FIELD This application relates to the field of stem cells, specifically to methods of producing and differentiating muscle stem cells.

BACKGROUND Many people die each year of congestive heart failure. Heart failure may occur from a variety of causes, including cardiomyopathy, myocardial ischemia, congenital heart disease, and valvular heart disease, resulting in cardiac cell death and myocardial dysfunction. As cardiomyocytes are not replaced in adult myocardial tissue, physiologic demands on the existing, healthy, cardiomyocytes leads to their hypertrophy. Heart transplant has been the only recourse for patients in end-stage heart disease, however the United Network of Organ Sharing (UNOS) has reported that although more than 40,000 patients were waiting for heart transplants as of February 2000, only 2,345 people received a donated heart in 1998. Furthermore, heart transplants are complicated by the incompatibility between the transplanted donor tissue and the recipient's immune system, which requires life-long immunosuppression. Yet another drawback of heart transplants is their high cost.

An alternative approach to heart transplantation is to generate cardiomyocytes from stem cells in vitro that can be used in the treatment of heart failure, and other cardiac diseases characterized by myocardial cell death or dysfunction. This approach is based on the ability of stem cells to both self-renew and differentiate into one or more mature cell types, including cardiomyocytes. Stem cells may be obtained from an individual suffering from heart disease and then used to generate cardiomyocytes in vitro in order to repair the damaged myocardium. This approach avoids problems inherent with heart transplantation, such as lack of a suitable heart for transplant or immune rejection of a transplanted heart.

Embryonic stem (ES) cells, derived from the inner cell mass of the blastocyst, are the most primitive stem cell, as disclosed in WO 01/11011 A2. These cells have unlimited self-renewal capability, and because they can differentiate into several cell lineages and repopulate tissues upon transplantation, they have multipotent differentiative potential.

Although protocols are available for differentiating embryonic stem cells into beating cardiomyocytes, there is an ongoing ethical debate about the use of human embryos for medical research.

Lineage specific stem cells, identified in most organ tissues, have less self-renewal capability than ES cells and their differentiative ability is limited to tissues of that lineage.

Of the lineage specific stem cells, the hematopoietic stem cell (HSC), derived from bone marrow, blood, cord blood, fetal liver and yolk sack, is the best characterized. These cells are defined by the expression of cell surface markers, such as c-kit (c-kit+), and can terminally differentiate into all the hematopoietic cell types. HSC have been shown to contribute to the formation of functional cardiac tissue in vivo (Jackson et al., J. Clin.

Invest., 107: 1395-1402,2001). Mesenchymal stem cells (MSC) are pluripotent progenitor cells derived from tissues of mesodermal origin (U. S. Patent No. 5,486, 359). These cells are most often obtained from bone marrow, although they can be obtained from other sources, such as blood or dermis. These cells have been shown to differentiate to form muscle, bone, cartilage, fat, marrow stroma and tendon, but have not been shown to differentiate into cardiomyocytes. In addition, progenitor cells, termed satellite cells (Cornelison and Wold, Dev. Biol. , 191: 270-283,1997), have been identified in skeletal muscle. These cells are characterized by the expression of the cell surface marker c-met (c- met+) in both its quiescent and activated states. When activated these cells re-enter the cell cycle, express myogenic regulatory factors, and differentiate into myoblasts.

However, despite the existence of a variety of stem cells, there is currently no pure population of stem cells that can be induced under defined conditions to differentiate into spontaneously beating cardiomyocytes in vitro. Thus, there remains a need in the art for isolated populations of stem cells that can be induced to differentiate into cardiomyocytes.

SUMMARY The methods and cells described herein are based on the ability of certain stem cells to differentiate in vitro to form fully functional cells of more than one given type.

Disclosed herein is a novel isolated population of stem cells, called spoc cells, that can be induced, either in vivo or in vitro, to differentiate into cardiomyocytes. Methods are disclosed herein to differentiate the spoc cells, and to utilize these spoc cells for screening agents that affect cardiomyocytes. Methods are also provided herein to utilize spoc cells in therapeutic applications.

The foregoing and other objects, features, and advantages will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a series of digital images of transmission electron micrographs demonstrating the progression of differentiation of CS (cardiac precursor from spoc cells) cells over time when cultured in differentiation medium. Figure lA is a digital image of CS cell at day 3 with disordered myosin filaments. Figure 1B is a digital image showing that at day 7 myosin filaments of characteristic 1.6 cm-length (top box) radiate outward and the cells contain dense bodies (lower box). Figure 1C and Figure IF are digital images of a cell at day 14, showing a single, central nucleus shows a stretching out of the dense bodies into an organizing sarcomere. Figure 1D shows that day 3 CS cells are round cells with copious mitochondria (box and detail). Figure 1E shows elongated day 7 cells contain dense bodies (arrowhead). Figure 1G shows that by day 56, a well-defined sarcomere (Figure 1G) is present, with identifiable A-and I-bands and Z-lines.

Figure 2 demonstrates the existence of calcium transients, in cardiomyocytes differentiated from CS cells. Figure 2A shows a graphical representation of the calcium transient in a beating CS cell-derived cardiomyocyte. Peak intensity and baseline are shown in Figure 2B and Figure 2C, respectively.

Figure 3 shows whole cell voltage recordings from spoc cell-derived cardiomyocytes demonstrate spontaneous action potential firing in a non-beating, teardrop shaped cell (Figure 3A). Figure 3B shows a representative action potential from recording in Fig. 3A on an expanded time scale; action potential threshold is-60 mV. Figure 3C shows the block of action potential firing in another cell upon bath perfusion with 0.5 mM CdCl2 (horizontal bar). Acceleration of action potential firing upon perfusion with 25 nM isoproterenol (horizontal bar) is demonstrated in Figure 3D, indicating the presence of adrenergic receptors on these cells. Skeletal myotube APs differ in that their frequency is unaffected by Cd2+ (Figure 3E) or isoproterenol (Figure 3F).

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS In order to facilitate review of the various embodiments disclosed herein, the following list of abbreviations and explanation of terms is provided:

I. Abbreviations and Terms A. Abbreviations CS: Cardiac precursors from spoc cells DNA: Deoxyribonucleic acid EGF: Epidermal growth factor EGFP: Enhanced green fluorescent protein ES: Embryonic stem FACS: Fluorescence activated cell sorting FBS: Fetal bovine serum FGF: Fibroblast growth factor HSC: Hematopoietic stem cell MI : Myocardial infarct MRNA: Messenger ribonucleic acid PBS: Phosphate buffered saline RNase: Ribonuclease RT-PCR: Reverse transcriptase-polymerase chain reaction SP: side population SPOC: Skeletal-based precursors of cardiomyocytes B. Terms Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes Vl, published by Oxford University Press, 1997 (ISBN 0-19-857778-8); Kendrew et al. <BR> <BR> <P>(eds. ), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. , 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology : a Comprehensive Desk Reference, published by VCH Publishers, Inc. , 1995 (ISBN 1- 56081-569-8) Adult : A fully developed and physically mature subject, having attained full size and strength.

Animal : Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.

Cardiac: Pertaining to the heart.

Cardiac dysfunction: Any impairment in the heart's pumping function. This includes, for example, impairments in contractility, impairments in ability to relax (sometimes referred to as diastolic dysfunction), abnormal or improper functioning of the heart's valves, diseases of the heart muscle (sometimes referred to as cardiomyopathy), diseases such as angina and myocardial ischemia and infarction characterized by inadequate blood supply to the heart muscle, infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (such as may occur in some kinds of cardiomyopathy or systemic hypertension), and abnormal communications between chambers of the heart (for example, atrial septal defect). For further discussion, see Braunwald, Heart Disease: a Textbook of Cardiovascular Medicine, 5th edition 1997, WB Saunders Company, Philadelphia PA (hereinafter Braunwald).

Cardiac muscle : The heart is made of specialized muscle tissue with some similarities to both smooth and skeletal muscle. It is involuntary and most cardiac myocytes are mononucleate as is smooth muscle. Cardiac muscle is striated like skeletal muscle, which means that it has microscopically visible myofilaments arranged in parallel including the sarcomere. These filaments slide along each other during the process of contraction in the same manner as occurs in skeletal muscle. Cardiac muscle also differs from skeletal muscle in that the fibers in cardiac muscle branch and usually have a single centrally located nucleus. Another difference in cardiac muscle is the presence of intercalated discs which serve as specialized connections between cardiac muscle cells. These tight connections allow for almost completely free movement of ions so that action potentials can freely pass from one cell to another. This arrangement makes cardiac muscle tissue a functional syncytium. When one cell is excited the resultant action potential is spread to all of them.

This is an important feature in that it allows the atrial or ventricular muscle to contract as a unit to forcefully pump blood. Cardiac muscle can generate its own excitatory impulses from the sino-atrial node, which acts like a biological pacemaker. In this manner, the contracting signal for cardiac muscles originates in the heart itself. However, the autonomic nervous system (for example through the vagus nerve) can exert control over how fast the signals form and propagate through the heart, which regulates the rate of myocardial contraction. A"cardiomyocyte"is a cell of the cardiac muscle.

Cardiac precursors from spoc cells (CS cells) : When spoc cells are isolated from skeletal muscle and are cultured under growth conditions designed to promote their growth, spoc cells undergo several rounds of division. During this proliferative phase they become clusters of floating round cells with an increased diameter as compared to spoc cells. These round cells, with an increased diameter, are referred to as CS cells. In one embodiment, a

diameter of a CS cell is from about 10 to about 14 llm. When placed in growth promoting conditions in vitro (such as the examples described below) CS cells differentiate into spontaneously beating cardiomyocytes.

Cardiomyopathy: Any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and/or stiffened. As a result, the heart muscle's ability to pump blood is usually weakened. The disease or disorder can be, for example, inflammatory, metabolic, toxic, infiltrative, fibrotic, hematological, genetic, or unknown in origin. There are two general types of cardiomyopathies : ischemic (resulting from a lack of oxygen) and nonischemic. Ischemic cardiomyopathy is a chronic disorder caused by ischemia secondary to coronary artery disease-a disease in which there is atherosclerotic narrowing or occlusion of the coronary arteries on the surface of the heart.

Coronary artery disease often leads to episodes of cardiac ischemia, in which the heart muscle is not supplied with enough oxygen-rich blood. Eventually, the heart muscle is weakened because of areas that no longer function due to myocyte death from ischemia.

Larger volumes of blood are needed to fill the heart to augment output from the failing heart. This eventually leads to dilatation and worsening cardiac function.

Nonischemic cardiomyopathy is generally classified into three groups based primarily on clinical and pathological characteristics: (1) dilated cardiomyopathy, a syndrome characterized by cardiac enlargement and impaired systolic function of one or both ventricles; (2) hypertrophic cardiomyopathy, herein defined as (a) global or regional increase in thickness of either ventricular wall or the interventricular septum, or (b) an increased susceptibility to global or regional increase in thickness of either ventricular wall or the interventricular septum, such as can occur in genetic diseases, hypertension, or heart valve dysfunction; or (3) restrictive and infiltrative cardiomyopathies, a group of diseases in which the predominant clinical feature is usually impaired ability of the heart to relax (diastolic dysfunction), and is often characterized by infiltration of the heart muscle with foreign substances such as amyloid fibers, iron, or glycolipids.

