Title of Invention

A METHOD FOR MANUFACTURING A MEDICAMENT FOR TREATING NON-ISCHEMIC HEART DISEASE

Abstract

The present invention relates to the differentiation of cardiomyocytes from a human embryonic stem cell line, for example the ReliCell®hES1 cell line. The present invention in particular relates to the delivery of these cells via non-invasive routes like intravenous routes and its potential applications in cardiac disorders. The present invention has demonstrated that hESC-derived cardiomyocytes in combination with a cytokine can enhance the efficacy of cellular cardiomyoplasty in idiopathic dilated cardiomyopathy (IDCM). The present invention provides new avenues pertaining to the potential of tissue specific stem cells in therapeutic applications.

Full Text

FORM 2 THE PATENTS ACT, 1970 {39 of 1970) & THE PATENT RULES , 2003 PATENT OF ADDITION (See Section 54) "CARDIOMYOCYTE LIKE CELLS FROM HUMAN EMBRYONIC STEM CELLS" RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Chitrakoot, 2nd Floor, Shree Ram Mills Compound, Ganpath Rao Kadam Marg, Worli, Mumbai -400 013, Maharashtra, India. The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:- RELATED REFERENCE: The present application is a patent of addition to Indian Patent number 595/MUM/2005 filed on May 17, 2005. The present application further claims priority to U.S. Serial No. 11/436,193, filed on May 17, 2006, and PCT application No. PCT/IN2006/00169, filed on May 16, 2006. The contents of each of the parent applications are incorporated herein by reference. FIELD OF THE INVENTION: The present invention relates to the derivation of cardiac progenitors and their methods and applications in treatment of cardiac disorder or diseases. The present invention in particular relates to the use of cardiac progenitors derived from human ES cells in nonischemic cardiomyopathy and the delivery of the cells through intravenous route. BACKGROUND OF THE INVENTION Cardiac muscle is a type of involuntary mononucleated, or uninucleated, striated muscle found exclusively within the heart. Its function is to "pump" blood through the circulatory system by contracting. A single cardiac muscle cell, if left without input, will contract rhythmically at a steady rate; if two cardiac muscle cells are in contact, whichever one contracts first will stimulate the other to contract, and so on. This inherent contractile activity is heavily regulated by the autonomic nervous system. If synchronization of cardiac muscle contraction is disrupted for some reason (for example, in a heart attack), uncoordinated contraction known as fibrillation can result. This transmission of impulses makes cardiac muscle tissue similar to nerve tissue, although cardiac muscle cells are notably connected to each other by intercalated discs. Intercalated discs conduct electrochemical potentials directly between the cytoplasms of adjacent cells via gap junctions, in contrast to the chemical synapses used by neurons. The destruction of heart muscle cells, cardiomyocytes, can be the result of hypertension, chronic insufficiency in the blood supply to the heart muscle caused by coronary artery disease, or a heart attack, the sudden closing of a blood vessel supplying oxygen to the heart. Despite advances in surgical procedures, mechanical assistance devices, drug -2- therapy, and organ transplantation, more than half of patients with congestive heart failure die within five years of initial diagnosis. Research has shown that therapies such as clot-busting medications can reestablish blood flow to the damaged regions of the heart and limit the death of cardiomyocytes. Researchers are now exploring ways to save additional lives by using replacement cells for dead or impaired cells so that the weakened heart muscle can regain its pumping power. Over two million people die of coronary diseases in India annually. Currently, India is home to over 60 million coronary heart patients and more than two million patients are succumbing to the disease every year, According to a World Health Organization (WHO) estimate, India's economic loss due to heart related disease could be $236 billion till 2015. Over 17 million people died of cardiovascular diseases such as heart attack or stroke in 2005, WHO says. The WHO has warned that Indians, being genetically prone to cardiac disorders, are likely to constitute about 60 percent of the world's cardiac patients by 2010. Statistics show an alarming incidence of heart diseases among youngsters in India. Cases of heart diseases (per 1,000,000) increased from 145 males and 126 females in 1985 to 253 males and 204 females in 2000. It is calculated that the prevalence of coronary heart diseases among the urban population was more than three times compared to the rural populace. Nearly, 1,000,000 children were born every year with congenital heart diseases. Researchers are building their knowledge base about how stem cells, and particularly embryonic stem cells, are directed to become specialized cells. One important type of cell that can be differentiated from stem cells is the cardiomyocyte, the heart muscle cell that contracts to eject the blood out of the heart's main pumping chamber (the ventricle). Two other cell types that are important to a properly functioning heart are the vascular endothelial cell, which forms the inner lining of new blood vessels, and the smooth muscle cell, which forms the wall of blood vessels. The heart has a large demand for blood flow, and these specialized cells are important for developing a new network of arteries to bring nutrients and oxygen to the cardiomyocytes after a heart has been damaged. The potential capability of both embryonic and adult stem cells to develop into these cells types in the damaged heart is now being explored as part of a strategy to -3- restore heart function to people who have had heart attacks or have congestive heart failure. Researchers now know that under highly specific growth conditions in laboratory culture dishes, stem cells can be coaxed into developing as new cardiomyocytes and vascular endothelial cells. Scientists are interested in exploiting this ability to provide replacement tissue for the damaged heart. This approach has immense advantages over heart transplant, particularly in light of the paucity of donor hearts available to meet current transplantation needs. Recently, Orlic and colleagues [(2001). Bone marrow cells regenerate infarcted myocardium. Nature. 410, 701-705] reported on an experimental application of hematopoietic stem cells for the regeneration of the tissues in the heart. When injected into the damaged wall of the ventricle, these cells led to the formation of new cardiomyocytes, vascular endothelium, and smooth muscle cells, thus generating de novo myocardium, including coronary arteries, arterioles, and capillaries. The newly formed myocardium occupied 68 percent of the damaged portion of the ventricle nine days after the bone marrow cells were transplanted, in effect replacing the dead myocardium with living, functioning tissue. The researchers found that mice that received the transplanted cells survived in greater numbers than mice with heart attacks that did not receive the mouse stem cells. Follow-up experiments are now being conducted to extend the posttransplantation analysis time to determine the longer-range effects of such therapy. The partial repair of the damaged heart muscle suggests that the transplanted mouse hematopoietic stem cells responded to signals in the environment near the injured myocardium. The cells migrated to the damaged region of the ventricle, where they multiplied and became "specialized" cells that appeared to be cardiomyocytes. A second study, by Jackson et al. ( Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J. Clin. Invest. 