Indian Patents. 241512:A NOVEL PROCESS FOR DERIVING CARDIOMYOCYTE PRECURSORS FROM BONE-MARROW STEM CELLS

Title of Invention	A NOVEL PROCESS FOR DERIVING CARDIOMYOCYTE PRECURSORS FROM BONE-MARROW STEM CELLS
Abstract	This invention involves a method and process of deriving cardiomyocyte, by co-culturing bone - marrow derived mesenchymal stem cells with atrial condition media, in vitro. The condition media has specific factors that are potent transdifferentiators of the bone marrow derived stem cells.

Full Text	FIELD OF INVENTION: This invention relates to the field of deriving cardiomyocyte precursors from bone-marrow stem cells and specifically to the process of co-culturing mesenchymal stem cells, in vitro. More specifically, this invention provides controlled differentiation of human pluripotent stem cells to form cardiomyocytes precursors, using special culture conditions and selection techniques. BACKGROUND OF INVENTION: Most cardiac diseases if remain untreated eventually turn into heart failure. In the western world, cardiovascular diseases account for about 43% of deaths and for about 50% of hospitalizations. Cardiac diseases account for 80% - 85% of cardiovascular death. Researchers have identified a number of factors that put human heart at risk. Some of the factors are smoking, high blood cholesterol, high blood pressure, increasing age, diabetes, obesity, lack of exercise etc. There are low density and high-density lipoproteins in the blood. They are the fat or lipid content of the blood and especially the low-density lipoprotein is responsible for focal accumulation of lipids in the luminal layer of coronary artery. This process is called atherosclerosis. The most common form of arteriosclerosis is a process that in coronary arteries, as in other blood vessels consists of focal inner layer accumulations of lipids, complex carbohydrates, blood and blood products, fibrous tissue and calcium deposits and associated changes in the middle layer of the coronary artery wall, thus producing thickening of the inner layer and middle layer compromising the lumen. Myocardial infarction in human beings is caused due to the narrowing of the coronary artery and thereby reduction of blood flow to the heart muscle Producing ischemia followed by infarction. Ischemic heart disease is caused by an imbalance between the myocardial blood flow and metabolic demand of the myocardium. Reduction in coronary blood flow is related to progressive atherosclerosis with increasing occlusion of coronary arteries. Blood flow can be further decreased by superimposed events such as vasospasm, thrombosis or circulatory changes leading to hypoperfusion. The myocardial infarct signifies death of cardiac muscles in the areas of ischemia due to nonavailability of blood and oxygen. The cardiomyocyte responsible for mechanical pump function are post mitotical terminally differentiated cells that can no more divide and thus react to increased workload with hypertrophy, if they are alive to compensate for the dead cardiac muscle. The cardiomyocytes are post. mitotic and do not have any power of regeneration. Therefore, they are replaced by the scar tissue composed of fibroblast if there is myocardial infarction and necrosis of the cardiac muscle around the site of compromised blood flow. The fibroblasts are connective tissues and do not have the ability to polarization, depolarization and contractility. As a result of multiple infarcts the heart becomes very weak, especially the left ventricle which is required to pump blood into the aorta. There is a possibility for this scar tissue to balloon out and form aneurysm where the blood get sequestered and does not effectively reach the aorta; as a consequence all the organs of the body gets very less blood supply for them to function adequately including the heart itself. Heart gradually dilates because of scar tissue and aneurysm formation as it loses its elasticity and contractility. Laplace's law does not hold good anymore and leads to heart failure or pump failure that subsequently leads to death. Conventionally for treating myocardial injuries cell transplantation techniques were used. Since heart dysfunction after myocardial injury is due to the loss of cardiac cells and scar expansion, replacing lost cardiac cells with muscle cells By cell transplantation may limit scar expansion and prevent heart failure. The transplanted cardiomyocyte may proliferate in vivo and grow to form myocardial tissue. At present there is no permanent solution to prevent heart from failure, however with the advent of the stem cell regenerative therapy it is possible to regenerate the lost heart muscle and thus treat these conditions. Stem cells have the ability to divide indefinitely in culture and give rise to specialized cells constituting a tissue upon stimulation by a specific differentiation stimulus. Stem cells are two types: embryonic stem cells (ES cells) and adult stem cells. ES cells are isolated from the inner cell mass (ICM) of embryos at the blastocyst stage and are totipotent, i. e. , they are capable of differentiating into virtually every type of cells found in an organism, but once they start producing a specific cell type their cell division can become uncontrolled giving rise to tumour formation. Though there are methods for induction of differentiation of embryonic stem cells into cardiomyocytes, the transplanted ES cells can potentially form teratomas is some undifferentiated totipotent cells are still present. In addition, recipients must receive immunosuppressants because ES are allogenic. In contrast, mesenchymal stem cells do not carry any inherent risk of tumour formation and are syngeneic. In contrast, tissue-specific adult stem cells are generally committed to give rise to cells constituting a specific tissue line. These tissue-specific stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically. Representative tissue-specific stem cells include hematopoietic stem cells and mesenchymal stem cells present in bone marrow. Hematopoietic stem cells give rise to various blood cells such as erythrocytes and leukocytes ; and Mesenchymal stem cells, to the cells of connective tissues, e. g., osteoblasts, chondroblasts, adipocytes and myoblasts. Recently, clinical applications of the stem cells have drawn an increasing interest since the successful isolation of human embryonic stem cell. But due to uncontrolled cell division and to other ethical issues embryonic stem cell is not being pursued much in regenerative therapy. The most noticeable potential application of the adult stem cells is their use as a perfect source for a cell replacement therapy, if they can be directed properly to grow the specific tissue. Hardly curable diseases, e. g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction and liver cirrhosis, are caused by the disruption and permanent functional disorder of the cells constituting an organ and the cell replacement therapy, wherein the loss of cells is replenished from the outside, has been presented as an effective remedy. However, notwithstanding the obvious benefit of the cell replacement therapy, there exist many limitations in its clinical applications. Specifically, the conventional method', wherein fully differentiated cells isolated from the tissues of a donor are transplanted into a patient, has the problem of antigenicity. It requires life long immunosuppressive therapy and very often rejection of the transplanted tissue becomes the ultimate outcome. Over and above all these surgical and medical therapies carry a very high expenditure and requires continuous monitoring; of course patient is also subjected to the adverse effects of immunosuppression. That is why worldwide search has been focused on autologous stem cell therapy in regenerative medicine. In the past decade, clinical observations have shown that bone marrow derived stem cells can migrate to the site of infarct and repair the damaged heart by transdifferentiating into cardiomyocytes. However this repair is not enough to Improve the clinical conditions; the amount of stem cell induced cardiomyocytes produced are very small and as they are cardiomyocytes they can no longer multiply, and if the blood supply is not adequate they do not survive long. Therefore there is need for a process to differentiate bone marrow derived mesenchymal stem cells into cardiomyocyte precursor cells and implant these into the infracted area of the heart to derive healthy cardiomyocytes and thus treat the clinical conditions. Existing methods for deriving cardiomyocytes from bone marrow stem cells use a combination of fibroblast growth factor and 5-azacytidine. This method is very expensive due to the use of recombinant growth factors and also does not yield optimum numbers of cardiomyocyte precursors. Mesenchymal stem cells are capable of differentiating into more than one type of mesenchymal cell lineage. Mesenchymal stem cells have been identified and cultured from avian and mammalian species including mouse, rat, rabbit, dog and human (see Caplan 1991, Caplan et al 1993 and U. S patent no. 5486359). Isolation, purification and culture expansion of human mesenchymal cells is described in detail therein. A cardiomyogenic cell line was developed from bone marrow stroma, and cultured for more than 4 months. To induce cell differentiation, cells were treated with 5- azacytidine for 24 hours, which caused 30% of the cells to form myotube-like structures, acquire cardiomyocyte markers, and begin beating. Most established cardiomyocyte lines have been obtained from animal tissue. There are no established cardiomyocyte cell lines that are approved