See Wynne and Braunwald, The Cardiomyopathies and Myocarditities, Chapter 41, supra.

CD34: A cell surface antigen formerly known as hematopoietic progenitor cell antigen 1, and MY10, is a known marker of human hematopoietic stem cells. The human CD34 gene, which maps to chromosome lq32, spans 26 kb and has 8 exons. CD34 is a 67 kDa transmembrane glycoprotein. CD34 is expressed selectively on human hematopoietic

progenitor cells and such a cell is CD34 positive (CD34+). A cell that does not express CD34 is CD34 negative (CD34-). The biological function of CD34 is still unknown.

Cell surface marker: A protein, glycoprotein, or other molecule expressed on the surface of a cell, which serves to help identify the cell. A cell surface marker can generally be detected by conventional methods. Specific, non-limiting examples of methods for detection of a cell surface marker are immunohistochemistry, fluorescence activated cell sorting (FACS), or an enzymatic analysis. c-kit: A proto-oncogene that encodes a transmembrane receptor with intrinsic tyrosine-specific protein kinase activity in its intracellular domain. It is the cellular homologue of the viral kit oncogene of HZ4-FSV (Hardy-Zuckerman 4 feline sarcoma virus). The kit receptor is also known as CD117. The ligand for the kit receptor is Stem Cell Factor.

Human c-kit maps to chromosome 4ql 1-ql2 in the same region also encoding one of the platelet derived growth factor (PDGF) receptors (PDGFRA). The two genes have been located on a DNA fragment of approximately 700 kb. The human kit gene has a length of more than 70 kb and contains 21 exons. The longest transcript is 5230 bp and is alternatively spliced. A c-kit negative (c-kit) cell is a cell that does not express c-kit. A cell that expresses c-kit is c-kit positive (c-kit+). c-met: A proto-oncogene that encodes a transmembrane receptor tyrosine kinase that transduces mitogenic, migratory, or morphogenic signals in a variety of non-muscle tissues during development and wound healing in response to its ligand hepatocyte growth factor (HGF) /scatter factor. HGF is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. In muscle development HGF/c-met signaling is essential for proper emigration of muscle precursor cells of the axial lineage during embryogensis. The c-met gene product is synthesized as a 190kDa precursor and is proteolytically processed into an amino terminal 50 KDa alpha chain and a carboxy terminal 140KDa beta chain. c-met is expressed in quiescent satellite cells but not in muscle-derived fibroblasts or other mononucleated cells from healthy muscle explants (Cornelison and Wold, Developmental Biology, 191: 270,1997). Met plays a role in normal hematopoiesis, and is expressed in various lymphoid and leukemic cell lines. It also contributed to the malignant progression of gliomas. A c-met negative (c-met) cell is a cell that does not express c-met. A cell that expresses c-met is c-met positive (c-met+).

Congenital heart disease: A heart-related problem that is present since birth and often as the heart is forming even before birth. Congenital heart disease may affect the

heart, the heart's valves, the veins leading to, or the arteries leading away, from the heart, or the connections between these parts of the body.

Differentiation: The process whereby relatively unspecialized cells (e. g. , stem cells) acquire specialized structural and/or functional features characteristic of mature cells.

Similarly, "differentiate"refers to this process. Typically, during differentiation, cellular structure alters and tissue-specific proteins appear. The term"differentiated muscle cell" refers to cells expressing proteins characteristic of the specific muscle cell type. A differentiated muscle cell includes a skeletal muscle cell, a smooth muscle cell, and a cardiac muscle cell.

Differentiation Medium: A synthetic set of culture conditions with the nutrients necessary to support the growth or survival of cultured cells, and which allows the differentiation of stem cells into differentiated cells.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic <BR> <BR> acid (RNA) ). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Epidermal growth factor (EGF): In particular examples, EGF is a globular protein of 6.4 kDa consisting of 53 amino acids. It contains three intramolecular disulfide bonds essential for biological activity. EGF proteins are evolutionarily closely conserved.

Human EGF and murine EGF have 37 amino acids in common. Approximately 70 percent homology is found between human EGF and EGF isolated from other species. Mammalian EGF includes, but is not limited to, murine, avian, canine, bovine, porcine, equine, and human EGF. The amino acid sequences and methods for making these EGF polypeptides are well known in the art.

The gene encoding the EGF precursor has a length of approximately 110 kb, and contains 24 exons. Fifteen of these exons encode protein domains that are homologous to domains found in other proteins. The human EGF gene maps to chromosome 4q25-q27.

EGF is a strong mitogen for many cells of ectodermal, mesodermal, and endodermal origin. EGF controls and stimulates the proliferation of epidermal and epithelial cells, including fibroblasts, kidney epithelial cells, human glial cells, ovary granulosa cells, and

thyroid cells in vitro. EGF also stimulates the proliferation of embryonic cells. However, the proliferation of some cell lines has been shown to be inhibited by EGF.

EGF is also known to act as a differentiation factor for some cell types. It strongly influences the synthesis and turn-over of proteins of the extra-cellular matrix including fibronectin, collagen, laminin, and glycosaminoglycans, and has been shown to be a strong chemoattractant for fibroblasts and epithelial cells.

EGF can be assayed in a cell-based assay wherein the proliferation of a cell population is assessed. EGF can also be assayed by an immunoassay, such as an ELISA assay.

Fragments of EGF, smaller than the full-length sequence can also be employed in methods disclosed herein. Suitable biologically active variants can also be utilized. One specific, non-limiting example of an EGF variant of use is an EGF sequence having one or more amino acid substitutions, insertions, or deletions, wherein a biological function of EGF is retained. Another specific, non-limiting example of an EGF variant is EGF as wherein glycosylation or phosphorylation is altered, or a foreign moiety is added, so long as a biological function of EGF is retained. Methods for making EGF fragments, analogues, and derivatives are available in the art. Examples of EGF variants are known in the art, for example U. S. Patent No. 5,218, 093 and WO 92/16626A1. Examples of EGF from many different species are disclosed in WO 92/16626A1, as are examples of variants, and strategies for producing them.

As used herein,"EGF'refers to naturally occurring EGF, and variants and fragments that perform the same function of EGF in the culture media disclosed herein.

Embryonic stem (ES) cells : Totipotent cells isolated from the inner cell mass of the developing blastocyst and can generate all of the cells present in the body (for example, bone, muscle, brain cells, etc. )."ES cells"can be derived from any organism, for example from mammals such as humans.

Fibroblast growth factor (FGF): Any suitable fibroblast growth factor, derived from any animal, and functional variants and fragments thereof. A variety of FGFs are known and include, but are not limited to, FGF-1 (acidic fibroblast growth factor), FGF-2 (basic fibroblast growth factor, bFGF), FGF-3 (int-2), FGF-4 (hst/K-FGF), FGF-5, FGF-6, FGF-7, FGF-8, and FGF-9. FGF refers to a fibroblast growth factor protein such as FGF-1, FGF-2, FGF-4, FGF-6, FGF-8, or FGF-9, or a biologically active fragment or mutant thereof. The FGF can be from any animal species. In one embodiment the FGF is mammalian FGF including but not limited to, rodent, avian, canine, bovine, porcine, equine,

and human. The amino acid sequences and method for making many of the FGFs are well known in the art.

Fragments of FGF that are smaller than those described can also be employed.

Suitable biologically active variants can be FGF analogues or derivatives. An analogue of FGF is either FGF or an FGF fragment that includes a native FGF sequence and structure having one or more amino acid substitutions, insertions, or deletions. Analogs having one or more peptoid sequences (peptide mimic sequences) are also included (see e. g.

International Publication No. WO 91/04282). By"derivative"is intended any suitable modification of FGF, FGF fragments, or their respective analogues, such as glycosylation, phosphorylation, or other addition of foreign moieties, so long as the FGF activity is retained. Methods for making FGF fragments, analogues, and derivatives are available in the art.

Growth factor: A substance that promotes cell growth, survival, and/or differentiation. In general, growth factors stimulate cell proliferation or maturation when they bind to their receptor. In one embodiment, growth factors are a complex family of polypeptide hormones or biological factors that control growth, division, and maturation of muscle cells. In another embodiment a growth factor can be used to promote the proliferation of muscle stem cells and maintain the stem cells in an undifferentiated state. A growth factor can be a naturally occurring factor or a factor synthesized using molecular biology techniques. Examples of growth factors include platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, insulin, somatomedin, stem cell factor, vascular endothelial growth factor, granulocyte colony stimulating factor, and transforming growth factor-beta, amongst others. A muscle cell growth factor is a growth factor that effects the development (maturation), differentiation, division, or proliferation of muscle cells.

Growth medium: A synthetic set of culture conditions with the nutrients necessary to support the growth or survival of microorganisms or culture cells.

Heart: The muscular organ of an animal that circulates blood. The walls of the heart are comprised of working muscle, or myocardium, and connective tissue.

Myocardium is comprised of myocardial cells, which are also referred to herein as cardiac cells, cardiac myocytes, cardiomyocytes and/or cardiac fibers. Cardiomyocytes may be cells of the atrium or cells of the ventricle.

Heart failure : The inability of the heart to supply sufficient oxygenated blood to meet the metabolic needs of the tissues and cells in a subject. This can be accompanied by circulatory congestion, such as congestion in the pulmonary or systemic veins. As used

herein, the term heart failure encompasses heart failure from any cause, and is intended herein to encompass terms such as"congestive heart failure,""forward heart failure," "backward heart failure, ""high output heart failure, ""low output heart failure, "and the like. See Chapters 13-17 in Braunwald for a detailed discussion. Conditions that could lead to heart failure include, but are not limited to, coronary artery disease, cardiomyopathy, or congenital heart disease.

Heterologous: A heterologous sequence is a sequence that is not normally (i. e. in the wild-type sequence) found adjacent to a second sequence. In one embodiment, the sequence is from a different genetic source, such as a virus or organism, than the second sequence.

Isolated: An"isolated"biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i. e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been"isolated"include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Lineage specific marker: A marker that is expressed by a specific population of cells. In one embodiment, the cells are cells of a muscle, other than satellite cells, such as spoc cells.

Mammal : This term includes both human and non-human mammals. Similarly, the term"subject"includes both human and veterinary subjects.

Muscle cell : Includes skeletal, cardiac or smooth muscle tissue cells. This term is synonymous with myocyte, and encompasses those cells which differentiate to form more specialized muscle cells (e. g. myoblasts). "Cardiomyocyte"refers to a cardiac muscle cell.

Myocardial injury: Damage to the muscle or the"myocardium"in the wall of the heart as a result of disease or trauma. Myocardial injury can be attributed to many things such as, but not limited to, cardiomyopathy, myocardial infarction, or congenital heart disease.

