phosphate buffered solution (PBS) until all the red blood cells and debris were removed. The cut ventricles were placed in a solution containing 18 ml of PBS and 1% glucose and 1 ml of 2% DNase / collagenase solution was added. The mixture was incubated on a shaker for 30 minutes at 37 ° C. The supernatant was discarded and the process repeated once more.

After incubation, the supernatant (~ 20 ml) was transferred to a tube containing 20 ml of DF 20 (Dulbecco's Modified Eagle's Medium Mix / Ham Nutrient F12, 1: 1 (GIBCO-BRL, Gaithersburg, MD) and 20% fetal bovine serum). After mixing, the tubes were centrifuged at 1650 rpms in a Beckman CS-6R centrifuge (Beckman, Fullerton, CA) at 4 ° C for 10 minutes. The supernatant was discarded and the pellet resuspended in DF 10 (10% FBS). The cells were kept cool and centrifuged again and resuspended in 40 ml of DF 10. The cell mixture was passed over a 40 μl filter (Becton Dickinson, Detroit, MI) and counted using a hemacytometer. The cells were incubated in FITC-labeled zFGF5 at 4 ° C for 30 minutes at a concentration of 2 x 106 cells / 1 μg of zFGF5. After incubation, the cells were centrifuged at 1600 rpm in a Beckman SC-6R (Beckman) centrifuge for 5 minutes. The supernatant was discarded and the pellet was washed once with 10 ml of DF 10 and resuspended in 4 ml of DF10. 10 μl of anti-DITC MACS microbeads (Miltenyi Biotech, Auburn, CA) were mixed with 107 cells in 4 ml of DF10 and incubated at 4 ° C for 30 minutes. LSS positive separation columns of MACS positive selection (Miltenyi Biotech) were washed with 3 ml of MAC buffer (PBS, 5% BSA, 2 mM EDTA) and the cell / bead mixture was washed in 10 ml of MAC buffer and Then it was resuspended in 6 ml of MAC buffer. The cell / bead mixture was divided between the two columns and the first negative fraction was discarded. 1.5 ml of 0.6 M NaCl was added to each column and eluted but not collected. The columns were then washed with 1.5 ml of MAC buffer. Cells bound with FITC-labeled cFGF5 were harvested by adding 3 ml of MAC buffer, removing the column from the magnet and washing the positive cells using the piston. The fraction of positive cells was cultured in a T75 flask and 50 ml of culture medium (DF with 15% FDS and antibiotics) was added. The cells were incubated for 37 ° C for one week and counted. The yield of positive cells was approximately 0.1% of the original total cells counted. Cells bound to FITC-labeled zFGF5 were examined by transmission electron microscopy (TEM). The cells were between 3-5 microns in diameter. The nuclei of the cells occupied the majority of cell volume, and few cytoplasmic organelles were evident. The phenotype was identified by TEM identifying the cells isolated by zFGF5 as primitive mesenchymal undifferentiated cells. 8. Study of In Vivo Cardiomyopathic Rats Rats infused subcutaneously with epinephrine for 2 weeks developed a cardiomyopathy very similar to human idiopathic dilated cardiomyopathy (Deisher et al., Am. J. Cardiovasc. Pa thol. 5 (l): 79-88, 1994 and Deisher et al., Pharmacol. Exp. Ther 266 (1): 262-269, 1993). The effect of cardiac derived MSCs on the onset and progression of catecholamine-induced heart disease was evaluated by administering cardiac, derived MSCs isolated from healthy rats with similar genetic backgrounds by intrapericardial, intracoronary, intraarterial, intraventricular or intravenous injection to rats receiving subcutaneous infusions of epinephrine or saline. In one protocol, rats (300 gm) were implanted with epinephrine in an osmotic pump and injected with derived, cardiac, or control MSCs and mortality was verified for 2 weeks, at the end of which each of the rats was sacrificed , hearts were weighed wet, and fixed in buffered formalin, 10% neutral, for histological purposes. Before the sacrifice, cardiac function was measured. The measurement included body weight, heart weight, cardiac fibrosis and cardiohistomorphometry in rats infused with epinephrine. Cardiac fibrosis was determined in cardiohistomorphometry in rats with infused epinephrine.