for widespread use in human cardiac therapy. Therefore there is need for a reliable process for transdifferentiating stem cells, both adult as well as embryonic stem cells into cardiomyocyte precursor. OBJECT OF INVENTION: The objective of this invention is to find a novel and enhanced process for transdifferentiating stem cells, from adUt tern rPll. s intn rardinmvnr ; vtP nrPrmsnrs Another objective of the invention is to identify, isolate and express as recombinant product, novel factors which can induce stem cells to differentiate cardiomyocyte precursors. Yet another objective of this invention is to provide an inexpensive process of differentiating stem cells to cardiomyocytes precursor. Mesenchymal stem cells are used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration. The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. The stem cells are co-cultured with atrial tissues. BRIEF DESCRIPTION OF THE DRAWINGS: The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the drawing, which respectively show: Fig. 1: a photomicrograph; (hereinafter, same magnification is applied) shows spindle shaped cells with intercalated disc like structure, resembling cardiomyocyte. Fig 2 is a block diagram explaining the various steps involved in the process of co-culturing bone marrow stem cells and atrial condition media Fig 3 shows the picture of human long-term bone marrow stroma (21 days old), from which the stem cells are obtained. DETAILED DESCRIPTION OF THE INVENTION: The present invention provides method for differentiating mesenchymal stem cells isolated from bone-marrow into cardiomyocytes precursor. The autologous mesenchymal stem cells differentiate into cardiac muscle cells and integrate with the healthy tissue of the recipient to replace the function of the dead or damaged cells, thereby regenerating the cardiac muscle as a whole. Cardiac muscle does not normally have reparative potential. The mesenchymal stem cells are used, for example, in cardiac muscle regeneration for a number of principal indications (i) ischemic cardiomyopathy, (ii) therapy for heart failure patients and (iii) reduction of scar tissue area in aneurysmal heart wall undergoing coronary artery bypass graft. The proper environmental stimuli convert mesenchymal stem cells into cardiac myocytes. Differentiation of mesenchymal stem cells to the cardiac lineage is controlled by factors present in the cardiac environment. Exposure of mesenchymal stem cells to a simulated cardiac environment directs these cells to cardiac differentiation as detected by expression of specific cardiac muscle lineage markers. Local chemical, electrical and mechanical environmental influences alter pluripotent mesenchymal stem cells and convert the cells grafted into heart into cardiac lineage. In order to derive cardiomyocyte precursors from mesenchymal stem cell, it is preferred to c-culture the mesenchymal stem cells with a condition medium. The condition medium has specific factors that are potent transdifferentiators of the bone marrow derived stem cells. Mesenchymal stem cell (MSC) therapy can serve as a means to deliver high densities of repair-competent cells to a defect site when adequate numbers of mesenchymal stem cell and mesenchymal stem cell lineage-specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly Producing large numbers of MSC are necessary. While MSC have been exposed to a number of growth factors in vitro, only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra), while none have been demonstrated to independently induce differentiation. Methods that increase the ex vivo proliferation and differentiation of MSC will greatly increase the utility of MSC therapy. Similarly, methods that increase in vivo proliferation and differentiation of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site. Furthermore, methods that enhance the proliferation of lineage-specific descendants of MSC, including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites. Since adult stem cell banking has come into existence, specific conditioning media and/or the factors isolated from it along with 5-Azacytidine and angiogenic agents may protect the human heart whenever necessary. Thus, there exists a need in the art for methods that increase the proliferation and differentiation of hematopoietic and mesenchymal pluripotent and lineage-specific cells that are useful in rapidly providing a large population of such cells for use in cell therapy and for making a large population of transfected cells for use in gene therapy. The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. Bone-marrow aspirates are collected in sterile containers containing preservative free heparin. The bone marrow is diluted with sterile tissue culture medium containing autologous serum and plated into sterile tissue culture petri dishes Under a laminar airflow hood. These plates are incubated in 5%-7% carbon dioxide incubator at 33-37 degree centigrade for two hours. This procedure removes the adherent neutrophils and monocytes which inhibits the growth of other cells. The cell suspension is again replated into fresh dishes and incubated as before for 24 hours. The non-adherent cells are transferred to fresh dishes and incubated as before. The adherent cells are again fed with fresh medium containing 10% v/v of atrial-conditioned medium. This procedure is repeated till three plates are made from the starting plate. Incubation with conditioned medium is for 7 days, with a weekly feeding with fresh medium. The plates are incubated for a total of three weeks. At the end of three weeks all the plates are treated with 5-Azacytidine for 24 hours before termination of cultures. The cells are fixed for immunocytochemistry and electron microscopy. The condition media is made by mincing autologous atrial tissue in tissue culture medium and incubating it for three days at 5% carbon dioxide and at 33-37 degree centigrade temperature. The cell free supernatant fluid is collected by centrifuging the culture at 800g-1000g for 20 - 30 minutes, at room temperature. Atrium has more precursors similar to that of cardiac muscle. As the atrial crush is autologous this avoids the occurrence of harm to the patient. The cells used in the process are autologous hence it is safe for clinical use, no antigenic reaction, no immunosuppression and thereby ethical issues do not arise. The atrial tissues have specific protein factors which lead to deriving of cardiomyocyte by co-culturing, with mesenchymal stem cells. The present invention is potent in inducing cardiomyocytes precursors from other methods of deriving cardiomyocytes. The present invention along with angiogenic therapy may create a fool-proof treatment for ischemic myocardial failure in"no option"ischemic heart diseases. Isolation and identification of the atrial conditioning media may be helpful for intravenous delivery of the media, thereby enhancing the in vivo process of cardiomyocyte precursor to differentiate into cardiomyocyte whenever such situation warrants. The mesenchymal therapy of the invention can be provided by several routes of administration such as Intracardiac muscle injection, which avoids the need for an open surgical procedure, can be used where the mesenchymal stem cells along with the condition medium are in an injectable liquid suspension preparation or when they are in a biocompatible medium which is injectable in liquid form and becomes semi-solid at the site of damaged myocardium. A conventional intracardiac syringe or a controllable arthroscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter that shear forces will not damage the mesenchymal stem cells and the condition media. The injectable liquid suspension preparations can also be administered intravenously, either by continuous drip or as a bolus. Culturing Mesenchymal Stem Cells : A needle was punctured to the bone and used to aspirate the bone marrow and approximately lgm of specific tissue that has factors, which are potent transdifferentiators of the bone marrow derived stem cells. Different cells present at different portions of the bone marrow sample are obtained. The sediment cells constitute majorly RBCs and the supernatant especially that of its central region posses the desired stem cells. The particular part of the bone marrow aspirate was collected carefully in a sterile container containing preservative free heparin. The collected bone marrow aspirates were added into a petridish in which 5 ml of DMEM media with autologous serum in ratio of 1: 3 were already added. The cells were incubated with 95% air and 5% C02 at 33-37 C for 24 hours. The procedure was repeated for two or more times and all the petridish were incubated for 7 days in the incubator. After every 7th day the cells grown in petridishes were passaged. To test the effect of cell passaging on cardiomyocyte differentiation of mesenchymal stem cells, samples of MSCs, prepared as described above were passaged once prior to preparing of cell cultures described below. In this case, the term passaging refers to the distribution of cultured cells growing in the culture dish into new culture dishes after the cells reach confluence i. e. when the cells contact each other and cover the surface of the dish. The passaging process was repeated every 3 weeks. Culturing condition media: The 1 gm of specific tissue which has factors which are potent transdifferentiators of the bone marrow derived stem cells, were cut into Icm3 and washed with gentamycin containing the medium which is obtained from the plasma extracted from autologous blood. The tissue condition media taken from transport medium are chopped into