Nucleotide:"Nucleotide"includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

Pax: A member of a family of evolutionary conserved transcription factors, termed "paired box transcription factors"There are nine members of this family, each of which contain a conserved DNA binding domain of 128 amino acids, known as the paired domain.

The Pax gene family of nuclear transcription factors function during embryogenesis to regulate the temporal and position-dependent differentiation of cells. In addition, the family is involved in a variety of signal transduction pathways in the adult organism. For example, Pax3 transcription factor is thought to specify dorsal cell fate in the neural tube.

Pax7 is expressed within the muscle satellite cell lineage, and skeletal muscle from Pax7~'~ mice is completely ablated of satellite cells. Mutations in the Pax family of proteins have been linked to disease and cancer in humans. For example, several Pax genes have been identified as targets of specific tumor associated chromosomal translocations.

A cell that is Pax (3/7)- does not express Pax3 or Pax7, as detected by a Pax (3/7) antibody. This antibody is reactive with both Pax-3 and Pax-7 but does not cross-react with other members of the Pax transcription factor family. A cell that is Pax (3/7) +, as detected by the Pax (3/7) antibody, expresses either Pax3, or Pax7, or both Pax3 and Pax7.

Pharmaceutically acceptable carriers: Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co. , Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of stem cells herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a

cell. "Incubating"includes exposing a target to an agent for a sufficient period of time for<BR> the agent to interact with a cell. "Contacting"includes incubating an agent in solid or in liquid form with a cell.

Polypeptide A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred.

The terms"polypeptide"or"protein"as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide"is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.

The term"polypeptide fragment"refers to a portion of a polypeptide which exhibits at least one useful epitope. The term"functional fragments of a polypeptide"refers to all fragments of a polypeptide that retain an activity of the polypeptide. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An "epitope"is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants of the insulin, are thus included as being of use.

The term"soluble"refers to a form of a polypeptide that is not inserted into a cell membrane.

The term"substantially purified polypeptide"as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.

Conservative substitutions replace one amino acid with another amino acid that is similar in size, hydrophobicity, etc. Examples of conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln ; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu Variations in the cDNA sequence that result in amino acid changes, whether conservative or not, are usually minimized in order to preserve the functional and immunologic identity of the encoded protein. The immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide. Variant amino acid sequences may, for example, be 80,90 or even 95% or 98% identical to the native amino acid sequence.

Programs and algorithms for determining percentage identity can be found at the NCBI website.

Precursor Cell : A cell that can generate a fully differentiated functional cell of at least one given cell type. Generally, precursor cells can divide. After division, a precursor cell can remain a precursor cell, or may proceed to terminal differentiation. A"muscle precursor cell"is a precursor cell that can generate a fully differentiated functional muscle cell, such as a cardiomyocyte or a skeletal muscle cell. One specific, non-limiting example of a muscle precursor cell is a"cardiomyocyte precursor cell, "which is a cell that gives rise to cardiac muscle cells.

Progenitor Cell : A cell that gives rise to progeny in a defined cell lineage. A "muscle progenitor cell"is a cell that gives rise to cells of the muscle lineage. One specific, non-limiting, example of a skeletal muscle progenitor cell is a"satellite cell, "which gives rise to immature and mature skeletal muscle cells.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e. g. , by genetic engineering techniques. Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule.

Side population (SP) cells : A collection of cells that does not express the same complement of markers as the cells of interest. In one specific non-limiting example, a side population includes cells that express Sca-1. In another non-limiting example, the side population cells are c-kit c-met CD34-Pax (3/7)-Sca-1+.

Skeletal muscle : Skeletal muscle makes up most of the body's muscle and does not contract without nervous stimulation. It is under voluntary control and lacks anatomic cellular connections between fibers. The fibers (cells) are multinucleate and appear striated due to the arrangement of actin and myosin protein filaments. Each fiber is a single cell, long, cylindric and surrounded by a cell membrane. The muscle fibers contain many myofibrils that are made of myofilaments. These myofilaments are made up of contractile proteins. The key proteins in muscle contraction are myosin, actin, tropomyosin and troponin.

Skeletal-based precursor of cardiomyocytes (Spoc) cells : Stem cells derived from skeletal muscle, which do not express the cell surface markers c-met, c-kit, or CD34, that can be differentiated into cardiomyocytes. Spoc cells can be further purified by sorting for the Sea-) cell surface marker (found on hematopoietic cells). Spoc cells are thus Sca-1-, distinguishing them from side population (SP) cells. In addition, spoc cells do not express the Pax3 and Pax 7 transcription factors (Pax (3/7) ). Thus, in one non-limiting example, spoc cells are c-kifc-met-CD34'Sca-l-Pax (3/7)-. In another non-limiting example spoc cells are muscle derived precursor cells that are about 4 um in diameter when cultured in vitro.

These cells remain in suspension and proliferate when cultured in the presence of a growth factor. Specific, non-limiting examples of growth factors of use in propagating spoc cell are FGF, EGF, or a combination thereof.

In one embodiment, spoc cells can differentiate into spontaneously beating cardiomyocytes in vitro. During a proliferative phase (e. g. about 7 days after being maintained in vitro in the presence of a growth factor), spoc cells cluster and increase in size to about 10-14 pm in diameter. The cells in these clusters, referred to as CS cells, have the ability to differentiate into mature cardiac muscle cells when cultured in the absence of growth factors. Methods for isolating and differentiating spoc cells are disclosed herein.

Spontaneous: Arising from an internal cause, resulting from internal or natural processes, with no apparent external influence. A"spontaneously beating cardiomyocyte" is a cell that begins to beat as a result of internal signals.

Stem cell : A cell that can generate a fully differentiated functional cell of more than one given cell type. The role of stem cells in vivo is to replace cells that are destroyed during the normal life of an animal. Generally, stem cells can divide without limit. After division, the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation. Although appearing morphologically unspecialized, the stem cell may be considered differentiated where the possibilities for further differentiation are limited. A"muscle stem cell"is a stem cell derived from muscle or that gives rise to muscle cells after differentiation. One specific, non-limiting example of a muscle stem cell is a cell that gives rise to cardiac muscle cells.

Stem cell antigen-1 (Sca-1): A murine cell surface antigen also called also Ly- 6A/E. Sca-1 is expressed on immature hematopoietic progenitor cells and stem cells.

Antibodies directed against Sca-1 have been used to fractionate early hematopoietic progenitor cells that have the ability to repopulate bone marrow in vivo. A cell that does not express Sca-1 is Sca-1 negative (Sca-1'), whereas a cell that expresses Sca-1 is Sca-1 positive (Sca-l+). In specific, non-limiting examples, expression of Sca-1 (or any other cell marker) can be measured by fluorescence activated cell sorting (FACS) or immunohistochemistry.

Subject: Any mammal, such as humans, non-human primates, pigs, sheep, cows, rodents and the like which is to be the recipient of the particular treatment. In one embodiment, a subject is a human subject or a murine subject.

Suspension: A dispersion of solid particles, such as a cell, throughout the body of a liquid, such as a culture medium or an isotonic (physiologically compatible) buffer.

Therapeutic agent: Used in a generic sense, it includes treating agents, prophylactic agents, and replacement agents.

Therapeutically effective amount: The amount of agent that is sufficient to prevent, treat, reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease. In one embodiment, a"therapeutically effective amount"is sufficient to reduce or eliminate a symptom of a cardiac disease. In another embodiment, a therapeutically effective amount is an amount sufficient to overcome the disease itself.

A therapeutically effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the agent will be dependent on the subject being treated, the severity and type of

the condition, and the manner of administration of the compound. "Administering"can be accomplished by introducing the therapeutically effective amount locally or systemically into the subject. Systemic introduction can be accomplished by using an intravenous, intramuscular, transcutaneous or subcutaneous means. Such means could include introducing the therapeutically effective amount via injection, or via catheter.

The general term"administering a therapeutically effective amount to the subject" is understood to include all animals (e. g. humans, apes, dogs, cats, horses, and cows) that have or may develop some form of cardiac dysfunction.

Therapeutically effective amount of a cell : An amount of a spoc cell (e. g. a c-kit- c-met CD34-Sca-l-Pax (3/7)- cell) that can be determined by various methods, including generating an empirical dose-response curve, predicting potency and efficacy of using modeling, and other methods used in the biological sciences. In general, a therapeutically effective amount ofac-kit'c-met'CD34'Sca-l'Pax (3/7)' cell is an amount sufficient to prevent, treat, reduce, eliminate and/or ameliorate a symptom and/or the underlying causes of a cardiac disease. In one embodiment, a therapeutically effective amount is an amount sufficient to overcome a cardiac disorder.

The therapeutically effective amount of c-kit-c-met-CD34-Sca-l-Pax (3/7)-cells will be dependent on the subject being treated (e. g. the species or size of the subject), the type of cardiac dysfunction suffered by the subject, and the location of administration of the cells (e. g. intravenously, locally, etc). In one embodiment, a therapeutically effective amount of cells is an amount of cells sufficient to treat a subject suffering from a myocardial injury. In specific, non-limiting examples, a therapeutically effective amount of c-kit c-met CD34- Sua-Pax (3/7)-cells is more than about 100 cells, more than about 1000 cells, more than about 10,000 cells, more than about 100,000 cells, more than about 250,000 cells, more than about 1,000, 000 cells, or between about 250,000 cells and about 1,000, 000 cells.

Specific assays for determining the therapeutically effective amount of c-kit'c-met' CD34-Sca-l-Pax (3/7)- cells are provided herein. The methods disclosed in the present invention have equal application in medical and veterinary settings. Therefore, the general term"subject being treated"is understood to include all animals (e. g. humans, apes, dogs, cats, mice, rats, rabbits, sheep, pigs, mice and cows) and treatment of myocardial injury is monitored using assays that measure cardiac function. Other assays can determine the success of engraftment of c-kit-c-met-CD34-Sca-I-Pax (3/7)- cardiomyocyte precursor cells following transplantation.

Transfected: A transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transduction

encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Transplantation: The transfer of a tissue or an organ, or a portion thereof, from one body or part of the body to another body or part of the body.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used, suitable methods and materials are described below. In case of conflict, the present specification, including the explanation of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Spoc Cells Stem cells derived from skeletal muscle (spoc cells) are disclosed herein. Spoc cells do not express the cell surface markers c-met, CD34, Sca-1, or c-kit. In addition, spoc cells do not express the Pax3 and Pax7 transcription factors. Thus, in one example, spoc cells are termed c-met~c-kit~CD34~Sca-l~Pax (3/7) ~. Spoc cells can be isolated from any age mammal, either human or non-human. Thus spoc cells can be obtained from a fetus, a child or an adult of any mammalian species. In one embodiment, a spoc cell is a human or murine c- met c-kit CD34-Sca-l-Pax (3/7)- cell that can be differentiated into a cardiomyocyte in vitro.

In one embodiment, the spoc cell is between about 311m and logm in diameter, or are about 4 pm in diameter.