2001 107, 1-8), demonstrated that cardiac tissue can be regenerated in the mouse heart attack model through the introduction of adult stem cells from mouse bone marrow. In this model, investigators purified a "side population" of hematopoietic stem cells from a genetically altered mouse -4- strain. These cells were then transplanted into the marrow of lethally irradiated mice approximately 10 weeks before the recipient mice were subjected to heart attack via the tying off of a different major heart blood vessel, the left anterior descending (LAD) coronary artery. At two to four weeks after the induced cardiac injury, the survival rate was 26 percent. As with the study by Orlic et al, analysis of the region surrounding the damaged tissue in surviving mice showed the presence of donor-derived cardiomyocytes and endothelial cells. Thus, the mouse hematopoietic stem cells transplanted into the bone marrow had responded to signals in the injured heart, migrated to the border region of the damaged area, and differentiated into several types of tissue needed for cardiac repair. This study suggests that mouse hematopoietic stem cells may be delivered to the heart through bone marrow transplantation as well as through direct injection into the cardiac tissue, thus providing another possible therapeutic strategy for regenerating injured cardiac tissue. More evidence for potential stem cell-based therapies for heart disease is provided by a study that showed that human adult stem cells taken from the bone marrow are capable of giving rise to vascular endothelial cells when transplanted into rats (Kocher, A.A., Schuster, M.D., Szabolcs, M.J., Takuma, S., Burkhoff, D., Wang, J., Homma, S., Edwards, N.M.,.and Itescu, S. (2001). Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7, 430-436). As in the Jackson study, these researchers induced a heart attack by tying off the LAD coronary artery. They took great care to identify a population of human hematopoietic stem cells that give rise to new blood vessels. These stem cells demonstrate plasticity meaning that they become cell types that they would not normally be. The cells were used to form new blood vessels in the damaged area of the rats' hearts and to encourage proliferation of preexisting vasculature following the experimental heart attack. Like the mouse stem cells, these human hematopoietic stem cells can be induced under the appropriate culture conditions to differentiate into numerous tissue types, including cardiac muscle [Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., -5- Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143-147]. When injected into the bloodstream leading to the damaged rat heart, these cells prevented the death of hypertrophied or thickened but otherwise viable myocardial cells and reduced progressive formation of collagen fibers and scars. But this evidence is not complete; the mouse hematopoietic stem cell populations that give rise to these replacement cells are not homogenous. Rather, they are enriched for the cells of interest through specific and selective stimulating factors that promote cell growth. Thus, the originating cell population for these injected cells has not been identified, and the possibility exists for inclusion of other cell populations that could cause the recipient to reject the transplanted cells. This is a major issue to contend with in clinical applications, but it is not as relevant in the experimental models described here because the rodents have been bred to be genetically similar. . There are some practical aspects of producing a sufficient number of cells for clinical application. The repair of one damaged human heart would likely require millions of cells. The unique capacity for embryonic stem cells to replicate in culture may give them an advantage over adult stem cells by providing large numbers of replacement cells in tissue culture for transplantation purposes. Given the current state of the science, it is unclear how adult stem cells could be used to generate sufficient heart muscle outside the body to meet patients' demand. Exciting new advances in cardiomyocyte regeneration are being made in human embryonic stem cell research. Because of their ability to differentiate into any cell type in the adult body, embryonic stem cells are another possible source population for cardiac-repair cells. Itskovitz-Eldor et al. [Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, 0., Amit, M., Soreq, H., and Benvenisty, N. (2000). Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol. Med. 6, 88-95] demonstrated that human embryonic stem cells can reproducibly differentiate in culture into embryoid bodies made up of cell types from the -6- body's three embryonic germ layers. Among the various cell types noted were cells that had the physical appearance of cardiomyocytes, showed cellular markers consistent with heart cells, and demonstrated contractile activity similar to cardiomyocytes when observed under the microscope. In a continuation of this early work, Kehat et al. [Kehat, I., Kenyagin-Karsenti, D., Druckmann, M, Segev, H., Amit, M., Gepstein, A., Livne, E., Binah, 0., Itskovitz-Eldor, J., and Gepstein, L. (2001). Human embryonic stem cells can differentiate into myocytes portraying cardiomyocytic structural and functional properties. J. Clin. Invest. 108, 407-414] displayed structural and functional properties of early stage cardiomyocytes in the cells that develop from the embryoid bodies. The cells that have spontaneously contracting activity are positively identified by using markers with antibodies to myosin -. heavy chain, alpha-actinin, desmin, antinaturietic protein, and cardiac troponin—all proteins found in heart tissue. These investigators have done genetic analysis of these cells and found that the transcription-factor genes expressed are consistent with early stage cardiomyocytes. Electrical recordings from these cells, changes in calcium-ion movement within the cells, and contractile responsiveness to catecholamine hormone stimulation by the cells were similar to the recordings, changes, and responsiveness seen in early cardiomyocytes observed during mammalian development. A next step in this research is to see whether the experimental evidence of improvement in outcome from heart attack in rodents can be reproduced using embryonic stem cells. Recently, Cho et al., BBRC 340:573-582 (2006) reported that GCSF administration can enhance the efficacy of cellular cardiomyoplasty with ES-cell-derived cardiomyocytes in rat models of infarcted myocardium. Thus, GCSF may promote neovascularization in ischemic myocardial tissues. Although there is much excitement because researchers now know