thin pieces of Imm3 size. Prior to the process of chopping, any additional fat present is separated from tissue. The chopped tissues are transferred to the petridishes containing 5ml of DMEM (5- 10% of autologous serum or umbilical cord blood serum) and left for incubation for 3 days at 33-37'C in C02 incubation (5% C02). Preparation of condition media: The atrial tissues, which were grown in the petridishes after incubation period of 3 days, were used for preparation of condition media. The media along with the cells in it were mixed thoroughly for 3-4 times and then supernatant was collected in a centrifuge tube. The centrifugation process is performed to separate bone marrow cells and red blood cells. The collected media was centrifuged at 800-1000rpm for 5mins. After the centrifugation process the residue was left and the supernatant was Collected and used as condition media. These petridishes were subjected to centrifugation. After the condition media preparation was completed, it was stored at 0°C until the stem cells were ready for treatment. At the end of 3 weeks, the stem cells, which were grown in final passaging, were used. These petridishes were mixed thoroughly for 2-3 times and the supernatant was discarded. To. this fresh medium of about 5ml and 100). iL of the above-prepared condition media was added. These plates were left for incubation at 37°C for three days in 5% C02 incubator Two major types of cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells, were isolated from percoll gradients of bone marrow cells. The MSCs were spindle- shaped, attached to the culture dish tightly and proliferated in the culture medium. The hematopoietic stem cells were round-shaped, did not attach to culture dish, and were washed away with the culture medium changes. The Atrial condition medium promoted the growth of bone marrow mesenchymal cells. The morphology of mesenchymal cells grown in the presence of atrial tissue condition media resembled the cardiomyocytes. Qualitative analysis of the cell samples from the co- culturing experiments indicated that mesenchymal stem cells co-cultured with atrial tissues and 5-azacytidine formed more small striped, spindle shaped intercalated disc (cardiomyocyte precursors) as shown in Fig. 1 than those mesenchymal stem cells which were cultured with 5- azacytidine and ventricular cardiomyocytes. Mesenchymal stem cells did not form any cardiomyocyte precursors when cultured alone. The present invention successfully proves that mesenchymal stem cells can be directed to differentiate into cardiomyocyte precursors by co culturing with atrial tissues. The present method that result into formation of cardiomyocyte precursors is more effective as compared to the existing prior methods that use Ventricular tissues for co-culturing as it leads to an increased amount of cardiomyocyte precursor. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth. WE CLAIM 1. A method and process of deriving cardiomyocyte from bone-marrow stem cells, by co-culturing bone - marrow derived mesenchymal stem cells with atrial condition media, in vitro wherein the stem cells are treated with 5- azacytidine. 2. A method and process of deriving cardiomyocytes from bone marrow stem cells of claim 1, wherein the said co-culturing comprises of stem cell culturing and tissue culturing. The steps involved in culturing are as follows : a. The atrial crush containing the tissue condition medium is centrifuged at predetermined speed and temperature, to obtain atrial condition medium, b. The bone marrow derived mesenchymal stem, cells are incubated and co- cultured with atrial condition medium, c. The cultured condition medium is incubated with 5% of C02 and then treated with 5- azacytidine at predetermined concentration to derive the cardiomyocyte precursors. 3. The method and process of claim 1, wherein said mesenchymal stem cells (sterile aspirate) are obtained from autologous bone marrow. 4. The method and process of claim 2, wherein said mesenchymal stem cells are enriched on density gradient centrifugation. 5. The method and process of claim 2, wherein said enriched mesenchymal stem cells are expanded in presence of growth factor. 6. The method and process of claim 2, wherein said mesenchymal stem cells have been co-cultured with condition medium, in vitro. 7. The method and process of claim 2, wherein said mesenchymal stem cells have been co-cultured with condition medium that have specific protein. 8. The method and process of claim 1, wherein said condition medium is prepared by culturing atrial tissues in a petridish containing 5ml of DMEM (5- 10% of autologous serum or umbilical cord blood serum), in vitro. 9. The method and process of claim 1, wherein said condition medium is prepared by culturing the tissue in a petridish containing 5ml of DMEM in 1: 3 ratio. 10. The method and process of claim 6, wherein said specific tissue are potent in inducing cardiomyocytes from other stem cells including Embryonic Stem cells. 11. The method and process of claim 1, wherein said mesenchymal stem cells are exposed to 5- azacytidine. 12. The method and process of claim 1, wherein said 5- azacytidine is present at a concentration of 5-10 millimoles 13. The method and process of claim 1, wherein said mesenchymal stem cells have been cultured for atleast 3-4 weeks. 14. The method and process of claim 2, wherein the said centrifugation of atrial crush is performed at a speed of 800-1000 rpm. 15. The method and process of claim 1, wherein said mesenchymal stem cells are administered by injection. A novel process and method for deriving cardiomyocyte precursors from bone-marrow stem cells 5 FIELD OF INVENTION This invention relates to the field of deriving cardiomyocyte precursors from bone-marrow stem cells and specifically to the process of co-culturing mesenchymal stem cells, in 10 vitro. More specifically, this invention provides controlled differentiation of human pluripotent stem cells to form cardiomyocytes precursors, using special culture 15 conditions and selection techniques. BACKGROUND OF INVENTION Most cardiac diseases if remain untreated eventually turn 20 into heart failure. In the western world, cardiovascular diseases account for about 43% of deaths and for about 50% of hospitalizations. Cardiac diseases account for 80% - 85% of cardiovascular death. Researchers have identified a number of factors that put human heart at risk. Some of 25 the factors are smoking, high blood cholesterol, high blood 1 Pressure, increasing age, diabetes, obesity, lack of exercise etc. There are low density and high-density lipoproteins in the blood. They are the fat or lipid content of the blood and especially the low-density lipoprotein is responsible for focal accumulation of lipids in the luminal layer of coronary artery. This process is called atherosclerosis. The most common form of arteriosclerosis is a process that in coronary arteries, as in other blood vessels consists of focal inner layer accumulations of lipids, complex carbohydrates, blood and blood products, fibrous tissue and calcium deposits and associated changes in the middle layer of the coronary artery wall, thus producing thickening of the inner layer and middle layer compromising the lumen. Myocardial infarction in human beings is caused due to the narrowing of the coronary artery and thereby reduction of blood flow to the heart muscle producing ischemia followed by infarction. Ischemic heart disease is caused by an imbalance between the myocardial blood flow and metabolic demand of the myocardium. Reduction in coronary blood flow is related to progressive atherosclerosis with increasing occlusion of coronary arteries. Blood flow can be further decreased by Superimposed events such as vasospasm, thrombosis or circulatory changes leading to hypoperfusion. The myocardial infarct signifies death of cardiac muscles in the areas of ischemia due to non-availability of blood and oxygen. The cardiomyocyte responsible for mechanical pump function are post mitotical terminally differentiated cells that can no more divide and thus react to increased workload with hypertrophy, if they are alive to compensate for the dead cardiac muscle. The cardiomyocytes are post mitotic and do not have any power of regeneration. Therefore, they are replaced by the scar tissue composed of fibroblast if there is myocardial infarction and necrosis of the cardiac muscle around the site of compromised blood flow. The fibroblasts are connective tissues and do not have the ability to polarization, depolarization and contractility. As a result of multiple infarcts the heart becomes very weak, especially the left ventricle which is required to pump blood into the aorta. There is a possibility for this scar tissue to balloon out and form aneurysm where the blood get sequestered and does not effectively reach the aorta; as a consequence all the organs of the body gets very less blood supply for them to function adequately including the Heart itself. Heart gradually dilates because of scar tissue and aneurysm formation as it loses its elasticity and contractility. Laplace's law does not hold good anymore and leads to heart failure or pump failure that subsequently leads to death. Conventionally for treating myocardial injuries cell transplantation techniques were used. Since heart dysfunction after myocardial injury is due to the loss of cardiac cells and scar expansion, replacing lost cardiac cells with muscle cells by cell transplantation may limit scar expansion and prevent heart failure. The transplanted cardiomyocyte may proliferate in vivo and grow to form myocardial tissue. At present there is no