Culture conditions for spoc cells (e. g. c-kit'c-met CD34-Sca-l-Pax (3/7)- cells) have been identified and are disclosed herein. In one embodiment, spoc cells do not adhere to the culture dish but remain in suspension when cultured in the presence of at least one growth factor. In one specific, non-limiting example, the growth factor is EGF. In another specific, non-limiting example, the growth factor is FGF.

Culture conditions are also disclosed herein (see below) for differentiating spoc cells. The differentiation of spoc cells into cardiomyocytes can be assessed by observing morphological changes. In some examples, differentiated spoc cells are spontaneously beating cardiomyocytes. In several embodiments, organized gap junctions and sarcomeres with clear Z-lines and A-and 1-bands, are observed in the differentiated spoc cells. In addition, certain examples of the differentiated spoc cells may be mono-or multi-nucleate.

In one specific, non-limiting example, the cells are bi-nucleate.

The isolated spoc cell (e. g. a c-kit'c-met CD34-Sca-l-Pax (3/7) cell) can be transduced using standard procedures known in molecular biology in order to introduce a nucleic acid molecule of interest into the cell. In one embodiment, the nucleic acid molecule encodes a polypeptide. The polypeptide encoded by the nucleic acid molecule can be from the same species as the cells (homologous), or can be from a different species (heterologous). For example, a nucleic acid molecule can be utilized that supplements or replaces deficient production of a peptide by the tissue of the host wherein such deficiency i. s a cause of the symptoms of a particular disorder. In this case, the cells act as a source of the peptide. In one specific, non-limiting example the polypeptide is the cardiac specific transcription factor GATA-4.

In one embodiment, the nucleic acid molecule of interest encodes a polypeptide involved in growth regulation or neoplastic transformation of cardiac cells. Specific, non- limiting examples of nucleic acids sequences of interest are SV40 Tag, p53, myc, src, and bcl-2. In another embodiment, the nucleic acid sequence of interest encodes an enzyme.

Specific, non-limiting examples of enzymes are proteins involved in the conversion of a pro-drug to a drug, or growth factors that promote the expansion, differentiation, or survival of cardiac progenitor cells, such as EGF, FGF, or atrial natriuretic factor. In yet another embodiment, the nucleic acid sequence of interest encodes a transcriptional regulator.

In one embodiment, the nucleic acid sequence of interest is operably linked to a regulatory element, such as a transcriptional and/or translational regulatory element.

Regulatory elements include elements such as a promoter, an initiation codon, a stop codon, mRNA stability regulatory elements, and a polyadenylation signal. A promoter can be a constitutive promoter or an inducible promoter. Specific non-limiting examples of promoters include the CMV promoter, an atrial natriuretic factor promoter, and promoters including TET-responsive element for inducible expression of transgene. In another embodiment, the nucleic acid sequence of interest is inserted into a vector, such as an expression vector. Procedures for preparing expression vectors are known to those of skill in the art and can be found in Sambrook et al., Molecular Cloning : A Laboratory Manual,

2nd Ed. , Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (1989). Expression of the nucleic acid of interest occurs when the expression vector is introduced into an appropriate host cell.

In yet another specific, non-limiting example, a nucleic acid sequence can be introduced to decrease rejection. For example, the immunogenicity of a cell may be suppressed by deleting genes that produce proteins that are recognized as"foreign"by the host (a knock-out), or by introducing genes which produce proteins, such as proteins that are native to the host and recognized as"self'proteins by the host immune system.

Thus in one embodiment, a spoc cell (e. g. a c-kit'c-met'CD34'Sca-l'Pax (3/7)' cell) may be transfected with a nucleic acid molecule designed to functionally delete or"knock- out"a gene of interest. In this method, the nucleic acid molecule of interest is a nucleic acid molecule that undergoes homologous recombination and is inserted into the genome of the spoc cell. Methods for producing"knock-outs"in ES cells are known to one of skill in the art (e. g. see U. S. Patent No. 5,939, 598; W000214495A2).

According to this example, cells are cultured in vitro as described herein and an exogenous nucleic acid is introduced into the cells by any method known to one of skill in the art, for example, by transfection or electroporation. The transfected cultured cells can then be studied in vitro or can be administered to a subject (see below). Methods for the introduction of nucleic acid sequences into stem cells are known in the art (e. g. , see U. S. Patent No. 6,110, 743).

Methods of Isolating and Expanding Muscle Stem Cells A method of isolating a spoc cell (e. g. a c-met'c-kit CD34-Sca-l-Pax (3/7)- cardiomyocyte precursor cell of muscular origin) is described herein. In this method, spoc cells are separated by size from a suspension of muscle cells and the cells are cultured on a solid substrate. The cells that remain in suspension in the culture medium are isolated.

The method of isolation of the spoc cells (e. g. c-kitc-metCD34'Sca-l'Pax (3/7)' cells) includes obtaining the cells from the muscle of a subject. Muscle tissue can be prepared for the purpose of isolating or obtaining individual spoc cells by using methods well known to one of skill in the art. Examples of methods of tissue preparation include enzymatic digestion with enzymes such as collagenase, mechanical disruption using instruments such as hand-held or motor-driven homogenizers, or by chemical disruption using, for example, chelators of calcium and magnesium.

The preparation of muscle cells can be sorted by any method that separates cells on the basis of cell size. In one embodiment, the spoc cells (e. g. c-kit~c-met~CD34~Sca-1~ Pax (3/7) cells) are isolated by passing digested skeletal muscle through a series of filters of

varying pore size. The muscle cells are passed through two filters, where a first filter has a pore size of about 50-200 m, about 60-150 um, about 80-100 m, or about 100 tm and a second filter has a pore size of about 10-50 m, 20-40 zm, or about 40 um. In one embodiment the isolated cells are less than 40 Rm in diameter. In other embodiments, isolated cells are between about 3 llm and 10 im in diameter. In another embodiment the isolated cells are about 4 m in diameter.

The muscle cells can be also sorted by size by passing them through size-exclusion columns. In one such embodiment, the cells are eluted along a size gradient such that the largest cells are eluted first and the smallest cells are eluted last. The cells can also be sorted by size using FACS. Cells of about 3 Rm to 10 llm in diameter, or of about 4 um in diameter, are isolated.

Once the muscle cells are sorted by size the muscle cells are further selected and then expanded in culture medium. In one embodiment the cells are cultured on a solid substrate that permits the adhesion of a subpopulation of cells in the presence of a culture medium. In one embodiment, the solid substrate is a container, such as a tissue culture dish.

In another embodiment, the solid substrate is in the form of beads designed for tissue culture. The medium can be a growth medium, or any buffer that maintains the viability of the cells. A variety of culture media are known and are suitable for use. Generally, the growth medium includes a minimal essential medium. In one embodiment, the medium is (Dulbecco's Modified Eagle's Medium (DMEM) and/or F12, or a combination of DMEM and F12 (at a ratio between about 1 : 1 to about 10: 1).

The growth medium may be supplemented with serum. Specific, non-limiting examples of serum are horse, calf or fetal bovine serum. The medium can have between about 3% by volume to about 10% by volume serum, or about 5% by volume serum.

In one embodiment, the medium contains one or more additional additives such as nutrients. Specific, non-limiting examples of these nutrients are shown in the table below: Additive Exemplary Concentration serum About 3% to about 10% insulin About 5 Fg/ml to about 10 Zg/ml transferrin About S llg/ml to about 10 llg/ml selenium About 6 ng/ml ethanolamine About 2 ug/ml EGF About 5 ng/ml to about 10 ng/ml FGF About 5 ng/ml to about 10 ng/ml gentamycin About 25zg/ml to about 50 g/ml fungizone About 0. 2 gg/n-d to about 2.5 Fg/ml

The muscle stem cell growth media can also be supplemented with growth factors.

In one embodiment, the growth medium includes basic fibroblast growth factor (bFGF). In one specific example, the growth medium includes between about 2 ng/ml to about 100 ng/ml of bFGF, such as for example between about 5 ng/ml to about 50 ng/ml, between about 8 ng/ml to about 20 ng/ml, or between about 5 to about 10 ng/ml bFGF. In yet another example, the medium includes about 10 ng/ml bFGF. In another embodiment, the growth medium includes epidermal growth factor (EGF). In one specific example, the growth medium includes between about 2 ng/ml to about 100 ng/ml of EGF, such as for example between about 5 ng/ml to about 50 ng/ml, between about 8 ng/ml to about 20 ng/ml, or between about 5 ng/ml to about lOng/ml EGF. In yet another example, the medium includes about 10 ng/ml EGF. Thus in one specific, non-limiting example, the growth medium is 1: 1 DMEM/F12 and includes 5% fetal bovine serum, 10 ng/ml FGF, 10 ng/ml EGF, 5 Rg/ml insulin, 5 Fg/ml transferrin, 6 ng/ml selenium, 2 ßg/ml ethanolamine.

In one specific, non-limiting example the cells are cultured in the growth medium for about 4 days to about 8 days. In another specific, non-limiting example, the cells are cultured in the growth medium for about 6 days to about 7 days.

During the period that the cells are cultured in the presence of growth factors, the cells cluster and increase in size. Within the clusters the cells are between about 5-20 Hm in diameter, or between about 10-14 m in diameter.

A method is also provided for isolating spoc cells (e. g. c-kit~c-met CD34~Sca-1~ Pax (3/7)' cells) wherein the spoc cells are identified using specific binding agents, such as antibodies, for example monoclonal antibodies that recognize cell surface markers. This particular method of isolation of the spoc cells includes obtaining the cells from the muscle of a subject, as described above. In one embodiment, the cells are selected by size (see above) and then the spoc cells are identified using the specific binding agents, such as antibodies that recognize the c-met, c-kit, CD34, Sca-1 cell surface markers.

In one embodiment the c-met, Sca-1, CD34, and c-kit antibodies are immobilized.

A particular embodiment uses magnetic column cell sorting. This method involves a combination of monoclonal antibodies which are covalently bound to the surface of magnetic beads and which are directed to cell surface markers which are absent from the cells being selected. For example, to isolate a spoc cell that does not express c-met, c-kit, CD34, or Sca-1 (c-met~c-kit~CD34~Sca-1~), monoclonal antibodies to c-met, c-kit, CD34, and Sca-1 bound to magnetic beads are used. All cells expressing either CD34, Sca-1, c-met, or c-kit, or any combination of these cell surface markers, will be bound by the antibodies and retained by the beads. Since the cells bound to the magnetic beads are immobilized by the

magnet, the c-met~c-kit~CD34~Sca-1~ cells that remain in suspension can be isolated from the other cells. In a further embodiment, cells that are initially isolated because they are c-kit-c- met CD34'can be further sorted into Sca-1'and/or Sca-1 c-kit'c-met'CD34 cells.

In another embodiment, purified populations of cells are isolated via FACS.