that adult and embryonic stem cells can repair damaged heart tissue, many questions remain to be answered before clinical applications can be made. -7- Medical researchers believe that stem cell treatments have the potential to change the face of human disease and alleviate suffering. A number of current stem cell treatments already exist, although they are not commonly used because they tend to be experimental and not very cost-effective. Furthermore, many technical difficulties remain which hinder the ultimate goals in stem cell therapeutics. Expanding stem cell populations extracted from patients remains a large problem. Also, even once these populations are expanded, implanted stem cells may not expand or grow efficiently enough to add enough corrective factor to be beneficial for treatment. These and other technical problems remain to be solved. Using the patient's own bone marrow derived stem cells or more recently, peripheral blood-derived stem cells, has shown a dramatic increase in ejection fraction for patients with congestive heart failure. US Patent Number 6671558 is directed to a method of preventing heart failure after a myocardial infarction through the administration of stem cells, together with a device that induces electrical signals wherein the stem cells are transfected with genetic materia! to modify functions (i.e., increased contractility: transfect with SERCA-2, increase angiogenesis: transfect with FGF, etc). The use of genetically modified stem cells is not always compatible for application in human beings, since vectors and constructs commonly used for genetic modification are largely derived from bacteria or viruses. US Patent Number 6805860 relates to the temporary occlusion of a vessel for delivery of stem cells to the site of the tissue to be repaired. US patent Number: 6775574 relates to the implantation of a stem cell source, or genes associated with stem cells and regeneration, together with a pace-maker type device, into a patient whose heart is in need of regeneration. The implantation technique requires a special device for the delivery of the stem cells, which is similar to organ transplant assisted by replacement of cells. -8- US Patent Number: 6690970 is directed to the use of stem cells and products of stem cells as "biological pacemakers" so as to ensure that the electrical pulses of the heart are appropriately coordinated. US patent 6878371 relates to a disclosure of bone marrow autologous stem cells which stimulate angiogenesis in to the heart of patients. Although this is an autologous approach and also shows enhanced angiogenesis, it does not necessarily cause the regeneration of heart muscle cells. US Patent 7097833 relates to the regeneration of cardiac tissue with the use of a variety of various peripheral blood and bone marrow stem cell sources for implantation into diseased, damaged or subfiinctional cardiac tissue. US Patent 7,091,310 relates to stem cell mobilization, stem cell homing, as well as a variety of other applications involving various chemokines. This is a case of facilitating recruitment of endogenous stem cells taking advantage of the inflammatory response a heart injury evokes. US Patent 6991787 relates to using gene-transfected bone marrow cells as a way of delivering the gene of interest to a tissue that needs it. US Patent 6737054 is directed to the administration of stem cells, or cardiomyocytes, into areas of the heart that have been injured, including by infarction by grafting of the cells. US Patent 6156733 relates to how to treat heart failure by administration of antagonists to endothelin and LIF. US Patent 6387369 is directed to the use of mesenchymal stem cells for cardiac repair, specifically after myocardial infarction. Mesenchymal stem cells are currently in clinical trials for this application. -9- US Patent 6551338 is directed to ways of entering the myocardium through transthoracic means in order to administer angiogenic stimuli, which may include stem cells, or activators of stem cells that are associated with angiogenesis. The patent is also directed to various administration devices. This patent focuses on different modes of activating mechanisms that may auger localized heart repair. US Patent 6534052 relates to how to administer embryonic stem cells, and differentiated cells thereof, into the area of infarcted myocardium so as to prevent future heart failure. The method is a surgical technique, which introduces and implants mammalian embryonic stem cells into the infarcted area of the myocardium. US Patent 6652583 relates to how to use stem cells and other differentiated cells in order to generate a heart valve that can be used for replacement of defective valves in patients suffering from various valvular abnormalities. The advantage of this biological valve is that it conceptually is longer lasting than completely artificial or xenogeneic valves. US Patent 6435190 relates to what appears to be a surgical method for producing an autograft that can cover lesions in the heart. This is important since after a heart attack, there is scar tissue formation, and the heart starts to lose function. The patent also is directed to ways in which autologous stem cells may be administered. Despite glorious advances in disease management, myocardial infarction and/or heart failure remains a leading cause of morbidity and mortality worldwide. Owing to the limited capacity of native cardiomyocytes, the myocardium is particularly vulnerable to irreversible injury and poor outcome. Current efforts to reduce the extent of heart damage rely on the available palliative therapeutic modalities that do not adequately repair cardiac scarring and are unable to reverse progressive organ failure after myocardial infarction. This lack of curative therapy warrants the establishment of approaches capable of effectively regenerating at least part of the dysfunctional myocardium, and securing fresh heart muscle necessary for salvage of organ function. -10- Recently, cell transplantation has evolved as a promising therapy for end-stage heart failure and has been under rigorous experimentation especially in ischemic hearts, more specifically with hESC-derived functional cardiomyocytes. As described above, until now there has been remarkable progress in myocardial cell replacement therapy using skeletal myoblasts, neonatal/fetal cardiomyocytes and adult stem cells. However, neither skeletal myoblasts nor bone marrow-derived cells including bone-marrow mulitpotent stem cells can effectively differentiate into cardiac cells. The phenotypic changes often arise as a result of cell fusion instead of integration. At present, there is a great need for alternative treatments of non-ischemic heart diseases such as idiopathic dilated cardiomyopathy (IDCM). IDCM is a syndrome characterized by cardiac enlargement and congestive heart failure. Although no etiology is definable in most cases, IDCM is believed to represent the result of myocardial damage caused by toxic, metabolic or infectious agents. Genetic factors also appear to play a role in IDCM. Diagnosis of this disease generally depends on the exclusion of other possible causes at a late stage of the disorder. Notably, IDCM accounts for approximately one-third