permanent solution to prevent heart from failure, however with the advent of the stem cell regenerative therapy it is possible to regenerate the lost heart muscle and thus treat these conditions. Stem cells have the ability to divide indefinitely in culture and give rise to specialized cells constituting a tissue upon stimulation by a specific differentiation stimulus. Stem cells are two types: embryonic stem cells (ES cells) and adult stem cells. ES cells are isolated from the inner cell mass (ICM) of embryos at the blastocyst stage and are totipotent, i.e., they are capable of differentiating into Virtually every type of cells found in an organism, but once they start producing a specific cell type their cell division can become uncontrolled giving rise to tumour formation. Though there are methods for induction of differentiation of embryonic stem cells into cardiomyocytes, the transplanted ES cells can potentially form teratomas is some undifferentiated totipotent cells are still present. In addition, recipients must receive immunosuppressants because ES are allogenic. In contrast, mesenchymal stem cells do not carry any inherent risk of tumour formation and are syngeneic. In contrast, tissue-specific adult stem cells are generally committed to give rise to cells constituting a specific tissue line. These tissue-specific stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically. Representative tissue-specific stem cells include hematopoietic stem cells and mesenchymal stem cells present in bone marrow. Hematopoietic stem cells give rise to various blood cells such as erythrocytes and leukocytes; and mesenchymal stem cells, to the cells of connective tissues, e.g., osteoblasts, chondroblasts, adipocytes and myoblasts. Recently, clinical applications of the stem cells have drawn an increasing interest since the successful isolation of Human embryonic stem cell. But due to uncontrolled cell division and to other ethical issues embryonic stem cell is not being pursued much in regenerative therapy. The most noticeable potential application of the adult stem cells is their use as a perfect source for a cell replacement therapy, if they can be directed properly to grow the specific tissue. Hardly curable diseases, e.g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction and liver cirrhosis, are caused by the disruption and permanent functional disorder of the cells constituting an organ and the cell replacement therapy, wherein the loss of cells is replenished from the outside, has been presented as an effective remedy. However, notwithstanding the obvious benefit of the cell replacement therapy, there exist many limitations in its clinical applications. Specifically, the conventional method, wherein fully differentiated cells isolated from the tissues of a donor are transplanted into a patient, has the problem of antigenicity. It requires life long immunosuppressive therapy and very often rejection of the transplanted tissue becomes the ultimate outcome. Over and above all these surgical and medical therapies carry a very high expenditure and requires continuous monitoring; of course Patient is also subjected to the adverse effects of immunosuppression. That is why worldwide search has been focused on autologous stem cell therapy in regenerative medicine. In the past decade, clinical observations have shown that bone marrow derived stem cells can migrate to the site of infarct and repair the damaged heart by transdifferentiating into cardiomyocytes. However this repair is not enough to improve the clinical conditions; the amount of stem cell induced cardiomyocytes produced are very small and as they are cardiomyocytes they can no longer multiply, and if the blood supply is not adequate they do not survive long. Therefore there is need for a process to differentiate bone marrow derived mesenchymal stem cells into cardiomyocyte precursor cells and implant these into the infracted area of the heart to derive healthy cardiomyocytes and thus treat the clinical conditions. Existing methods for deriving cardiomyocytes from bone marrow stem cells use a combination of fibroblast growth factor and 5-azacytidine. This method is very expensive due to the use of recombinant growth factors and also does not yield optimum numbers of cardiomyocyte precursors. Mesenchymal stem cells are capable of differentiating into more than one type of mesenchymal cell lineage. Mesenchymal stem cells have been identified and Another objective of the invention is to identify, isolate and express as recombinant product, novel factors which can induce stem cells to differentiate cardiomyocyte precursors. Yet another objective of this invention is to provide an inexpensive process of differentiating stem cells to cardiomyocytes precursor. Mesenchymal stem cells are used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration. The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. The stem cells are co-cultured with atrial tissues. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the drawing, which respectively show: Fig. 1: a photomicrograph; (hereinafter, same magnification is applied) shows spindle shaped cells with intercalated disc like structure, resembling cardiomyocyte. Fig 2 is a block diagram explaining the various steps involved in the process of co-culturing bone marrow stem cells and atrial condition media Fig 3 shows the picture of human long-term bone marrow stroma (21 days old), from which the stem cells are obtained. DETAILED DESCRIPTION OF THE INVENTION The present invention provides method for differentiating mesenchymal stem cells isolated from bone - marrow into cardiomyocytes precursor. The autologous mesenchymal stem cells differentiate into cardiac muscle cells and integrate with the healthy tissue of the recipient to replace the function of the dead or damaged cells, thereby regenerating the cardiac muscle as a whole. Cardiac muscle does not normally have reparative potential. The mesenchymal stem cells are used, for example, in cardiac muscle regeneration for a number of principal indications (i) - ischemic cardiomyopathyIk(ii) therapy for heart failure patients and (iii) reduction of scar tissue area in aneurysmal heart wall undergoing coronary artery bypass graft. The proper environmental stimuli convert mesenchymal stem cells into cardiac myocytes. Differentiation of mesenchymal stem cells to the cardiac lineage is controlled by factors present in the cardiac environment. Exposure of mesenchymal stem cells to a simulated cardiac environment directs these cells to cardiac differentiation as detected by expression of specific cardiac muscle lineage markers. Local chemical, electrical and mechanical environmental influences alter pluripotent mesenchymal stem cells and convert the cells grafted into heart into cardiac lineage. In order to derive cardiomyocyte precursors from mesenchymal stem cell, it is preferred to co-culture the mesenchymal stem cells with a condition medium. The condition medium has specific factors that are potent transdifferentiators of the bone marrow derived stem cells. Mesenchymal stem cell (MSC) therapy can serve as a means to deliver high densities of repair- competent cells to a defect site when adequate numbers of mesenchymal stem cell and mesenchymal stem cell lineage - specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly producing large numbers of MSC are necessary. While MSC have been exposed to a number of growth factors in Vitro, only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra), while none have been demonstrated to independently induce differentiation. Methods that increase the ex vivo proliferation and differentiation of MSC will greatly increase the utility of MSC therapy. Similarly, methods that increase in vivo proliferation and differentiation of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site. Furthermore, methods that enhance the proliferation of lineage-specific descendants of MSC, including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites. Since adult stem cell banking has come into existence, specific conditioning media and/or the factors isolated from it along with 5-Azacytidine and angiogenic agents may protect the human heart whenever necessary. Thus, there exists a need in the art for methods that increase the proliferation and differentiation of Hematopoietic and mesenchymal pluripotent and lineage-specific cells that are useful in rapidly providing a large population of such cells for use in cell therapy and for making a large population of transfected cells for use in gene therapy. The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. Bone-marrow aspirates are collected in sterile containers containing preservative free heparin. The bone marrow is diluted with sterile tissue culture medium containing autologous serum and plated into sterile tissue culture petri dishes under a laminar airflow hood. These plates are incubated in 5%-7% carbon dioxide incubator at 33 - 37 degree centigrade for two hours. This procedure removes the adherent neutrophils and monocytes which inhibits the growth of other cells. The cell suspension is again replated into fresh dishes and incubated as before for 24 hours. The non-adherent cells are transferred to fresh dishes and incubated as before. The adherent cells are again fed with fresh medium containing 10% v/v of atrial-conditioned medium. This procedure is repeated till three plates are made from the starting plate. Incubation with conditioned medium is for 7 days, with a weekly feeding with fresh medium. The plates Are incubated for a total of three weeks. At the end of three weeks all the plates are treated