Fluorescent-tagged antibodies against the c-met, c-kit, CD34, and Pax (3/7) markers can be used to distinguish c-kit c-met CD34 Pax (3/7)' cells from cells that express any combination of the c-met, c-kit, CD34, and Pax (3/7) markers. In a further embodiment, the c-kit c-met CD34'Pax (3/7)- cells can be further sorted into Sca-1-or Sca-l+cells. Thus, cells are isolated that are either c-kit c-met CD34-Pax (3/7)-Sca-1- or c-kit-c-met-CD34-Pax (3/7)-Sca-l'.

In other embodiments a single antibody, or a combination of antibodies, can be covalently bound to inert beads, such as sepharose beads. The beads can be packed in a column or maintained as a slurry. The cells expressing one or more of the cell surface markers are recognized by one or more of the antibodies, thus becoming bound to the beads, thereby identifying a subpopulation of unbound cells that does not express the combination of cell surface markers.

In another embodiment the antibodies are not immobilized. In a particular embodiment the addition of the antibodies to a mixture of cells causes the aggregation of cells expressing the cell surface markers recognized by the antibodies. The cells not expressing the cell surface markers are excluded from the aggregates and can be isolated.

Spoc cells isolated by these or other methods can be maintained in culture. The spoc cells can further be differentiated into cardiomyocytes.

Methods of Differentiating Muscle Stem Cells A method is disclosed herein for differentiating a spoc cell (e. g. a c-kit c-met CD34- Sca-l-Pax (3/7)- cell) into a cardiomyocyte. In a particular example, the cardiomyocyte is a spontaneously beating cardiomyocyte.

In one embodiment, differentiation into cardiomyocytes is induced by culturing cells in medium similar to the growth medium, but which does not include at least one growth factor. Thus, a specific, non-limiting example of a differentiation medium is a growth medium that lacks at least one growth factor. Growth factors removed from the medium include, but are not limited to, bFGF or EGF, or a combination of bFGF and EGF.

Removal of at least one growth factor causes the cells to adhere to the tissue culture dish and acquire characteristics of a differentiated cardiomyocyte. Differentiation refers to the process whereby relatively unspecialized cells, such as spoc cells (e. g. c-met-c-kit-CD34'

Sca-l'Pax (3/7)- muscle-derived stem cells), acquire specialized structural and/or functional features characteristic of mature cells, such as cardiomyocytes.

Differentiation of spoc cells (e. g. c-met'c-kit'CD34'Sca-l'Pax (3/7)' cells) into cardiomyocytes, such as spontaneously beating cardiomyocytes, can be measured by any method known to one of skill in the art. Specific, non-limiting examples are immunohistochemical analysis to detect expression of cardiac polypeptides (e. g. troponin-T, L-type calcium channel, or cardiac-specific transcription factors GATA-4, or Nkx2.5), or assays such as ELISA assay and Western blot analysis. Differentiation of cells can also be measured by assaying the level of mRNA coding for cardiac polypeptides using techniques such as Northern blot, RNase protection and RT-PCR. In another embodiment, the number of spontaneously beating cells is assessed.

Calcium transients, or the flux in intracellular calcium concentrations, can be used as a measure of cardiomyocyte differentiation. In one embodiment calcium imaging is used to measure calcium transients. For example, ratiometric dyes, such as fura-2, fluo-3, or fluo-4 are used to measure intracelluar calcium concentration. The relative calcium levels in a population of cells treated with a ratiometric dye can be visualized using a fluorescent microscope or a confocal microscope. In other embodiments, the membrane potential across the cell membrane is monitored to assess calcium transients. For example, a patch clamp is used. In this method, an intracellular microelectrode is inserted into the cardiomyocyte.

In one embodiment, calcium transients can be seen before observable contractions of the cardiomyocytes. In other embodiments calcium transients are seen either during, or after, observable contractions of cardiomyocytes. In another embodiment the cells are cultured in the presence of conditions wherein the cells do not beat, such as in the presence of a calcium chelator (e. g. EDTA or EGTA) and the calcium transients are measured.

Methods for Treatment of Cardiac Diseases or Disorders In other embodiments, methods are provided for treating a subject suffering from a disease or a disorder, such as myocardial injury, or alleviating the symptoms of such a disorder, by administering cells isolated and cultured according to the methods disclosed.

In one embodiment, spoc cells (e. g. c-met~c-kit~CD34~Sca-l~Pax (3/7) ~ cells) are isolated as described herein and a therapeutically effective amount of spoc cells is administered to the subject. In another embodiment, a mixture of a therapeutically effective amount of c-kit c-met CD34-Pax (3/7)-Sca-1- cardiomyocyte precursor cells and a therapeutically effective amount of c-kit c-met CD34-Pax (3/7)-Sca-1+ cells is administered

to the subject. In a further embodiment, spoc cells are isolated and differentiated into cardiomyocytes, as disclosed above, and a therapeutically effective amount of the differentiated cells are administered to a subject, such as a human. The cells may be administered in any fashion, for example in a dose of, for example 0.25-1. 0 x 106 cells.

Different dosages can of course be used depending on the clinical circumstances. The cells may be administered systemically (for example intravenously) or locally (for example directly into a myocardial defect under echocardiogram guidance, or by direct application under visualization during surgery). In another example, the cells are administered in a gel matrix (such as Gelfoam from Upjohn Company) which polymerizes to form a substrate in which the administered cells can grow.

In one embodiment the subject has a myocardial injury. The myocardial injury may be due to trauma that occurred as the result of an object or projectile, such as a knife or a bullet, having penetrated the myocardium, or as a consequence of surgery to remove, for example, a tumor. Myocardial injury may also result from diseases such as cardiomyopathy, myocardial infarction, or congenital heart disease. In one specific, non- limiting example, a therapeutically effective amount of spoc cells (e. g. c-met-c-kit-CD34' Sca-l-Pax (3/7 cells) is administered to the subject with a myocardial injury. In another specific, non-limiting example, a mixture of a therapeutically effective amount of c-kit c- met'CD34-Pax (3/7)-Sca-l- cardiomyocyte precursor cells and a therapeutically effective amount of c-kit-c-met-CD34-Pax (3/7)-Sca-l' cells is administered to the subject with a myocardial injury.

In another embodiment the subject is suffering from cardiac dysfunction which includes, for example, abnormal or improper functioning of the heart valves, or abnormal communication between the chambers of the heart. In one specific, non-limiting example, a therapeutically effective amount of spoc cells (e. g. c-met'c-kit'CD34'Sca-l'Pax (3/7)' cells) is administered to the subject with a cardiac dysfunction. In another specific, non-limiting example, a mixture of a therapeutically effective amount of c-kit c-met CD34'Pax (3/7)'Sca- 1-cardiomyocyte precursor cells and a therapeutically effective amount of c-kit c-met'CD34- Pax (3/7)-Sca-1+ cells is administered to the subject with a cardiac dysfunction.

In one embodiment the spoc cells (e. g. c-met'c-kit'CD34'Sca-l'Pax (3/7)' cells) or differentiated cardiomyocytes are administered systemically by injection. In another embodiment, a mixture of a therapeutically effective amount of c-kit-c-met-CD34-Pax (3/7)- Sca-1 cardiomyocyte precursor cells and a therapeutically effective amount of c-kit-c-met- CD34-Pax (3/7)-Sca-1+ cells is administered systemically by injection. Specific, non-limiting examples of administration by injection include administration by subcutaneous injection,

intramuscular injection, or intravenous injection. If administration is intravenous, an injectible liquid suspension of spoc cells can be prepared and administered by a continuous drip or as a bolus.

In one embodiment, the spoc cells (e. g. c-met-c-kit-CD34-Sca-l-Pax (3/7)- cells) or differentiated cardiomyocytes are administered locally. In another embodiment, a mixture of a therapeutically effective amount of c-kit~c-met~CD34~Pax (3/7) ~Sca-1~ cardiomyocyte precursor cells and a therapeutically effective amount of c-kit c-met CD34-Pax (3/7)-Sca-1+ cells is administered locally. One specific, non-limiting example of local administration is intra-cardiac muscle injection. For intra-cardiac injection, the spoc cells are in an injectible liquid suspension preparation or in a biocompatible medium which is injectible in liquid form and becomes semi-solid at the site of damaged myocardium. A conventional intra- cardiac syringe or a controllable endoscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter (e. g. 30 gauge or larger) that shear forces will not damage the spoc cells.

In other embodiments the spoc cells (e. g. c-met'c-kit'CD34'Sca-l'Pax (3/7)' cells), differentiated cardiomyocytes, or a mixture of a therapeutically effective amount of c-kit-c- met~CD34~Pax (3/7) ~Sca-l~ cardiomyocyte precursor cells and a therapeutically effective amount of c-kit-c-met-CD34-Pax (3/7)-Sca-l' cells are administered locally on a support medium. One specific, non-limiting example of a support medium is a sterile mesh, or matrix, upon which the cells are cultured. A layer of cardiomyocytes, for example a confluent layer of cardiomyocytes, cultured on such a matrix can be applied locally, or grafted at or near, a site of myocardial injury. In one embodiment the support medium is a biodegradable mesh. In another embodiment the support medium is not biodegradable. The size of the mesh, and the density of cells on it, can vary depending on the myocardial defect being treated.

In another embodiment the cells are encapsulated prior to administration, such as by co-incubation with a biocompatible matrix known in the art. A variety of encapsulation technologies have been developed (e. g. Lacy et al., Science 254: 1782-84,1991 ; Sullivan et al., Science 252: 7180712, 1991 ; WO 91/10470; WO 91/10425; U. S. Patent No. 5,837, 234; U. S. Patent No. 5,011, 472; U. S. Patent No. 4,892, 538). During open surgical procedures, involving direct physical access to the heart, all of the described forms of spoc cell delivery preparations are available options.

In one embodiment, transformed spoc cell lines (e. g. c-met~c-kit~CD34~Sca-1~ Pax (3/7)- cells) beat and divide continuously. In specific, non-limiting examples, transformed spoc cells, transformed differentiated cardiomyocyte cells, or a mixture of

transformed c-kit-c-met-CD34-Pax (3/7)-Sca-l- cardiomyocyte precursor cells and transformed c-kit~c-met~CD34~Pax (3/7) ~Sca-l+ cells are used to obtain enough cells to produce a"pumping patch"to repair a diseased and failing heart. In one embodiment, transformed spoc cells derived ex vivo from a subject are introduced into a subject through the circulation. In another embodiment, transformed spoc cells derived ex vivo from a subject are introduced into a subject directly into the heart using a guided catheter. In several embodiments, transformed spoc cells derived ex vivo from a subject are used together with endothelial cell precursors, muscle fibroblasts, or other support cells from a subject to produce a patch of vascularized beating cells that is placed in the heart as an assist device. The patch is synchronized to the rest of the heart either by capturing the intrinsic cardiac pacing current or by the use of a pacemaker implant that entrains the entire heart.

The cells of the disclosure can be repeatedly administered at intervals until a desired therapeutic effect is achieved.