of the heart failure cases. Approximately half the patients receiving heart transplants suffer from non-ischemic heart disease such as IDCM. Further, most of the studies delivered donor cells through intramyocardial injection after cardiac surgery. The advantage of this approach is that it traps the implanted cells in selected injured areas of the heart. However, the procedure of intramyocardial injection is invasive and therefore might not be suitable for patients with acute MI or severe congestive heart failure. Hence looking into the requirement of an effective administration of cells that would result in high efficacy and reproducible effects, the present invention has developed a method for delivery of cardiac cells derived from human embryonic stem cells through cell transplantation via the intravenous route. Unlike ischemic forms, which are amenable to procedures like neovascularisation and remodeling operations, non-ischemic cardiomyopathies, once they have reached an end -11- stage of drug refractoriness, can be treated radically only by heart transplantation. The clinical relevance of this condition can be drawn from the high incidence of anthracyclin-induced heart failure in patients receiving chemotherapy for malignant blood diseases. The present invention has developed the intravenous mode of delivery of cells and this method of delivery has been successful in a sub-acute model of non-ischemic dilated cardiomyopathy. Further, in order to improve the efficiency of tissue regeneration, the present invention employs angiogenic cytokines, such as, for example, granulocyte stimulating factor (GCSF), which help mobilize endothelial progenitor cells from bone marrow into blood circulation. OBJECT OF THE INVENTION The present invention aims to provide cardiomyocytes derived from a human embryonic stem cell line with certain genetic characteristics that will allow the derived cardiomyocytes to be more effectively utilized to the advantage of the Indian population, for example the human embryonic stem cell line, ReliCelftiESl cell line. The present invention aims to provide methods of deriving cardiac progenitors and differentiation of those progenitors from a human embryonic stem cell line. The present invention aims to provide compositions for the therapeutic delivery of cardiomyocytes derived from human embryonic stem cells. The present inventions aims to provide a method of delivering hES-derived-cardiomyocytes to a patient in need thereof via the intravenous route. The present invention aims to provide the method of providing hES-derived-■ cardiomyocytes for treatment of cardiac disorders, conditions and diseases. -12- The present invention aims to provide hES-derived-cardiomyocytes to enhance the efficacy of cellular cardiomyoplasty in idiopathic dilated cardiomyopathy (IDCM). The present inventions aim to provide the co-administration of an angiogenic cytokine with the hES-derived-cardiomyocytes for mobilization of resident stem cells and/or hES-derived-cardiomyocytes towards the damaged regions of the patient's heart. The present invention aims to provide the cardiomyocytes in combination with an angiogenic cytokine,thereby achieving mobilization of the resident stem cells towards the damaged regions of the heart and/or facilitating repair of the damaged heart. SUMMARY OF THE INVENTION The present disclosure provides cardiomyocytes derived from human embryonic stem cells, as well as methods of deriving such cells. The present disclosure also provides the compositions and delivery of such compositions for treatment of idiopathic dilated cardiomyopathy (IDCM), and has demonstrated the therapeutic potential of treating such cardiomyopathies. In one embodiment, the present invention provides methods of derivation of cardiomyocytes from human embryonic stem cell line, for example a human embryonic stem cell line with certain genetic characteristics that will allow the cardiomyocytes derived therefrom to be more effectively utilized to the advantage of the Indian population. In the preferred embodiment the embryonic stem cell line used is Relicell and an exemplary protocol for the derivation of cardiac progenitors from the ReliCell cell line is described in the parent application number -595/MUM/2005 filed on May 17, 2005. The present invention provides methods of differentiation of the cardiac progenitors from a human embryonic stem cell line and its applications in treatment of idiopathic dilated cardiomyopathy. The ability to direct differentiation of embryonic stem cells (ESC) towards a cardiomyogenic phenotype makes them a viable therapeutic option for cardiac repair, but species-specific and individual-specific immunological imprinting remains as -13- a potential hurdle. The present invention has evaluated whether intravenously infused cardiac-committed cells derived from human embryonic stem (hES) cells could facilitate regeneration of neo-myocardium in mice models of doxorubicin-induced cardiomyopathy following G-CSF administration. GCSF appears to help the mobilization of resident stem cells towards the damaged regions of the heart and thereby facilitates heart repair. Further, GCSF also appears to guide the migration of adult stem cells towards the site of injury in response to certain chemokines secreted due to inflammation post tissue damage. The aim of the Examples of the present application is to evaluate the preclinical efficiency of the cardiomyocytes derived from human embryonic stem cells in mice as models of idiopathic dilated cardiomyopathy. In one preferred embodiment, each SCID mice (Mus Musculus) received a total dosage of 9mg of doxorubicin over a period of 2 weeks followed by a regime of G-CSF for 5 days and 1.5-2 million cardiomyocyte cells derived from human embryonic stem cells transplanted via the tail vein. Three to six weeks after treatment, survival, engraftment of implanted cells, cardiac tissue repair, gap junction formation between host and graft tissues and apoptotic activity in damaged regions were evaluated by histopathological studies, immunohistochemistry and RT-PCR. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. Figure 1: Characterization of hESC-derived-cardiomyocytes. (A) Embryoid bodies at day 6 in suspension culture; Myotube-like cardiac structures were observed in differentiating EBs and stained positively for (B) cardiac troponin I (cTnl); (C) a-actinin and (D) p-MHC. The scale bars indicate 10 um. RT-PCR analysis for cardiomyocytes -14- expressed cardiac-specific cytoskeletal proteins (ANP, ct-MHC, MLC2V, cTnl) and a transcription factor (Nkx2.5). Figure 2: Detailed experimental / study plan elucidating the onset and regime of Doxorubicin treatment (9mg/animal) followed by 5 days of G-CSF administration (50ug/kg/day). Each animal that was not treated with doxorubicin treatment in the control group (CN), as well as each animal treated with doxorubicin in the transplant group (TX), received 1.5-2X106 cells (70ul) via tail vein, whereas the same volume of normal saline was injected into the doxorubicin-treated animals belonging to the sham control (SH) group through the same intravenous route. Hearts were harvested at day 7, 14 and 21, post transplantation. Figure 3: (A) Graphical representation