with 5-Azacytidine for 24 hours before termination of cultures. The cells are fixed for immunocytochemistry and electron microscopy. The condition media is made by mincing autologous atrial tissue in tissue culture medium and incubating it for three days at 5% carbon dioxide and at 33 - 37 degree centigrade temperature. The cell free supernatant fluid is collected by centrifuging the culture at 800g - 1000g for 20 - 30 minutes, at room temperature. Atrium has more precursors similar to that of cardiac muscle. As the atrial crush is autologous this avoids the occurrence of harm to the patient. The cells used in the process are autologous hence it is safe for clinical use, no antigenic reaction, no immunosuppression and thereby ethical issues do not arise. The atrial tissues have specific protein factors which lead to deriving of cardiomyocyte by co-culturing. with mesenchymal stem cells. The present invention is potent in inducing cardiomyocytes precursors from other methods of deriving cardiomyocytes. The present invention along with angiogenic therapy may create a fool-proof treatment for ischemic myocardial failure in "no option" ischemic heart diseases. Isolation and identification of the atrial conditioning media may be helpful for intravenous delivery of the media, thereby enhancing the in vivo process of cardiomyocyte precursor to differentiate into cardiomyocyte whenever such situation warrants. The mesenchymal therapy of the invention can be provided by several routes of administration such as Intracardiac muscle injection, which avoids the need for an open surgical procedure, can be used where the mesenchymal stem cells along with the condition medium are in an injectable liquid suspension preparation or when they are in a biocompatible medium which is injectable in liquid form and becomes semi-solid at the site of damaged myocardium. A conventional intracardiac syringe or a controllable arthroscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter that shear forces will not damage the mesenchymal stem cells and the condition media. The injectable liquid suspension preparations can also be administered intravenously, either by continuous drip or as a bolus. Culturing Mesenchymal Stem Cells: A needle was punctured to the bone and used to aspirate the bone marrow and approximately 1gm of specific tissue that has factors, which are potent transdifferentiators of the bone Marrow derived stem cells. Different cells present at different portions of the bone marrow sample are obtained. The sediment cells constitute majorly RBCs and the supernatant especially that of its central region posses the desired stem cells. The particular part of the bone marrow aspirate was collected carefully in a sterile container containing preservative free heparin. The collected bone marrow aspirates were added into a petridish in which 5 ml of DMEM media with autologous serum in ratio of 1:3 were already added. The cells were incubated with 95% air and 5% CO2 at 33-37°C for 24 hours. The procedure was repeated for two or more times and all the petridish were incubated for 7 days in the incubator. After every 7th day the cells grown in petridishes were passaged. To test the effect of cell passaging on cardiomyocyte differentiation of mesenchymal stem cells, samples of MSCs, prepared as described above were passaged once prior to preparing of cell cultures described below. In this case, the term passaging refers to the distribution of cultured cells growing in the culture dish into new culture dishes after the cells reach confluence i.e. when the cells Contact each other and cover the surface of the dish. The passaging process was repeated every 3 weeks. Culturing condition media: The 1gm of specific tissue which has factors which are potent transdifferentiators of the bone marrow derived stem cells, were cut into 1cm3 and washed with gentamycin containing the medium which is obtained from the plasma extracted from autologous blood. The tissue condition media taken from transport medium are chopped into thin pieces of 1mm3 size. Prior to the process of chopping, any additional fat present is separated from tissue. The chopped tissues are transferred to the petridishes containing 5ml of DMEM (5-10% of autologous serum or umbilical cord blood serum) and left for incubation for 3 days at 33-37'C in CO2 incubation (5% CO2). Preparation of condition media: The atrial tissues, which were grown in the petridishes after incubation period of 3 days, were used for preparation of condition media. The media along with the cells in it were mixed thoroughly for 3-4 times and then supernatant was collected in a centrifuge tube. The centrifugation process is performed to separate bone marrow cells and red blood cells. The Collected media was centrifuged at 800-1OOOrpm for 5mins. After the centrifugation process the residue was left and the supernatant was collected and used as condition media. These petridishes were subjected to centrifugation. After the condition media preparation was completed, it was .stored at 0°C until the stem cells were ready for treatment. At the end of 3 weeks, the stem cells, which were grown in final passaging, were used. These petridishes were mixed thoroughly for 2-3 times and the supernatant was discarded. To .this fresh medium of about 5ml and 100µL of the above- prepared condition media was added. These plates were left for incubation at 37°C for three days in 5% CO2 incubator Two major types of cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells, were isolated from percoll gradients of bone marrow cells. The MSCs were spindle-shaped, attached to the culture dish tightly and proliferated in the culture medium. The hematopoietic stem cells were round-shaped, did not attach to culture dish, and were washed away with the culture medium changes. The Atrial condition medium promoted the growth of bone marrow mesenchymal cells. The morphology of Mesenchymal cells grown in the presence of atrial tissue condition media resembled the cardiomyocytes. Qualitative analysis of the cell samples from the co-culturing experiments indicated that mesenchymal stem cells co-cultured with atrial tissues and 5-azacytidine formed more small striped, spindle shaped intercalated disc (cardiomyocyte precursors) as shown in Fig. 1 than those mesenchymal stem cells which were cultured with 5-azacytidine and ventricular cardiomyocytes. Mesenchymal stem cells did not form any cardiomyocyte precursors when cultured alone. The present invention successfully proves that mesenchymal stem cells can be directed to differentiate into cardiomyocyte precursors by co culturing with atrial tissues. The present method that result into formation of cardiomyocyte precursors is more effective as compared to the existing prior methods that use ventricular tissues for co-culturing as it leads to an increased amount of cardiomyocyte precursor. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the Invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth. We Claim: 1. A method and process of deriving cardiomyocyte from bone-marrow stem cells, by co-culturing bone - marrow derived mesenchymal stem cells with atrial condition media, in vitro wherein the stem cells are treated with 5-azacytidine. 2. A method and process of deriving cardiomyocytes from bone marrow stem cells of claim 1, wherein the said co-culturing comprises of stem cell culturing and tissue culturing. The steps involved in culturing are as follows: a. The atrial crush containing the tissue condition medium is centrifuged at . predetermined speed and temperature, to obtain atrial condition medium. b. The bone marrow derived mesenchymal stem.cells are incubated and co-cultured with atrial condition medium. c. The cultured condition medium is incubated with 5% of CO2 and then treated with 5- azacytidine at predetermined concentration to derive the cardiomyocyte precursors 3. The method and process of claim 1, wherein said mesenchymal stem cells (sterile aspirate) are obtained from autologous bone marrow. 4. The method and process of claim 2, wherein said mesenchymal stem cells are enriched on density gradient centrifugation. 5. The method and process of claim 2, wherein said enriched mesenchymal stem cells are expanded in presence of growth factor. 6. The method and process of claim 2, wherein said mesenchymal stem cells have been co-cultured with condition medium, in vitro. 7. The method and process of claim 2, wherein said mesenchymal stem cells have been co-cultured with condition medium that have specific protein. 8. The method and process of claim 1, wherein said condition medium is prepared by culturing atrial tissues in a petridish containing 5ml of DMEM (5- 10% of autologous serum or umbilical cord blood serum), in vitro. 9. The method and process of claim 1, wherein said condition medium is prepared by culturing the tissue in a petridish containing 5ml of DMEM in 1:3 ratio. 10. The method and process of claim 6, wherein said specific tissue are potent in inducing Cardiomyocytes from other stem cells including Embryonic Stem cells. 11. The method and process of claim 1, wherein said mesenchymal stem cells are exposed to 5- azacytidine. 12The method and process of claim 1, wherein said 5- azacytidine is present at a concentration of 5 - 10 millimoles 13. The method and process of claim 1, wherein said mesenchymal stem cells have been cultured for atleast 3-4 weeks. 14. The method and process of claim 2, wherein the said centrifugation of atrial crush is performed at a speed of 800 -1000 rpm. 15. The method and process of claim 1, wherein said mesenchymal stem cells are administered by injection.