Three cuts were recorded for each heart, and the average record was taken. Positive results indicate that infusion of cardiac derived MSCs may be beneficial for the establishment of heart failure of various etiologies, which may include myocardial infarction (MI), idiopathic dilated cardiomyopathy (IDCM), hypertrophic cardiomyopathy, viral myocarditis , congenital abnormalities, and obstructive diseases. It is evident from the foregoing that the invention includes a number of uses, some of which may be expressed concisely as follows. The invention provides the use of non-differentiated, derived, cardiac, non-adherent, or undifferentiated, derived, cardiac, adherent cells in the treatment of diseases and / or discovery of drugs for use therein. The invention further provides the use of undifferentiated, derived, cardiac, non-adherent or adherent cells in the manufacture of a medicament for treating diseases. Although the invention has been described in some detail for purposes of clarity and understanding, it should be clear to one skilled in the art upon reading this description that various changes in form and detail can be made without departing from the true scope of the invention. invention. All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document was so denoted individually.

Claims (39)

CHAPTER CLAIMEDICATORÍO Having described the invention, it is considered as a novelty and, therefore, the content is claimed in the following CLAIMS:

1. An undifferentiated human, derived, cardiac, pluripotent, non-adherent, isolated cell characterized in that after proliferation and differentiation it produces progenitor cells comprising a cell type selected from the group consisting of an undifferentiated, derived, cardiac, adherent, cells a fibroblast, a smooth muscle cell, a skeletal muscle cell, a cardiocyte, a keratinocyte, an osteoblast and a -chondrocyte.

2. The non-differentiated, derived, cardiac, non-adherent, isolated cell according to claim 1, characterized in that the differentiation produces progenitor cells that include at least two cells selected from the group.

3. The non-differentiated, derived, cardiac, non-adherent, isolated cell according to claim 1, characterized in that after proliferation and differentiation it produces a progenitor cell comprising all cell types selected from the group.

4. The non-differentiated, derived, cardiac, non-adherent, isolated cell according to claim 1, characterized in that it is immortalized.

5. The non-differentiated, derived, cardiac, non-adherent, isolated cell according to claim 1, characterized in that it is produced by propagating a population of cells derived from cardiac tissue in a liquid medium on a substrate, discarding the cells of the population that it adheres to the substrate and leaves a suspension of undifferentiated, derived, cardiac, non-adherent cells.

6. An undifferentiated, derived, cardiac, pluripotent, nonadherent, isolated cell, characterized because after proliferation and differentiation it produces progenitor cells comprising cardiocytes and chondrocytes or keratinocytes.

7. The undifferentiated cell, cardiac derivative, pluripotent, non-adherent, isolated, according to claim 6, characterized in that after proliferation and differentiation it produces progenitor cells that further comprise at least one type of cell selected from the group consisting of a undifferentiated, derived, cardiac, adherent cell, a fibroblast, a smooth muscle cells, and a skeletal muscle cell.

8. The non-differentiated, derived, cardiac, non-adherent, isolated cell according to claim 7, characterized in that the differentiation produces progenitor cells that include at least two cells selected from the group.

9. The non-differentiated, derived, cardiac, non-adherent, isolated cell according to claim 7, characterized in that after proliferation and differentiation it produces a progenitor cell comprising all cell types selected from the group.

10. The non-differentiated, derived, cardiac, non-adherent, isolated cell, according to claim 7, characterized in that it is a human undifferentiated cell.

11. The non-differentiated, derived, cardiac, non-adherent, isolated cell, according to claim 7, characterized in that it is an undifferentiated mouse cell.

12. The non-differentiated, derived, cardiac, non-adherent, isolated cell, according to claim 11, characterized in that it is from a mouse deficient in p53.

13. An undifferentiated, derived, cardiac, human, adherent, isolated cell characterized in that it proliferates and differentiates to produce progenitor cells comprising a cell type selected from the group consisting of a fibroblast, a smooth muscle cell, skeletal muscle, a cardiocyte, a chondrocyte, a keratinocyte and an osteoblast.

14. The non-differentiated, derived, cardiac, adherent, isolated cell according to claim 13, characterized in that it proliferates and differentiates to produce progenitor cells comprising at least two types of 1 cells selected from the group.

15. The undifferentiated cell, derived, cardiac, adherent, isolated, according to claim 13, characterized in that it proliferates and differentiates to produce progenitor cells comprising each type of cell selected from the group.

16. "The undifferentiated cell, derived, cardiac, adherent, isolated, characterized because it proliferates and differentiates to produce progenitor cells comprising a cardiocyte and a chondrocyte or a keratinocyte

17. The undifferentiated cell, derived, cardiac, adherent, isolated according to claim 16, characterized in that it proliferates and differentiates to produce progenitor cells further comprising one or more cell types selected from the group consisting of a fibroblast, a smooth muscle cell, a skeletal muscle cell, and a osteoblast.