Use of Spoc Cells Produced to Screen Agents that Affect Cardiomyocyte Differentiation or Function In other embodiments, methods are provided for screening agents that affect cardiomyocyte differentiation or function. According to this method, a population of spoc cells (e. g. c-met c-kit CD34-Sca-l-Pax (3/7)- cells) or differentiated cardiomyocytes is produced as described above. The population of cells is contacted with an agent of interest, and the effect of the agent on the cell population is then assayed. The effect on differentiation, survival, proliferation, or function of the cells is assessed.

The methods described herein can be used to assess the effect of an agent on cardiomyocyte differentiation. In order to assess the effect of a test agent on cardiomyocyte differentiation or function, the agent is contacted either to spoc cells (e. g. c-met c-kit'CD34 Sca-l-Pax (3/7)- cells) or CS cells. In several embodiments the spoc cells are maintained in medium including a growth factor between about 1 day to about 8 days, between about 4 days to about 7 days, or about 7 days before the addition of an agent.

In another embodiment the growth factor is removed from the medium, generating CS cells, at or before the agent is added. In several specific, non-limiting examples CS cells are maintained in the medium between about 1 day to about 56 days, between about 7 days to about 28 days, or between about 14 days to about 21 days before the addition of an agent.

Differentiation of spoc cells (e. g. c-met'c-kit'CD34 Sca-1 Pax (3/7)' cells) contacted with an agent can be assessed by any means known to one of skill in the art. In one embodiment the morphology is examined, for example electron microscopy is used to assess

the ultrastructure of the cells. Suitable parameters for evaluation include, but are not limited to the evaluation of gap junctions between contacting cardiomyocytes. In other embodiments, immunohistochemical or immunofluorescence techniques are used to assess differentiation. In yet another embodiment, differentiation is assessed by analysis expression of specific mRNA molecules expressed in cardiomyocytes. Suitable assay systems include, but are not limited to RT-PCR, in situ hybridization, Northern analysis, or RNase protection assays. In a further embodiment the levels of polypeptides expressed in differentiated cardiomyocytes are assayed. Specific, non-limiting examples of polypeptide assays of use include Western blot analysis, ELISA assay, or immunofluorescence.

Alternatively, calcium transients are measured, as described above.

The assay can also be used to screen the effect of an agent on cardiomyocyte function. Any method known to one of skill in the art can be utilized to assess cardiac function. In one embodiment the beating rate of a cardiomyocyte is assayed to identify agents that increase or decrease beating. One method for assessing the beating rate is to observe beating under a microscope. Agents that can be screened in this manner include inotropic drugs, such as sympathomimetic agents.

In one embodiment, cells contacted with the agent are compared with a control.

Suitable controls include spoc or CS cells not contacted with the agent, or contacted with vehicle alone. Standard values can also be used as a control.

In one embodiment, transformed cardiac cell lines are used to screen therapeutic agents, derived from combinatorial chemical libraries, in high throughput assays. In several embodiments, the transformed cardiac cell lines are made from skeletal muscle of transgenic, knockout, or knockin mouse cell lines that are produced to study genes that are important to cardiac function and development.

In another embodiment, the transcriptome of the spoc cells at different stages of differentiation or the transformed spoc cells are determined by RNA array techniques (cDNA arrays, gene chip oligonucleotide arrays, SAGE analysis, or subtraction libraries). A fingerprint consisting of subsets of the transcriptome of these different cell populations is used to screen (by PCR based or array analysis) candidates for analogous precursor and stem cells from human subjects. The selected cells can then be transformed or expanded in primary cell cultures.

Kits The cells described herein are ideally suited for the preparation of a kit. The kit can include a carrier means, such as a box, a bag, or plastic carton. In one embodiment the

carrier contains one or more containers such as vials, tubes, and the like that include a sample of spoc cells (e. g. c-met'c-kit'CD34'Sca-l'Pax (3/7)' cells) and/or a sample of c-met c-kit CD34-Sca-l+Pax (3/7)- cells. In another embodiment, the carrier includes a container with an agent that affects differentiation, a buffer, or a vehicle for the introduction of the cells. Instructions can be provided to detail the use of the components of the kit, such as written instructions, video presentations, or instructions in a format that can be opened on a computer (e. g. a diskette or CD-ROM disk). These instructions indicate, for example, how to administer the cells to treat a myocardial defect or how to use the cells to screen test agents of interest (such as inotropic drugs).

Without further elaboration, it is believed that one skilled in the art can, using this description, utilize the present invention to its fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES EXAMPLE 1 Method of Isolating and Expanding Cardiomyocyte Precursor Cells from Adult Mouse Skeletal Muscle Skeletal muscle tissue from hind legs of 6-10 week-old male C57B1/SJ6 mice was cut into small pieces and digested with collagenase for two hours at 37 °C. The digested tissue was cleared of cell debris and other undigested tissue fragments by passage through a 100 m filter and then through a 40 (im filter (Falcon). The cell suspension was centrifuged at low speed (1,400 rpm) to clear as much as of the small muscle fiber fragments as possible. The cells at this stage consisted mostly of clusters of small round cells approximately 4, um in diameter, called spoc (skeletal-based precursors of cardiomyocytes) cells. The cells were further fractionated using Sca-1 antibody on a Miltenyi magnetic column. The spoc cells eluted in the Sca-l-fraction and consisted of mostly non-adherent round cells, whereas the Sca-l+ fraction consisted mainly of adherent cells.

The spoc cells were plated at a density of approximately 105 cells per cm2 in regular tissue culture dishes in complete growth medium (1 : 1 DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 10 ng/ml human EGF, 10 ng/ml human bFGF (PeproTech, Inc.), 5 zg/ml insulin, 5 Stg/ml transferrin, 6 ng/ml selenium, 2 Zg/ml ethanolamine (ITS-X, Invitrogen Corporation), 25 llg/ml gentamicin and 2.5 g/ml fungizone (Life Technologies) ). After a few days, the culture consisted of a floating population of round cells and some adherent fibroblasts. The round cells enlarged as they underwent a few

rounds of cell division during which time they became clusters of floating round cells with an increased diameter of 10-14 m. The cells in these clusters were GATA-4 positive and were referred to as CS (cardiac precursors from spoc) cells.

EXAMPLE 2 Method of Differentiating Spoc Cells into Cardiomyocytes CS cells were gently collected after seven days of growth in complete growth medium. The cells were then plated in the same medium in the absence of EGF and bFGF (differentiation medium) and were maintained at 37 °C. To assess the progression of differentiation of the cells, the cultures were observed at various time points using an inverted light microscope. Beating frequency measurements of the cardiomyocytes were obtained by video microscopy.

Under the differentiation culture conditions the cells gradually began to attach to the culture dish, and elongate in shape, taking on the appearance of myoblasts. Within a few days of being maintained in the differentiation medium, the cells began spontaneously beating. Elongated uninucleate cells (60 um in length) and round uninucleate cells (15 u. m in diameter) both exhibited spontaneous beating. By four days post replating the beating cells were more numerous. The beating cells did not undergo any more cell divisions and were maintained in this medium for several weeks, with the maintenance of the spontaneous beating phenotype. Spontaneous beating was continuous and measured at a frequency of 1- 8 Hz. Small contractions observed in a day 14 cell (30 um in length) were likely the consequence of an immature contractile apparatus (Figure 1C). Cells kept at room temperature beat continuously for at least 3 hours. Cells continue to beat even after culture for 3 months. The isolation and differentiation of spoc cells from skeletal muscle is extremely reproducible and has been performed successfully more than seventy times.

EXAMPLE 3 Immunofluorescence of Spoc Cells Cultured cells derived from skeletal muscle were fixed in 4% paraformaldehyde at 4°C for 10 minutes, then washed in PBS for a total of 15 minutes (3 times). Cells were permeabilized with 0.2% Triton x-100 for 10 minutes. Blocking was performed for 30 minutes at room temperature with either 3% BSA in PBS, 5% goat serum in PBS, or 10% goat serum in PBS. Incubation with primary antibody was performed at 4°C overnight.

Primary antibodies used are directed against myoD (Novocastra), Sca-1 (Cedarlane), CD34 and CD45 (PharMingen). GATA4, Myf-5, myogenin, Pax 3/7, c-met, and cKit antibodies

are from Santa Cruz. RLCP antibody was generated by ResGen. Cells were then washed for a total of 15 minutes (5 times), in 1X PBS. Incubation with secondary antibody was done at room temperature for 1 hour. Afterwards, cells were washed 3 times with PBS.

Fluorescent mounting medium with DAPI (Vector) and cover slip were then placed over sample. Images of cells were obtained with a laser scanning confocal fluorescence microscope (Leica TCS-4D DMIRBE) equipped with argon and argon-krypton laser sources. Excitation wavelengths of 365 nm (DAPI), 488 nm (FITC), and 568 nm (rhodamine) were used to generate fluorescence emission in blue, green, and red, respectively.

The non-adherent subpopulation of cells that was isolated from skeletal muscle has been named"spoc"cells (skeletal-based precursors of cardiomyocytes) because of their capacity to develop into beating cardiomyocytes. Spoc cells are CD34-, CD45-, and c-kit-, differentiating them from other nonadherent cells previously described as derived from skeletal muscle (Deasy et al., Blood Cells Mol Dis 27,924, 2001). Spoc cells are distinguished from satellite cells by the following criteria: (1) Spoc cells do not express Pax-7 (Seale et al., Cell 102, 777,2000) or the surface marker c-met (Cornelison & Wold Dev Biol 191, 270,1997) ; (2) Approximately the same number of spoc cells are isolated from both young (less than 4-week-old) and older (12-16 week-old) mice, whereas satellite cells are only efficiently isolated from uninjured muscle of young mice (less than 4 weeks old); (3) Spoc cells are round, floating cells approximately 4 llm in diameter, while satellite cells are adherent in nature; and (4) Spoc cells are CD34-and MyfS~, excluding them from the class of quiescent satellite cells (Beauchamp et al., J Cell Biol 151, 1221,2000).

The three skeletal muscle super regulatory genes Myf5, MyoD and Myogenin are known to be present at some time in satellite cells (Cornelison & Wold Dev Biol 191, 270, 1997). Because all three markers are negative from day 0 throughout long-term culture, these cells are neither satellite cells nor further developed skeletal muscle cells.

Additionally, spoc cells do not survive when cultured under conditions that support satellite cell growth and differentiation into myotubes.