Full Text

FIELD OF INVENTION:
This invention relates to the field of deriving cardiomyocyte precursors from bone-marrow stem cells and specifically to the process of co-culturing mesenchymal stem cells, in vitro.
More specifically, this invention provides controlled differentiation of human pluripotent stem cells to form cardiomyocytes precursors, using special culture conditions and selection techniques.
BACKGROUND OF INVENTION:
Most cardiac diseases if remain untreated eventually turn into heart failure. In the western world, cardiovascular diseases account for about 43% of deaths and for about 50% of hospitalizations. Cardiac diseases account for 80% - 85% of cardiovascular death. Researchers have identified a number of factors that put human heart at risk. Some of the factors are smoking, high blood cholesterol, high blood pressure, increasing age, diabetes, obesity, lack of exercise etc.
There are low density and high-density lipoproteins in the blood. They are the fat or lipid content of the blood and especially the low-density lipoprotein is responsible for focal accumulation of lipids in the luminal layer of coronary artery. This process is called atherosclerosis. The most common form of arteriosclerosis is a process that in coronary arteries, as in other blood vessels consists of focal inner layer accumulations of lipids, complex carbohydrates, blood and blood products, fibrous tissue and calcium deposits and associated changes in the middle layer of the coronary artery wall, thus producing thickening of the inner layer and middle layer compromising the lumen.
Myocardial infarction in human beings is caused due to the narrowing of the coronary artery and thereby reduction of blood flow to the heart muscle

Producing ischemia followed by infarction. Ischemic heart disease is caused by an imbalance between the myocardial blood flow and metabolic demand of the myocardium. Reduction in coronary blood flow is related to progressive atherosclerosis with increasing occlusion of coronary arteries. Blood flow can be further decreased by superimposed events such as vasospasm, thrombosis or circulatory changes leading to hypoperfusion. The myocardial infarct signifies death of cardiac muscles in the areas of ischemia due to nonavailability of blood and oxygen.
The cardiomyocyte responsible for mechanical pump function are post mitotical terminally differentiated cells that can no more divide and thus react to increased workload with hypertrophy, if they are alive to compensate for the dead cardiac muscle.
The cardiomyocytes are post. mitotic and do not have any power of regeneration. Therefore, they are replaced by the scar tissue composed of fibroblast if there is myocardial infarction and necrosis of the cardiac muscle around the site of compromised blood flow. The fibroblasts are connective tissues and do not have the ability to polarization, depolarization and contractility. As a result of multiple infarcts the heart becomes very weak, especially the left ventricle which is required to pump blood into the aorta. There is a possibility for this scar tissue to balloon out and form aneurysm where the blood get sequestered and does not effectively reach the aorta; as a consequence all the organs of the body gets very less blood supply for them to function adequately including the heart itself. Heart gradually dilates because of scar tissue and aneurysm formation as it loses its elasticity and contractility. Laplace's law does not hold good anymore and leads to heart failure or pump failure that subsequently leads to death.
Conventionally for treating myocardial injuries cell transplantation techniques were used. Since heart dysfunction after myocardial injury is due to the loss of cardiac cells and scar expansion, replacing lost cardiac cells with muscle cells

By cell transplantation may limit scar expansion and prevent heart failure. The transplanted cardiomyocyte may proliferate in vivo and grow to form myocardial tissue. At present there is no permanent solution to prevent heart from failure, however with the advent of the stem cell regenerative therapy it is possible to regenerate the lost heart muscle and thus treat these conditions.
Stem cells have the ability to divide indefinitely in culture and give rise to specialized cells constituting a tissue upon stimulation by a specific differentiation stimulus.
Stem cells are two types: embryonic stem cells (ES cells) and adult stem cells. ES cells are isolated from the inner cell mass (ICM) of embryos at the blastocyst stage and are

totipotent, i. e. , they are capable of differentiating into virtually every type of cells found in an organism, but once they start producing a specific cell type their cell division can become uncontrolled giving rise to tumour formation.
Though there are methods for induction of differentiation of embryonic stem cells into cardiomyocytes, the transplanted ES cells can potentially form teratomas is some undifferentiated totipotent cells are still present. In addition, recipients must receive immunosuppressants because ES are allogenic. In contrast, mesenchymal stem cells do not carry any inherent risk of tumour formation and are syngeneic.
In contrast, tissue-specific adult stem cells are generally committed to give rise to cells constituting a specific tissue line. These tissue-specific stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically. Representative tissue-specific stem cells include hematopoietic stem cells and mesenchymal stem cells present in bone marrow. Hematopoietic stem cells give rise to various blood cells such as erythrocytes and leukocytes ; and