of the effect of doxorubicin treatment on heart weight in different experimental groups. Quantification of damaged area in the injured hearts 3 weeks after hESC-derived-cardiomyocyte transplantation with G-CSF treatment. H&E staining of heart tissue showing transverse sections in (A) CN; (B) SH and (C) TX groups; morphological changes in left ventricular wall in (D) CN; (E) SH and (F) TX groups. Masson's trichrome staining showing different levels of collagen tissue formation in (G) CN; (H) SH and (I) TX groups. The scale bars indicate 10 urn. Figure 4: Assessment of graft survival and evaluation of the fate of transplanted cells. Immunofluorescent staining for Human-nuclei (FITC labeled) and cTnl (Texas-red labeled) of the damaged areas of the injured hearts 3 weeks after hESC-derived-cardiomyocyte transplantation in (A-C) TX; (D-F) CN and (G-I) SH groups. The scale bars indicate 50 um. All photographs were taken with the same microscopic adjustments. (J) Identification of mRNA transcripts of human specific cardiac markers (Nkx2.5, cTnl and ANP) in mice hearts indicate integration and further differentiation of implanted human cardiac committed cells in vivo. Increased expression of VEGF confirms enhancement in formation of vasculature in the host heart post transplantation; (H) denotes native heart; p-actin and GAPDH were used as internal controls. -15- Figure 5: Determination of gap junction formation between transplanted and host cardiomyocytes. Immunofluorescent staining for DAPI (nuclear stain) and Cx-43 in the heart sections show positive green fluorescence in the myocardium of (A-B) TX group in comparison to (C-D) CN and (E-F) SH groups. Human LFLC- derived cardiac cells show a substantially high proliferative capacity. A 3-week old cardiac implant is immunostained for a human-specific antibody against the proliferative marker Ki67 (red intranuclear staining) in (G) TX; (H) CN and (I) SH groups. The scale bars indicate 50 um. Figure 6: The apoptotic activity of the injured myocardium 3 weeks after cell transplantation. Apoptotic-positive nuclei (black) in the border regions of the damaged hearts were stained with DAB by the TUNEL staining method in (D) TX; (E) CN and (F) SH groups. DETAILED DESCRIPTION OF THE INVENTION Definitions: The term "embryonic stem cell" as used herein refers to any mammalian embryonic stem cell and in particular the human embryonic stem cell, which has the ability to proliferate and form cells of more than one different phenotype and is capable of self- renewal under defined culture conditions. The term "embryoid bodies" or "Ebs" as used herein refers to the aggregate bodies of differentiated or undifferentiated cells that appear when the embryonic stem cells are maintained in suspension culture. The term "cardiomyocytes" as used herein refers to cardiac muscle cells which are characterized by the phenotype marker used for immunostainirig. The term "interleukin" as used herein refers to any of a class of proteins that are secreted mostly by macrophages and T lymphocytes and induce growth and differentiation of lymphocytes and hematopoietic stem cells, including but not limited to interleukin 1 (IL- -16- 1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 14 (IL-14), and interleukin 15 (IL-15), as well as all alpha, beta and gamma forms of the interleukins and interleukins include up to IL-30 The term "GCSF" as used herein refers to granulocyte colony stimulating factor, which may be obtained as a commercially available product, for example Neupogen from Roche, Germany. The term "SCF" as used herein refers to stem cell factor, which may be obtained as a commercially available product. The term "VEGF" as used herein refers to vascular endothelial cell growth factor, which may be obtained as a commercially available product. The term "interferon" as used herein refers to any of a class of proteins that are produced by the cells of the immune system in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells, including but not limited to interferon alpha (e.g., IFN-a-la, IFN-a-lb, IFN-a-2a, IFN-a-2b), interferon beta (e.g., IFN-p-la, IFN-P-lb, IFN-p-2a, IFN-p-2b), and interferon gamma (e.g., IFN-y-la, IFN-y-lb, IFN-y-2a, and IFN-y-2b). The term "TNF" as used herein refers to tumor necrosis factor, including but not limited to TNF-a and TNF-p, which may be obtained as a commercially available product. The present invention has demonstrated the methods of derivation and differentiation of cardiac progenitors from human ES cell, and the application of such cells in the treatment of a non-ischemic cardiomyopathic mouse model via the non-invasive intravenous route. -17- Processes for deriving cardiac progenitors from the human embryonic stem cell line Relicell have been described in the parent application number 595/MUM/2005, filed on May 17, 2005, which is incorporated herein in its entirety, as well as U.S. Serial No. 11/436,193, filed on May 17, 2006, and PCT application No. PCT/IN2006/00169, filed on May 16, 2006, also incorporated herein in their entirety. One aspect of the present invention is a modification or further improvement in that the cardiac progenitors were differentiated into cardiomyocytes. The present invention has provided the characteristics of the cardiac cells in terms of morphological, phenotypic and functional analysis. The present invention also has provided its application in treatment of cardiac conditions not limited to idiopathic myopathy has been demonstrated in the rat model. The present invention also aims to provide the cardiomyocytes derived from human embryonic stem cells in combination with a cytokine, such as an angiogenic cytokine. Examples of cytokines that may be combined for therapeutic use of hES-derived-cardiomyocytes include but are not limited to an interleukin, GCSF, SCF, VEGF, interferon, TNF-a, and TNF-p. These cytokines, such as GCSF, may provide for the mobilization of resident stem cells towards the damaged regions of the heart and thereby facilitate heart repair. Further, GCSF also guides the migration of adult stem cells towards the site of injury in response to certain chemokines secreted due to inflammation post tissue damage. TNF- a, which is upregulated in response to cardiac damage and dysfunction, has been shown to enhance the migration and survival of ES cells in vitro (Chen et al., (2003) FASEB J 17(15):2231-2239). Cardiomyocytes derived from human embryonic stem cells and transplanted intravenously into the non-ischemic cardiomyopathy mouse model, wherein the mice were also treated with GCSF, were found to survive, integrate, and differentiate into cardiomyocytes and participate in improvement / regeneration of the injured myocardium. The combination therapy also promoted angiogenesis and attenuated scar tissue formation and cell apoptosis. -18- Hence, hESC-derived cardiomyocytes in combination with a cytokine such as G-CSF can enhance the efficacy of cellular cardiomyoplasty in idiopathic dilated cardiomyopathy (IDCM). These results may open up new avenues pertaining to the potential of tissue specific stem cells in therapeutic applications. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1: DERIVATION OF CARDIOMYOCYTES FROM HUMAN EMBRYONIC STEM CELL LINE The age of the EBs for differentiation induction into different phenotypes belonging to separate germ layers was decided on the basis of the expression profile of the lineage specific markers in the EBs (Mandal et al., 2006; Differentiation 74: 81-90). 3-6 days old EBs were seeded onto 35 mm tissue culture dishes (Nunc, Germany) pre-coated with 0.1% gelatin (Sigma, USA) in DMEM (Gibco) media supplemented with 1% nonessential amino acid, 1 mM glutamine, 0.1% beta-mercaptoethanol and 25 ng/ml recombinant human bone morphogenetic protein, BMP-2 (R&D systems). Rhythmic beating of EBs appearing on 14-16th day of differentiation culture, indicative of cardiac muscle differentiation, was carefully monitored by daily observation of cultures under a phase contrast microscope for more than 30 days. To determine whether a functional cell lineage could be produced from the EBs generated, we scored the appearance of spontaneously beating outgrowths, which are -19- indicative of cardiomyocyte differentiation. 3-6 days old ReliCell hESl-EBs were cultured in DMEM media with a low concentration of serum (5% FBS) supplemented with 25 ng/ul BMP-2 to promote further differentiation. It is reasoned that the microenvironment provided by high concentration of FBS (20%) could be supplemented with exogenous application of BMP-2, keeping the FBS content to as low as 5%. Beating was first detected on day 15 in ReliCell®hESl-EBs. Beating EBs from both lines could still be detected on day 30, the longest time assessed. Total number of beating EBs consistently decreased during extended culture mostly due to degradation. EXAMPLE 2: Characterization of ReliCell®hESl-derived cardiomyocytes: Characterization of the differentiated cells was carried out at cellular and molecular level by immunfluorescence analysis and RT-PCR analysis. Cardiac muscle specific markers including a-actinin, cardiac troponin I (cTnl) and (3-MHC were tested by immunostaining and a group of genes such as hNkx2.5, GATA-4, hANP, hcTnl and hcc-MHC were evaluated, which are all associated with heart formation and maturation. Cardiomyocytes generated from hESC express markers specific to heart muscle including hNkx2.5, hMEF2, hGATA-4, hANP, hcTnl, ha-MHC and a-actinin, cTnl, and ANP, respectively (Fig. 1). Further, in the line of cardiac specification of cells, the expression of Oct-4- a transcription factor of undifferentiated cells- dropped significantly, indicating commitment of cells towards a cardiac lineage. Percentage of differentiated cells: The "cardiac bodies" were mechanically selected using a glass pulled pasteur pipette. The EBs with cardiac outgrowths were identified under a bright field microscope. Further, characterization of the selected cardiomyocytes s carried out using tissue specific molecular and protein markers which confirms the identity of these cells. Further methods were also followed wherein the differentiated cultures are treated initially with trypsin (0.05%) or collagenase IV for 3-5 mins. Once the EB outgrowths start loosening off from the substratum, it becomes easier to mechanically pick-up the colonies. A flow cytometric was performed for analysis of the isolated cardiomyocyte-like cells using -20- markers specific for cardiac progenitors to assess the percentage of cells actually committed to the cardiac lineage. It was also ensured that the selected population of cardiac cells does not contain undifferentiated stem cells by showing the absence of stem cell marker expression by RT-PCR. However, this method does not preclude the presence of a small number of other mesodermal lineage cells such as smooth muscle cells, skeletal myocytes in the population. Functional assessment of the cardiomyocytes Assessment of beat rate (beats/min) of single beating ReliCell®hESl-EBs at rest or stimulated with 10"6 M isoprenaline was measured at 37°C in differentiation medium, which contains 1.8 mM Ca2+ and 5.2 mM K+. In this way this study could roughly examine the percentage of functionally active cardiomyocytes derived from hESC. By day 28, a cumulative total of 8-25 out of 72 (ReliCell®hESl) beating EBs were recorded. The cumulative total represents -9-45% (ReIiCell®hESl) relative to total EBs scored on day 16. This is a conservative calculation because total number of EBs consistently decreased during extended culture mostly due to degradation. The beating rate of ReliCell®hESl-EBs derived from cultures on feeders was scored to be 24.2 beats/min (approx). The present invention has compared the results with the recent paper by Chris Denning in 2006 (Intl J Dev Biol. 50:27-37). In that paper, the author exhibits the potential of BG01 and HUES7 cell lines to differentiate into beating cardiomyocytes under common culture conditions. The percentage of cardiomyocytes obtained in either case ranges between 15-20%, which was shown to increase upon the addition of a beta-adrenergic receptor called isoprenaline. The authors also demonstrated that the density of feeders at which the undifferentiated hESC are grown and the composition of the culture medium at a micro-level contribute significantly towards the yield of the cardiac lineage. EXAMPLE 3: PRECLINICAL EVALUATION: A) Animal preparation: SCID mice (25-30gm) purchased from Charles River Laboratories (MA, USA), were used which were maintained in Laboratory animal -21- research services facility. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the animal experiments were undertaken with prior approval from Institutional Animal Ethics Committee (IAEC). B) Generation of doxorubicin-induced cardiomyopathy: The heart failure was induced with doxorubicin as described earlier. Briefly, doxorubicin-hydrochloride (Sigma) was administered in 6 equal injections (each containing 1.5mg/kg in 0.5ml saline) intraperitoneally to 12 mice during a 2-week period resulting to a total dose of 9mg/kg. C) COMPOSITIONS: The cardiac progenitor cells were harvested from live cultures by trypsinization (0.05%). At the dose mentioned earlier, a single cell suspension was then injected in the mice either with normal DMEM medium (Gibco) or sterile IX PBS (Gibco). After preparing cells for each batch, the viability of the cells in DMEM/PBS was observed to be high (92-96%) as detected by trypan blue staining. D) DELIVERY Cells were injected via the tail vein (intravenous) into the mice with a 27-gauge tuberculin syringe. The mortality rate for both the groups (SH and TX) was 16.7%. The doxorubicin treated animals (n=12) were randomly divided into 2 groups: TX (n=6) and SH (n=6). There was an additional group (n=6) without doxorubicin injection (CN). In transplant group (TX) and the control group (CN), cardiac progenitor cells derived from hESC (1.5-15X106/70-100ul) were injected via the tail vein into 12 (6+6) mice with a 27-gauge tuberculin syringe, whereas the sham control group did not receive any stem cell injection. In the sham control group (SH), 70ul PBS was injected through the same route. E) Cell transplantation and G-CSF administration: The doxorubicin treated animals (n=12) were randomly divided into 2 groups: TX (n=6) and SH (n=6) (Table I). There -22- was an additional group (n=6) without doxorubicin injection (CN). After preparing cells for each batch, the viability of