EXAMPLE 4 Detection of Cardiac-Specific Polypeptides by Immunofluorescence (1) The specimens (cultured cells derived from skeletal muscle) were air-dried for 30 minutes and then fixed in 4% paraformaldehyde at 4 °C followed by a rinse for 5 minutes

with phosphate buffered saline (PBS). They were blocked with goat serum for 30 minutes and then incubated overnight, at 4 °C, with either GATA-4 (mAb H-112, Santa Cruz Biotechnology), sarcomeric myosin (MF-20 Ab, ATTC), cardiac-specific troponin-T (mAb RDI-TRK4T19-lA11, Molecular Probes, Inc. ), cardiac L-type calcium channel (mAB<BR> AB5412-2000Ula, Chemicon Inc. ), cardiac-specific transcription factor Nkx2.5 (mAb N- 19, Santa Cruz Biotechnology), or connexin 43 (mAb 71-07000, Zymed Laboratories Inc.) (1: 200). Following the overnight incubation, the specimens were rinsed 3 times (5 minutes each) with PBS and blocked again with goat serum for 30 minutes. The specimens were then incubated at room temperature with a secondary antibody, conjugated with either Fluorescein Isothiocyanate (FITC), Texas Red, or Tetramethylrhodamine Isothiocyanate (TRITC), for 1 hour. They were again rinsed 3 times (5 minutes each) with PBS and then visualized with a laser confocal microscope (Leica) to detect fluorescent signals.

The earliest time of GATA-4 expression is after 3 days in culture in growth factor containing medium. Within 3 days after replating the cells in factor-containing medium (differentiation medium), some cells begin to express sarcomeric myosin. Cytospins of day 7 CS cells stained with monoclonal antibodies demonstrate the expression of cardiac- specific transcription factor GATA-4, sarcomeric myosin, and cardiac-specific troponin-T.

Day 14 cells stained for GATA-4 and sarcomeric myosin. Overlays of images of cells stained with GATA-4 and sarcomeric myosin demonstrated that they were co-localized in the cell. At this early stage in development some cells may either be positive for GATA-4 or sarcomeric myosin. By day 28, the majority of cells express both proteins. By day 21 the cells are positive for cardiac L-type calcium channel, cardiac-specific transcription factor Nkx2.5, and connexin 43.

EXAMPLE 5 Detection of Cardiac-Specific Polypeptides by Immunofluorescence (2) Cultured cells derived from skeletal muscle were fixed in 4% paraformaldehyde at 4°C for 10 minutes, then washed in PBS for a total of 15 minutes (3 times). Cells were permeabilized with 0.2% Triton x-100 for 10 minutes. Blocking was performed for 30 minutes at room temperature with either 3% BSA in PBS, 5% goat serum in PBS, or 10% goat serum in PBS. Incubation with primary antibody was performed at 4°C overnight.

Primary antibodies used are directed against myoD (Novocastra), MF-20 (ATTC), GFP (Clontech), cardiac L-type channel (US Biologicals), Sca-1 (Cedarlane), CD34 and CD45 (PharMingen), and cardiac troponin-T (Research Diagnostics). GATA4, Myf-5, myogenin, connexin 43, Nkx-2.5 Pax 3/7, c-met, and cKit antibodies are from Santa Cruz. RLCP

antibody was generated by ResGen. Cells were then washed for a total of 15 minutes (5 times), in 1X PBS. Incubation with secondary antibody was done at room temperature for 1 hour. Afterwards, cells were washed 3 times with PBS. Fluorescent mounting medium with DAPI (Vector) and cover slip were then placed over sample. Images of cells were obtained with a laser scanning confocal fluorescence microscope (Leica TCS-4D DMIRBE) equipped with argon and argon-krypton laser sources. Excitation wavelengths of 365 nm (DAPI), 488 nm (FITC), and 568 nm (rhodamine) were used to generate fluorescence emission in blue, green, and red, respectively.

During the first 7 days in factor-containing medium, spoc cells undergo several rounds of division, begin to express GATA4 (cardiac-specific transcription factor), and become clusters of floating round"CS"cells (cardiac precursors from spoc cells) with an increased diameter of 10-14 m. These cells go on to express other cardiac specific markers including cardiac troponin-T, Nkx-2. 5, and a cardiac specific L-type calcium channel. Even before they become adherent, some cells begin to beat, and by day 14 following replating, 10% of the cells in a confluent dish beat spontaneously. Spoc cells do not appear to be bone marrow cells sequestered in skeletal muscle and as they are cKit-, they are distinguished from the cKit+bone marrow cells and side population (SP) cells that have been used directly or indirectly in experiments to reconstitute infarcted heart (Jackson et al., J Clin Invest 107, 1395,2001 ; Orlic, et al., Nature 410,701, 2001). When marrow or dissociated total heart is co-cultured with GFP-labeled spoc cells either directly or in permeable membrane-separated compartments, only the labeled spoc cells develop into beating cardiomyocytes. Thus, bone marrow and heart do not contain a cell population that can be isolated in this manner and that is phenotypically similar to spoc cells.

EXAMPLE 6 Ultrastructure of differentiated cardiomyocytes For routine transmission electron microscopy, cultured cells derived from skeletal muscle were fixed in situ on Petri dishes with 1.25% glutaraldehyde in 0.1 M cacodylate buffer containing 1% CaCl2 at 4 °C for 2 hours. Following fixation, cells were washed three times in Sabatini's solution (0.1 M cacodylate buffer containing 6.8% sucrose), and post- fixed with 1% osmium tetroxide in cacodylate buffer for one hour. After three washes in Sabatini's solution, samples were dehydrated in alcohol and embedded in Scipoxy 812 (Energy Beam Sciences, Inc. Agawarm, MA). Polymerization was carried out at 37 °C for 24 hours and then at 60 °C overnight. Ultra-thin sections were cut with a Leica Ultracut

UCT ultramicrotome, stained with uranyl acetate and Reynold's lead citrate, and examined with a JEOL 1200 CXII transmission electron microscope.

In Figure 1, transmission electron micrographs show the progression of CS cells.

At day 3 following replating, when increasing numbers of cells show rhythmic beating, round cells with disordered myosin filaments (Figure 1A) and large central nuclei surrounded by copious mitochondria (Figure 1D, box and detail) exist. By day 7 elongated cells (Figure 1E) contain dense bodies (Figure 1E arrowhead and Figure 1B, lower box).

Myosin filaments of characteristic 1. 6, um-length (Figure 1B, top box) radiate outward. A day 14 cell (Figures 1C and F) with a single, central nucleus shows a stretching out of the dense bodies, which have begun to align with filaments coursing between. (Figure 1C).

These structures are nearly identical to those seen in embryoid body-derived developing cardiomyocytes (Westfall et al., Cell Motil Cytoskeleton 36, 43, 1997). By day 56, the electron-dense structures have progressed to a well-defined organized sarcomere (Figure I. G) with identifiable A-and I-bands and Z-lines. The beating cells also have one or two large centrally located nuclei surrounded by mitochondria, distinguishing them from skeletal myotubes, which have many subsarcolemmal nuclei.

EXAMPLE 7 Calcium Transients as a Measure of Cardiomyocyte Differentiation Cardiomyocytes derived from spoc cells were incubated for 30 minutes at 37 °C with fluo-3 or fluo-4 dye at a concentration of approximately 5-10 m in DMEM/F-12 (dyes dissolved in DMSO 1: 1 with pluronic solution). The cells were then washed with fresh DMEM/F-12. The images were collected with a Zeiss LSM-510 laser scanning confocal system and a C-Apochromat 63x objective (1.2 N. A. ). Fluo-3 and fluo-4 were excited at 488 nm with an argon laser and the emission light was collected using an LP 505 filter. The pinhole was adjusted to produce a 5 Zm slice to minimize the influence of axial movements with contraction on viewing the calcium transients. All transmitted light images were collected simultaneously using a transmitted light detector in conjunction with the 488 nm excitation light. Data depth for the images was 8-bit. The size of the images varied from 512 x 512 pixels to 128 x 128 pixels.

Calcium transients can be observed with confocal microscopy in fluo-3-and fluo-4- treated cells (Figure 2). Fluorescent intensity is proportional to the amount of calcium binding to fluo-3 dye upon release of calcium from the sarcoplasmic reticulum. Figure 2A shows a graphical representation of the calcium transient in a beating CS cell-derived cardiomyocyte. Peak intensity and baseline are shown in Figure 2B and Figure 2C,

respectively. In some CS cells, calcium transients can be seen by confocal microscopy even before observable contractions are noted, suggesting the development of cardiomyocyte excitation elements in advance of maturing contractile elements or restriction of cellular motility by the dense extracellular matrix.

EXAMPLE 8 Measurement of Action Potentials in Spoc Cell-Derived Cardiomyocytes The calcium transients indicate the existence of action potentials (APs), which have been characterized with patch recordings of single spoc cell-derived cardiomyocytes in culture. Current clamp recordings were carried out using the tight-seal whole cell patch technique at room temperature in Tyrode solution containing 136 mM NaCI, 5.4 mM KCI, 1 mM MgC12, 1.8 mM CaC12, 0.33 mM NaH2PO4, 10 mM glucose, and 10 mM HEPES (adjusted to pH 7.4 with NaOH). The pipette solution contained 20 mM KCI, 110 mM Postassium aspartate, 1 mM MgC12, 10 mM HEPES, 5 mM EGTA, 0.1 mM GTP, and 5 mM Mg2+/ATP (adjusted to pH 7. 2 with KOH). Voltages were filtered at 2 kHz (-3 dB; four-pole, low-pass Bessel filter). The resting membrane potential upon breaking in was- 39.8 1. 6 mV (n=9), but generally improved by 10 mV or more. In some cases, cells were hyperpolarized slightly to action potential threshold.

A variety of cardiac APs are observed in beating and non-beating cells (Fig. 3A, 3B), which both show a resting membrane potential of approximately-60 mV with a robust overshoot of 50-90 mV. The form and duration of the APs match the descriptions of adult murine cardiomyocytes APs, which lack the plateau phase seen in cardiomyocytes of other species (Wang et al., Circ Res 79,79, 1996). The beating cells differ from skeletal muscle in that the addition of 0.5 mM CdCl2, a non-specific blocker of L-type Ca'and Na' channels, abolishes the action potential as expected (Piper et al. in Isolated Adult Cardiomyocytes 30-65 (CRC Press, Inc. , Boca Raton, FL), 1989; Sperelakis, N. in Physiology and Pathophysiology of the Heart 101-114 (Kluwer Academic Publishers, Boston, MA), 1995) while having no effect on skeletal myotubes (Fig. 3C, 3E) (Mould and Dulhunty Pflugers Arch 437,197, 1999; Garcia et al., J Membr Biol 168, 141,1999).

Likewise, both beating and non-beating cardiac cells have an intact adrenergic pathway (Shumaker et al., Am J Physiol 261, H1937-44,1991 ; Tanaka et al., Proc R Soc Lond B Biol Sci 263,241, 1996; Giles et al., J Physiol 415,233, 1989), as shown by the increase in AP frequency with the addition of 25 nM isoproterenol (Fig. 3D). The expected lack of effect (Reinecke and Murry Cardiovasc Pathol 9,337, 2000) is observed in the skeletal myotubes (Fig. 3F). Uninucleate myoblasts with spontaneous calcium transients and action potentials

have not been described under standard culture conditions (Cognard et al., Development 117,1153, 1993) and even when they are contrived by arresting cell fusion, they retain the electrical activity characteristic of skeletal muscle cells, such as the lack of response to CdCl2 (Constantin et al., Exp Cell Res 217,497, 1995).