Mesenchymal stem cells, to the cells of connective tissues, e. g., osteoblasts, chondroblasts, adipocytes and myoblasts.
Recently, clinical applications of the stem cells have drawn an increasing interest since the successful isolation of human embryonic stem cell. But due to uncontrolled cell division and to other ethical issues embryonic stem cell is not being pursued much in regenerative therapy.
The most noticeable potential application of the adult stem cells is their use as a perfect source for a cell replacement therapy, if they can be directed properly to grow the specific tissue. Hardly curable diseases, e. g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction and liver cirrhosis, are caused by the disruption and permanent functional disorder of the cells constituting an organ and the cell replacement therapy, wherein the loss of cells is replenished from the outside, has been presented as an effective remedy.
However, notwithstanding the obvious benefit of the cell replacement therapy, there exist many limitations in its clinical applications. Specifically, the conventional method', wherein fully differentiated cells isolated from the tissues of a donor are transplanted into a patient, has the problem of antigenicity. It requires life long immunosuppressive therapy and very often rejection of the transplanted tissue becomes the ultimate outcome. Over and above all these surgical and medical therapies carry a very high expenditure and requires continuous monitoring; of course patient is also subjected to the adverse effects of immunosuppression. That is why worldwide search has been focused on autologous stem cell therapy in regenerative medicine.
In the past decade, clinical observations have shown that bone marrow derived stem cells can migrate to the site of infarct and repair the damaged heart by transdifferentiating into cardiomyocytes. However this repair is not enough to

Improve the clinical conditions; the amount of stem cell induced cardiomyocytes produced are very small and as they are cardiomyocytes they can no longer multiply, and if the blood supply is not adequate they do not survive long.
Therefore there is need for a process to differentiate bone marrow derived mesenchymal stem cells into cardiomyocyte precursor cells and implant these into the infracted area of the heart to derive healthy cardiomyocytes and thus treat the clinical conditions.
Existing methods for deriving cardiomyocytes from bone marrow stem cells use a combination of fibroblast growth factor and 5-azacytidine. This method is very expensive due to the use of recombinant growth factors and also does not yield optimum numbers of cardiomyocyte precursors. Mesenchymal stem cells are capable of differentiating into more than one type of mesenchymal cell lineage. Mesenchymal stem cells have been identified and cultured from avian and mammalian species including mouse, rat, rabbit, dog and human (see Caplan 1991, Caplan et al 1993 and U. S patent no. 5486359). Isolation, purification and culture expansion of human mesenchymal cells is described in detail therein.
A cardiomyogenic cell line was developed from bone marrow stroma, and cultured for more than 4 months. To induce cell differentiation, cells were treated with 5- azacytidine for 24 hours, which caused 30% of the cells to form myotube-like structures, acquire cardiomyocyte markers, and begin beating.
Most established cardiomyocyte lines have been obtained from animal tissue. There are no established cardiomyocyte cell lines that are approved for widespread use in human cardiac therapy.
Therefore there is need for a reliable process for transdifferentiating stem cells, both adult as well as embryonic stem cells into cardiomyocyte precursor.

OBJECT OF INVENTION:
The objective of this invention is to find a novel and enhanced process for transdifferentiating stem cells, from adUt tern rPll. s intn rardinmvnr ; vtP nrPrmsnrs Another objective of the invention is to identify, isolate and express as recombinant product, novel factors which can induce stem cells to differentiate cardiomyocyte precursors.
Yet another objective of this invention is to provide an inexpensive process of differentiating stem cells to cardiomyocytes precursor.
Mesenchymal stem cells are used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration. The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor.
The stem cells are co-cultured with atrial tissues. BRIEF DESCRIPTION OF THE DRAWINGS:
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the drawing, which respectively show: Fig. 1: a photomicrograph; (hereinafter, same magnification is applied) shows spindle shaped cells with intercalated disc like structure, resembling cardiomyocyte.
Fig 2 is a block diagram explaining the various steps involved in the process of co-culturing bone marrow stem cells and atrial condition media Fig 3 shows the picture of human long-term bone marrow stroma (21 days old), from which the stem cells are obtained.
DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides method for differentiating mesenchymal stem cells isolated from bone-marrow into cardiomyocytes precursor.
The autologous mesenchymal stem cells differentiate into cardiac muscle cells and integrate with the healthy tissue of the recipient to replace the function of the dead or damaged cells, thereby regenerating the cardiac muscle as a whole. Cardiac muscle does not normally have reparative potential. The mesenchymal stem cells are used, for example, in cardiac muscle regeneration for a number of principal indications (i) ischemic cardiomyopathy, (ii) therapy for heart failure patients and (iii) reduction of scar tissue area in aneurysmal heart wall undergoing coronary artery bypass graft.
The proper environmental stimuli convert mesenchymal stem cells into cardiac myocytes. Differentiation of mesenchymal stem cells to the cardiac lineage is controlled by factors present in the cardiac environment.
Exposure of mesenchymal stem cells to a simulated cardiac environment directs these cells to cardiac differentiation as detected by expression of specific cardiac muscle lineage markers. Local chemical, electrical and mechanical environmental influences alter pluripotent mesenchymal stem cells and convert the cells grafted into heart into cardiac lineage.
In order to derive cardiomyocyte precursors from mesenchymal stem cell, it is preferred to c-culture the mesenchymal stem cells with a condition medium. The condition medium has specific factors that are potent transdifferentiators of the bone marrow derived stem cells.
Mesenchymal stem cell (MSC) therapy can serve as a means to deliver high densities of repair-competent cells to a defect site when adequate numbers of mesenchymal stem cell and mesenchymal stem cell lineage-specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly

Producing large numbers of MSC are necessary. While MSC have been exposed to a number of growth factors in vitro, only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra), while none have been demonstrated to independently induce differentiation. Methods that increase the ex vivo proliferation and differentiation of MSC will greatly increase the utility of MSC therapy.
Similarly, methods that increase in vivo proliferation and differentiation of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site.
Furthermore, methods that enhance the proliferation of lineage-specific descendants of MSC, including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites.
Since adult stem cell banking has come into existence, specific conditioning media and/or the factors isolated from it along with 5-Azacytidine and angiogenic agents may protect the human heart whenever necessary.
Thus, there exists a need in the art for methods that increase the proliferation and differentiation of hematopoietic and mesenchymal pluripotent and lineage-specific cells that are useful in rapidly providing a large population of such cells for use in cell therapy and for making a large population of transfected cells for use in gene therapy.
The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. Bone-marrow aspirates are collected in sterile containers containing preservative free heparin. The bone marrow is diluted with sterile tissue culture medium containing autologous serum and plated into sterile tissue culture petri dishes