the cells was observed to be high (92-96%) as detected by trypan blue staining. In transplant group (TX) and the control group (CN), cardiac progenitor cells derived from hESC (1.5-2X106/70ul) was injected via the tail vein into 12 (6+6) mice with a 27-gauge tuberculin syringe where as the sham control group did not receive any stem cell injection. In the sham control group (SH), we injected 70ul PBS through the same route. Table I: Detailed study plan Description of the group (n=6) Doxorubicin treatment G-CSF administration hESC transplantation Group I (SH) Yes■ Saline Saline Group H (CN) No Yes Yes Group m(TX) Yes Yes Yes G-CSF (Neupogen, Roche) was injected subcutaneously for 5 days as follows: In Group I, G-CSF (50ug/kg/day) was administered from the day of termination of doxorubicin injection and in Group III, G-CSF (50ug/kg/day) was administered, starting from the same day as in Group I. Group II (sham control) received 0.1% saline in place of G-CSF injection. (Figure 2) EXAMPLE 4: POST TRANSPLANTATION EVALUATION No mice died in the control Group II (CN). During the 2-week period of the doxorubicin treatment regime, 1 animal from Group I (sham control) died and 1 animal died during the transplantation of cells in Group III (TX). So, the mortality rate for both the groups (SH and TX) was 16.7%. However, the mortality rate was found to be much higher when the animals were challenged with a total dose of 15mg/kg and hence we resorted to 9mg/kg. -23- At 4 weeks post transplantation, along with the examination of physical parameters like body weight and heart weight, histological and immunohistochemical analysis were used to evaluate the graft success in the TX (n=5)s SH (n=5) and CN (n=6) groups (Fig. 2). Body weight after doxorubicin treatment gradually decreased or stabilized. In no group did body weight change significantly from just before to 2 weeks post transplantation. The harvested hearts from the TX and CN group were slightly heavier than those from the SH group. The CN group did not differ significantly from the TX group. Histopathological analysis: At the indicated post-implantation time points, mice were sacrificed and hearts were immediately harvested and processed for histology and immunohistochemical staining. Briefly, the heart specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated with a graded ethanol series, embedded in paraffin, and cut into 6-um thick sections, which were stained with hematoxylin and eosin (H&E). Sections were also investigated for tumor formation. In H&E stained heart sections of SH group, left ventricular (LV) free wall with evident fibrosis and ventricular dilatation, rupture of blood vessels, nuclear degeneration was detected (Fig. 3). The TX group receiving cell therapy showed less necrosis. There was no tumor formation in animals with hES-derived-cardiomyocyte cell injections. Masson's Trichrome staining: 'Trichrome' stains are used primarily for distinguishing collagen from muscle tissue. In general, they consist of nuclear, collagenous and cytoplasmic dyes in mordants such as phosphotungstic or phosphomolybdic acid. The Accustain® Trichrome Stains (Masson; Sigma-Aldrich, St. Louis) were used according to the instruction manual (Protocol No HT15). Briefly, tissue sections are treated with Bouin's solution to intensify the final coloration, cytoplasm and muscle are then stained with Beibrich scarlet-acid fiichsin. After treatment with phosphotungstic and phosphomolybdic acid, collagen is demonstrated by staining with aniline blue. Rinsing in acetic acid after staining renders the shades of color more delicate and transparent. -24- Collagen staining by Masson's trichrome method showed that a larger area of viable tissues and a smaller area of fibrous tissues were present in the combined therapy (TX) than in controls (Fig. 3) Immunocytochemical staining: For immunohistochemical analysis, 5-6 \im thick slices of heart were washed with water, and the sections incubated with primary antibodies at 37°C for 1 hour. After incubation with first antibody, the section was washed with PBS and was incubated in suitable secondary antibody for 30 minutes at room temperature, rinsed and embedded. For double labeling, the treatment with second antibody and its conjugate follows the first one. DAPI was also used as a counter stain for the nuclei. The samples were evaluated and photographed with a Nikon E600 inverted microscope. The percentage of positively stained cells was estimated by fluorescent microscopy and calculated in 4 randomly selected fields for each section from 3 groups. In order to identify the intravenously infused hESC-derived cardiac committed cells, the heart sections were immunostained for mouse anti-human nuclei (Chemicon). Positively stained (red) cells along the periphery of the sections indicated survival of transplanted cells at 28 days post transplantation in the TX group Fig. 4, whereas very few positively stained cells were found in the CN and SH groups respectively Fig. 4. To more specifically address the cardiogenic potential of the hESC-derived cardiomyocytes, we looked at myofibrillo-genesis of engrafted differentiating ESC. We did immunofluorescence with an antibody against cTnl that recognizes embryonic human but not adult mouse troponin Fig. 4. Double staining revealed co-expression of human nuclei and cTnl in localized areas of the heart sections Fig. 4. This data suggests that infused cardiac committed cells not only survived in injured myocardium but also differentiate into cardiac tissue. The capability of the implanted cells to form gap junctions with the host tissue was evaluated by immunofluorescent staining to anti-human Cx-43 in the heart sections. The -25- positive expression of Cx-43 counter stained with DAPI shows neo-formation of gap junctions in the TX group Fig. 5 in comparison to the SH and CN groups Fig. 5. Immunofluorescent studies with a human-specific monoclonal antibody recognizing the nuclear proliferative marker Ki67, known to identify cells in all active phases of the cell cycle, was found to be positive in the regions of the mouse heart which showed cTnl staining in the TX group Fig. 5. This data indicates that cardiac progenitor cells were still in the process of differentiation while proliferating. RNA isolation from mice heart tissue and RT-PCR: Mice hearts were collected from all the three groups and performed gene expression analysis to identify human cardiomyocytes in the mouse heart post transplantation. RNA extraction and RT-PCR was carried out. The mRNA expression of a set of cardiac markers (hNkx2.5, hcTnl and hANP) and VEGF was analyzed in mice heart tissue using species specific primers. For all markers, PCR were performed for 35 cycles, consisting of an initial denaturation at 94°C for 1 min, then 94°C for 30 see, annealing temperature of the respective gene primer for 45 sec, 72°C for 1 min, and was terminated by final extension at 72°C for 5 min. mRNA transcripts of human cardiac markers were also detected like hANP, hNkx2.5 and hcTnl by RT-PCR, which confirms the presence of donor cells in the host heart tissue (Fig. 3). The combination of hESC transplantation and G-CSF administration resulted in more extensive vascularization in the damaged