18. A method for preparing a non-differentiated, derived, cardiac, nonadherent, isolated cell, characterized in that it comprises: centrifuging a suspension of cardiac tissue cells from a subject on a density gradient; isolate a band of cells comprising the monocytes; propagating the cells until the attached cardiocytes have dried or been discarded leaving cells in suspension; Cultivate the cells in suspension until a population of nonadherent cardiac cells is detected.

19. The method according to claim 18, characterized in that it further comprises: obtaining cardiac tissue from a subject; digest cardiac tissue with collagenase to produce cell suspension.

20. A method for preparing an undifferentiated, derived, cardiac, adherent cell, characterized in that it comprises: providing an undifferentiated, derived, cardiac, non-adherent cell according to claim 1; propagate the undifferentiated cell until adherent progenitor cells appear; identifying an adherent cell lacking markers of a cell selected from the group consisting of the group myoblasts, smooth muscle cells, skeletal muscle cells, cardiocytes, osteoblates, keratinocytes and chondroblasts, which in the proliferation and differentiation of a progenitor cell include at least one cell type of the group.

21. A method for preparing a cardiocyte, characterized in that it comprises providing an undifferentiated, derived, cardiac, nonadherent cell according to claim 1; propagating the cell under conditions in which the cell proliferates and differentiates to produce progenitor cells comprising adherent cells; identify an adherent cell with markers of differentiation characteristic of a cardiocyte.

22. A method for preparing a cardiocyte, characterized in that it comprises providing an undifferentiated, derived, cardiac, non-adherent cell according to claim 13 or 16; propagate the cell under conditions in which the cell proliferates and differentiates to produce adherent progenitor cells; identify an adherent cell with markers of differentiation characteristic of a cardiocyte.

23. A method for propagating an undifferentiated, derived, cardiac, non-adherent cell, characterized in that it comprises: culturing the cell in the presence of fibroblast growth factor, where the cell propagates.

24. An isolated population of cells comprising smooth muscle cells, skeletal muscle cells, cardiocytes, fibroblasts, keratinocytes, osteoblasts and chondrocytes.

25. A method for treating a patient suffering from necrotic cardiac tissue, characterized in that it comprises administering to the patient an effective dose of non-differentiated "non-adherent cells" according to claim 1, whereby the undifferentiated cells proliferate and differentiate to 26. Producing cardiocytes, which replace the necrotic tissue

26. The method according to claim 25, characterized in that the non-differentiated non-adherent cells are administered directly to the patient's heart

27. The method according to claim 25, characterized in that undifferentiated, nonadherent cells are administered intravenously.

28. The method according to claim 25, characterized in that it comprises administering FGF to the patient to stimulate the proliferation and / or differentiation of the non-adherent cells.

29. The method of compliance with the claim 25, characterized in that it also comprises administering undifferentiated cell factor to the patient to stimulate the differentiation of non-adherent cells to cardiocytes.

30. The method of compliance with the claim 18, characterized in that the patient has a congestive heart defect.

31. The method according to claim 25, characterized in that the undifferentiated cells are obtained from the patient's blood and propagated in vitro before being administered to the patient.

32. A method for treating a patient suffering from necrotic cardiac tissue, characterized in that it comprises administering to the patient an effective dose of adherent undifferentiated cells according to claim 13, whereby undifferentiated cells proliferate and differentiate to produce cardiocytes , which replace the necrotic tissue.

33. The method according to claim 32, characterized in that the non-differentiated non-adherent cells are administered directly to the heart of the patient.

34. The method according to claim 32, characterized in that it further comprises administering FGF to the patient to stimulate the proliferation of the adherent cells.

35. The method of compliance with the claim 32, characterized in that it comprises administering the factor of v the undifferentiated cell to the patient to stimulate the differentiation of adherent cells to cardiocytes.

36. A method for screening potential agents for their activity in promoting the proliferation and / or differentiation of undifferentiated, derived, cardiac cells by propagating the non-differentiated, cardiac, non-adherent derived cells according to claim 1 or claim 6 in the presence of a potential agent, verify a change in the stage of progenitor cell differentiation in relation to the non-differentiated, cardiac, non-adherent cells.

37. The method according to claim 36, characterized in that the change in the differentiation state is the adhesion of the progenitor cells.

38. The method according to claim 36, characterized in that the verification comprises verifying the appearance of the cardiocytes.

39. The method according to claim 36, characterized in that the verification comprises verifying the appearance of an undifferentiated, derived, cardiac, adherent cell.