EXAMPLE 9 Distinguishing Spoc Cells from Bone Marrow Cells Spoc cells are c-kit-, distinguishing them from the c-kit+ bone marrow cells that have been used directly or indirectly in experiments to reconstitute infarcted heart. Despite this, spoc cells could be derived from circulating bone marrow cells that become c-kit-after migration to skeletal muscle. In order to more fully evaluate this question, whole bone marrow was fractionated into c-kit+ and c-kit-populations. Both separate and combined populations were cultured under the same conditions as spoc cells. None of the 3 marrow cell populations developed into spontaneously beating cells.

To test whether marrow cells have the potential to differentiate into cardiomyocytes in the presence of soluble factors released from spoc cells, equal proportions of marrow and spoc cells were co-cultured in a Costar transwell system, in which the two chambers are separated by a 0. 4 um permeable membrane. Although the total number of cells increased in each compartment, the spoc cells alone differentiated into beating cells expressing cardiac markers.

In order to test if cell-cell contact between bone marrow and spoc cells would lead bone marrow cells to differentiate into cardiomyocytes, total bone marrow was mixed in equal proportion with EGFP-expressing spoc cells obtained from EGFP-expressing transgenic mice (ACTbEGFP, The Jackson Laboratory). In three separate experiments, under the same culture conditions, total cell number increased, but only EGFP-expressing cells developed into beating cells. The converse experiments showed a similar increase in cell number, but beating cells did not express EGFP. Taken together, these experiments show that bone marrow does not contain any cell population phenotypically similar to spoc cells isolated from skeletal muscle.

EXAMPLE 10 Examining the Ability of Spoc Cells to Reconstitute Bone Marrow Unlike the case with hematopoietic stem cells, no colony-forming units are generated when spoc cells are cultured in methylcellulose in the presence of erythropoietin, IL-3, IL-6, and SCF. To evaluate the ability of spoc cells to reconstitute bone marrow,

standard and competitive bone marrow transplantations with spoc cells were performed. In four mice, 3x106 bone marrow cells and 3x104 GFP + Sca-1-spoc cells were injected into each of the lethally irradiated mice. All four mice survived, however, only a rare GFP+ donor-derived cell was seen in the peripheral blood or bone marrow. The marrow of six lethally irradiated mice, injected with either 1. 5x105 Sca-1-spoc cells or 2x105 spoc cells unfractionated for Sca-1, could not be rescued, and all the mice died within two weeks.

EXAMPLE 11 Distinguishing Spoc Cells from Cells Derived from the Heart In order to determine if spoc cells can be isolated from heart, as well as skeletal muscle, 500 mg of heart or skeletal muscle from the same mouse were dissociated and used to isolate cells as described in Example 1, above. The tissue was taken from the mouse and cultured separately under the same conditions. Approximately 3 x 105 cells capable of developing into beating cells were observed by days 7-8 in the skeletal muscle-derived cells, while none were observed in the culture of heart-derived cells. Roughly 10% of the cells derived from skeletal muscle exhibited spontaneous beating. Thus, only the spoc cell preparation from skeletal muscle differentiated into beating cells expressing cardiac markers. Two replicate co-culture experiments of both cell populations in Costar transwell systems produced an increased number of cells in both chambers, but again, only the skeletal muscle-derived cells developed into beating cells expressing cardiac markers.

EXAMPLE 12 Distinguishing Spoc Cells from Mesenchymal Stem Cells To determine if spoc cells can be distinguished from mesenchymal stem cells (MSC), MSC were compared to spoc cells in culture. The MSC (Clonetics Corporation) were cultured in parallel with spoc cells generated from skeletal muscle as described in the methods above. The MSC adhered to the plate almost immediately upon plating, remained adherent throughout 12 days of observation, and did not show any sign of beating. In contrast, the cardiac progenitor cells from skeletal muscle were smaller in size, remained nonadherent while they developed into floating clusters of spoc cells, and they progressed to beating cardiac myocytes expressing cardiac markers. Spoc cells did not form in the MSC cultures. Thus, spoc cells are not MSC.

EXAMPLE 13 In Vivo Differentiation of Spoc Cells In order to determine if spoc cells engraft within a myocardial infarct and differentiate into mature cardiomyocytes, 1x105 EGFP+ spoc cells were injected into the peripheral circulation in acute myocardial infarct (MI) models. After 14 weeks, many donor-derived EGFP+ cells had engrafted. Of donor cells that had migrated to the infarct, 8% (63/782) had developed into cardiomyocytes.

To evaluate for similar effect in an older infarct, the same number of GATA-4- negative spoc cells was injected via tail vein into 2 mice (8 and 14 weeks status-post MI).

Two weeks following injection, the heart of the 8-week-old infarct model shows colocalization of EGFP and GATA4 in 3% (4/136) of donor cells that migrated to the peripheral region of the infarct. Five weeks following injection into the 14-week-old infarct model, an increased number of EGFP+/RLCP+ cells (7/102 donor-derived cells) in the infarct region are evident. Since spoc cells are not GATA-4 or RLCP positive these findings suggest that spoc cells either home to or are filtered out in an area of cardiac damage where they begin to differentiate into cardiomyocytes, as observed in vitro. Spoc cells that were partially differentiated by culturing for 7 days were injected into the hearts and tail veins of 3 mice with acute infarcts. No labeled cells were identified in the hearts at 7 days and later, compared to 2 control mice injected with saline at the time of infarct. This suggests that the undifferentiated cells more easily home to or are preferentially filtered out in the heart.

Further fractionation of spoc cells by sorting for the Sca-1 marker (surface antigen found on hematopoietic stem cells) demonstrates that the overwhelming majority of beating cells develop from the Sca-1 pool. Approximately 90% of the spoc cells are Sca-1+ cells that are rapidly adherent and do not develop into beating cells. With the Sca-1+ population removed, the remaining 10% Sca-1-population undergo 2-3 additional divisions as undifferentiated cells before beginning to differentiate. Some of these cells begin to beat while still floating. When a Sca-1-population that has reached the stage of CS cells is replated onto an adherent monolayer of Sca-1+ cells, the Sca-1 cells adhere to the monolayer within 24 hours, days before they would normally adhere. Within 3 days, they stretch out beyond the extent that they would otherwise and an increase in the proportion of beating cells is observed.

In order to determine if Sca-1~ spoc cells engraft within a myocardial infarct and differentiate into mature cardiomyocytes, anterior myocardial infarct (MI) models in C57B1/6J mice were created by ligation of the left coronary artery. When 1x105 Sca-1-

EGFP+ donor cells were injected via tail vein immediately following infarction, a low level of engraftment occurred, with only an occasional donor-derived cardiomyocyte found at the periphery of the infarct zone. No labeled cells, differentiated or undifferentiated, were observed in the normal portion of the infarcted heart. To overcome the low level of engraftment of the Sca-1-cells, a mixture of the Sca-1+ and Sca-1~ cells, in different proportions, is provided to the mouse model.

EXAMPLE 14 Method of Producing Transformed Cell Lines that Continue to Beat and Divide Transformed spoc cell lines that continue to beat and divide are generated. Spoc cells are transfected with plasmid expression vector harboring SV40 large T antigen or polyoma large T antigen with commercially available reagents. The cells are cultured in Teflon vessels and the growing population of cells is isolated and cloned. Single transformed cells are expanded to produce a clonal population. These cell lines are used to obtain enough cells to produce a"pumping patch"to repair a diseased and failing heart.

Transformed cells derived ex vivo from a subject are introduced into a subject either through the circulation or directly into the heart using a guided catheter. Transformed cells derived ex vivo from a subject are also used together with endothelial cell precursors, muscle fibroblasts and other support cells from a subject to produce a patch of vascularized beating cells that is placed in the heart as an assist device. The patch is synchronized to the rest of the heart either by capturing the intrinsic cardiac pacing current or by the use of a pacemaker implant that entrains the entire heart.

EXAMPLE 15 Method of Screening for Agents that Affect Cardiomyocyte Differentiation or Function A large variety of transformed cardiac cell lines are made from skeletal muscle of transgenic, knockout, or knockin mouse cell lines that were produced to study genes that are important to cardiac function and development. These cell lines are used to screen therapeutic agents, derived from combinatorial chemical libraries, in high throughput assays. In one embodiment, a clonal population of transformed cells can be treated with agents that affect calcium channels and other channels. Cells can be screened by various methods such as immunofluorescence or electrical activity to screen for agonists and antagonists that affect cardiomyocyte function. Examples of knockout mice that may be of interest include but are not limited to connexin-43 knockout and p53 knockout mice.

EXAMPLE 16 Distinguishing Spoc Cells from Satellite Cells Spoc cells and satellite cells are air-dried on glass slides for 30 minutes and then fixed in 4% paraformaldehyde at 4 °C followed by a rinse for 5 minutes with PBS. The cells are blocked with goat serum for 30 minutes and then incubated overnight, at 4 °C, with rabbit anti-met (1: 200, Santa Cruz Biotechnology). Following the overnight incubation, the slides are rinsed 3 times (5 minutes each) with PBS and blocked again with goat serum for 30 minutes. The cells are then incubated at room temperature with a secondary antibody, conjugated with Fluorescein Isothiocyanate (FITC) for 1 hour. They are again rinsed 3 times (5 minutes each) with PBS and then visualized with a laser confocal microscope (Leica) to detect fluorescent signals. Of the two cell types examined, only the satellite cells are positively stained with c-met indicating that satellite cells express c-met on their cell surface, whereas spoc cells do not.

EXAMPLE 17 Method of Isolating Cardiomyocyte Precursor Cells from Adult Human Skeletal Muscle Skeletal muscle tissue is surgically obtained from the deltoid muscle of an adult human, is cut into small pieces and is digested with collagenase for two hours at 37 °C. The digested tissue is cleared of cell debris and other undigested tissue fragments by passage through a 100 um filter and then through a 40 Zm filter. The cell suspension is centrifuged at low speed to clear as much as of the small muscle fiber fragments as possible. The cells at this stage consist mostly of clusters of small round cells approximately 4 m in diameter which are the human spoc cells. These cells do not express the satellite cell surface marker c-met.

The spoc cells are plated at a density of approximately 105 cells per cm2 in regular tissue culture dishes in complete growth medium (1: 1 DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 10 ng/ml human EGF, 10 ng/ml human bFGF (PeproTech, Inc.), 5 gg/n-d insulin, 5 llg/ml transferrin, 6 ng/ml selenium, 2 Zg/ml ethanolamine (ITS-X, Invitrogen Corporation), 25 gg/n-fl gentamicin and 2.5 llg/ml fungizone (Life <BR> <BR> Technologies) ). After a few days, the culture consists of a floating population of round cells and some adherent fibroblasts. The round cells enlarge as they undergo a few rounds of cell division during which time they become clusters of floating round cells with an increased

diameter of 10-14 m. The cells in these clusters were referred to as CS (cardiac precursors from spoc) cells.

In view of the many possible embodiments to which the principles of our disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as a limitation on the scope of the disclosure.

Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.