myocardium than stem cell therapy alone. This was confirmed by the enhanced expression of vascular endothelial growth factor (VEGF) in the TX group than in the CN and SH groups Fig. 5 as detected by RT-PCR. TUNEL staining: Apoptotic activity was determined by the terminal deoxynucleotide transferase-mediated deoxyuridine tri-phosphate nick-end labeling (TUNEL) method using a commercially available apoptosis detection kit (Promega). The tissue sections were counter-stained with hematoxylin. The number of TUNEL positive cells in the border zone of the injured myocardium is critical. -26- In the TX group, cell apoptosis was reduced in the border regions of the myocardium, as compared with the control groups Fig. 6. The number of TUNEL- positive cells was significantly smaller (p Cyclic AMP (low pH) immunoassay: In general, cells are treated with an agonist and intracellular cAMP is extracted. The mass of cAMP present in the cellular extracts is then determined by the following method. cAMP (low pH) assay kit (R&D Systems, Minneapolis, MN, USA) is a 3 hour competitive immunoassay designed to directly measure cAMP in tissues, cell lysates, and cell culture supernates. Prior to assay, samples are acidified, inhibiting phosphodiesterase activity. The assay is based on the competitive binding technique in which cAMP present in the sample competes with a fixed amount of alkaline phosphatase-labeled cAMP for sites on a rabbit polyclonal antibody. The enzyme activity is measured by reading absorbance at 405 nm. The intensity of the color is inversely proportional to the concentration of cAMP in the sample. Adenosine 3', 5'-cyclic monoposphate (cAMP) is one of the most important "second messangers" involved as a modulator of physiological processes and is also involved in cardiovascular functions. Changes in cellular cAMP levels can occur from increases or decreases in its biosynthesis (which results from the catalytic conversion of ATP to cAMP by the enzyme adenylyl cyclase) or by altering its degradation (which results from hydrolysis of the cyclic phosphodiester bond by cyclic nucleotide or cAMP phosphodiesterases). Adenylyl cyclase is an example of an effector enzyme whose activity can be stimulated and inhibited by different GTP-binding proteins. There remains a considerable interest in the measurement of intracellular cAMP in cell cultures since this may be a direct evidence of establishing functional efficiency of cardiomyocytes. An increase in the number of contracting EBs were shown accompanied with an elevated level of cAMP in the EBs in response to 10'6 M isoprenaline, a well-known betal-adrenoceptor. Although, the basal levels of intracellular cAMP detected was low, the increase post isoprenaline treatment of the cells was significant. -27- Safety studies on teratomas A safety study for teratoma formation was done successfully wherein no tumor formation was detected after 12-16 weeks of cell injection into the left thigh of SCID mice. The sections of the thigh muscle was further taken and analyzed by H&E staining to confirm the same. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and irr the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention.. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Dated this $TJ day of Mardx,2007 For Reliance Life Sciences Pvt. Ltd K. V. StrbTalnamam President -28- CLAIMS: 1. A method for manufacturing a medicament for treating non-ischemic heart disease in a mammal in need of such treatment, comprising adding a therapeutically effective amount of a population of cardiac precursor cells and/or terminally differentiated cardiomyocytes derived from mammalian embryonic stem cells to a first pharmaceutically acceptable carrier to produce a therapeutic composition and further comprising adding a cytokine to a second pharmaceutically acceptable carrier. 2. A method of claim 1, wherein the medicament is formulated for the treatment of idiopathic dilated cardiomyopathy (IDCM). 3. A method of claim 1, wherein the medicament is formulated for administration to a human. 4. A method of claim 1, wherein the pharmaceutically acceptable carrier is suitable for transplantation by a parenteral route. 5. A method of claim 1, wherein the pharmaceutically acceptable carrier is suitable for transplantation by an intravenous route. 6. A method of claim 1, wherein the population of cells is derived from human embryonic stem cells. 7. A method of claim 1, wherein the cytokine is contained in a separate therapeutic composition. 8. A method of claim 1, wherein the cytokine is a growth factor. 9. A method of claim 1, wherein the cytokine is granulocyte colony stimulating factor. 10. A method of claim 1, wherein the cytokine is an interleukin or an interferon. 11. A method of claim 1, wherein the cytokine is stem cell factor, vascular endothelial cell growth factor, tumor necrosis factor-alpha, or tumor necrosis factor-beta. 12. Use of a composition comprising a therapeutically effective amount of a population of cardiac precursor cells and/or terminally differentiated cardiomyocytes derived from mammalian embryonic stem cells, and a cytokine for the treatment of a mammal with non-ischemic heart disease. -29- 13. A method of as per claim 12, wherein the non-ischemic heart disease is idiopathic dilated cardiomyopathy (IDCM). 14. A method as per claim 12, wherein the mammal is human. 15. A method as per claim 12, wherein the population of cells is formulated for delivery by a parenteral route. 16. A method as per claim 12, wherein the preparation is formulated for delivery by an intravenous route. 17. A method as per claim 12, wherein the population of cells is derived from human embryonic stem cells. 18. A method as per claim 12, wherein the cytokine is delivered to the mammal after the population of cells is delivered to the mammal. 19. A method as per claim 12, wherein the cytokine is a growth factor. 20. A method as per claim 12, wherein the cytokine is granulocyte colony stimulating factor. 21. A method as per claim 12, wherein the cytokine is an interleukin or an interferon. 22. A method as per claim 12, wherein the cytokine is stem cell factor, vascular endothelial cell growth factor, tumor necrosis factor-alpha, or tumor necrosis factor-beta. 23. A population of cells and a cytokine and its use and method of manufacture according to the claims above substantially as herein described with reference to the examples and figures. Dated this 60 day of March,, 2007 For Reliance Life Sciences Pvt. Ltd K. V. Stlb*ramaniam President -30- ABSTRACT The present invention relates to the differentiation of cardiomyocytes from a human embryonic stem cell line, for example the ReliCeH hESl cell line. The present invention in particular relates to the delivery of these cells via non-invasive routes like intravenous routes and its potential applications in cardiac disorders. The present invention has demonstrated that hESC-derived cardiomyocytes in combination with a cytokine can enhance the efficacy of cellular cardiomyoplasty in idiopathic dilated cardiomyopathy (IDCM). The present invention provides new avenues pertaining to the potential of tissue specific stem cells in therapeutic applications.

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