Under a laminar airflow hood. These plates are incubated in 5%-7% carbon dioxide incubator at 33-37 degree centigrade for two hours. This procedure removes the adherent neutrophils and monocytes which inhibits the growth of other cells.
The cell suspension is again replated into fresh dishes and incubated as before for 24 hours. The non-adherent cells are transferred to fresh dishes and incubated as before.
The adherent cells are again fed with fresh medium containing 10% v/v of atrial-conditioned medium. This procedure is repeated till three plates are made from the starting plate. Incubation with conditioned medium is for 7 days, with a weekly feeding with fresh medium. The plates are incubated for a total of three weeks. At the end of three weeks all the plates are treated with 5-Azacytidine for 24 hours before termination of cultures. The cells are fixed for immunocytochemistry and electron microscopy. The condition media is made by mincing autologous atrial tissue in tissue culture medium and incubating it for three days at 5% carbon dioxide and at 33-37 degree centigrade temperature. The cell free supernatant fluid is collected by centrifuging the culture at 800g-1000g for 20 - 30 minutes, at room temperature. Atrium has more precursors similar to that of cardiac muscle. As the atrial crush is autologous this avoids the occurrence of harm to the patient. The cells used in the process are autologous hence it is safe for clinical use, no antigenic reaction, no immunosuppression and thereby ethical issues do not arise. The atrial tissues have specific protein factors which lead to deriving of cardiomyocyte by co-culturing, with mesenchymal stem cells.
The present invention is potent in inducing cardiomyocytes precursors from other methods of deriving cardiomyocytes.
The present invention along with angiogenic therapy may create a fool-proof treatment for ischemic myocardial failure in"no option"ischemic heart diseases.

Isolation and identification of the atrial conditioning media may be helpful for intravenous delivery of the media, thereby enhancing the in vivo process of cardiomyocyte precursor to differentiate into cardiomyocyte whenever such situation warrants.
The mesenchymal therapy of the invention can be provided by several routes of administration such as Intracardiac muscle injection, which avoids the need for an open surgical procedure, can be used where the mesenchymal stem cells along with the condition medium are in an injectable liquid suspension preparation or when they are in a biocompatible medium which is injectable in liquid form and becomes semi-solid at the site of damaged myocardium. A conventional intracardiac syringe or a controllable arthroscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter that shear forces will not damage the mesenchymal stem cells and the condition media. The injectable liquid suspension preparations can also be administered intravenously, either by continuous drip or as a bolus.
Culturing Mesenchymal Stem Cells : A needle was punctured to the bone and used to aspirate the bone marrow and approximately lgm of specific tissue that has factors, which are potent transdifferentiators of the bone marrow derived stem cells. Different cells present at different portions of the bone marrow sample are obtained.
The sediment cells constitute majorly RBCs and the supernatant especially that of its central region posses the desired stem cells.
The particular part of the bone marrow aspirate was collected carefully in a sterile container containing preservative free heparin. The collected bone marrow aspirates were added into a petridish in which 5 ml of DMEM media with autologous serum in ratio of 1: 3 were already added. The cells were incubated with 95% air and 5% C02 at 33-37 C for 24 hours.

The procedure was repeated for two or more times and all the petridish were incubated for 7 days in the incubator.
After every 7th day the cells grown in petridishes were passaged.
To test the effect of cell passaging on cardiomyocyte differentiation of mesenchymal stem cells, samples of MSCs, prepared as described above were passaged once prior to preparing of cell cultures described below. In this case, the term passaging refers to the distribution of cultured cells growing in the culture dish into new culture dishes after the cells reach confluence i. e. when the cells contact each other and cover the surface of the dish. The passaging process was repeated every 3 weeks.
Culturing condition media: The 1 gm of specific tissue which has factors which are potent transdifferentiators of the bone marrow derived stem cells, were cut into Icm3 and washed with gentamycin containing the medium which is obtained from the plasma extracted from autologous blood.
The tissue condition media taken from transport medium are chopped into thin pieces of Imm3 size. Prior to the process of chopping, any additional fat present is separated from tissue. The chopped tissues are transferred to the petridishes containing 5ml of DMEM (5- 10% of autologous serum or umbilical cord blood serum) and left for incubation for 3 days at 33-37'C in C02 incubation (5% C02).
Preparation of condition media: The atrial tissues, which were grown in the petridishes after incubation period of 3 days, were used for preparation of condition media. The media along with the cells in it were mixed thoroughly for 3-4 times and then supernatant was collected in a centrifuge tube. The centrifugation process is performed to separate bone marrow cells and red blood cells. The collected media was centrifuged at 800-1000rpm for 5mins. After the centrifugation process the residue was left and the supernatant was

Collected and used as condition media. These petridishes were subjected to centrifugation.
After the condition media preparation was completed, it was stored at 0°C until the stem cells were ready for treatment. At the end of 3 weeks, the stem cells, which were grown in final passaging, were used. These petridishes were mixed thoroughly for 2-3 times and the supernatant was discarded. To. this fresh medium of about 5ml and 100). iL of the above-prepared condition media was added. These plates were left for incubation at 37°C for three days in 5% C02 incubator Two major types of cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells, were isolated from percoll gradients of bone marrow cells. The MSCs were spindle- shaped, attached to the culture dish tightly and proliferated in the culture medium. The hematopoietic stem cells were round-shaped, did not attach to culture dish, and were washed away with the culture medium changes.
The Atrial condition medium promoted the growth of bone marrow mesenchymal cells. The morphology of mesenchymal cells grown in the presence of atrial tissue condition media resembled the cardiomyocytes.
Qualitative analysis of the cell samples from the co- culturing experiments indicated that mesenchymal stem cells co-cultured with atrial tissues and 5-azacytidine formed more small striped, spindle shaped intercalated disc (cardiomyocyte precursors) as shown in Fig. 1 than those mesenchymal stem cells which were cultured with 5- azacytidine and ventricular cardiomyocytes. Mesenchymal stem cells did not form any cardiomyocyte precursors when cultured alone.
The present invention successfully proves that mesenchymal stem cells can be directed to differentiate into cardiomyocyte precursors by co culturing with atrial tissues. The present method that result into formation of cardiomyocyte precursors is more effective as compared to the existing prior methods that use

Ventricular tissues for co-culturing as it leads to an increased amount of cardiomyocyte precursor.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

WE CLAIM
1. A method and process of deriving cardiomyocyte from bone-marrow stem
cells, by co-culturing bone - marrow derived mesenchymal stem cells with
atrial condition media, in vitro wherein the stem cells are treated with 5-
azacytidine.
2. A method and process of deriving cardiomyocytes from bone marrow stem
cells of claim 1, wherein the said co-culturing comprises of stem cell culturing
and tissue culturing. The steps involved in culturing are as follows : a. The
atrial crush containing the tissue condition medium is centrifuged at
predetermined speed and temperature, to obtain atrial condition medium, b.
The bone marrow derived mesenchymal stem, cells are incubated and co-
cultured with atrial condition medium, c. The cultured condition medium is
incubated with 5% of C02 and then treated with 5- azacytidine at
predetermined concentration to derive the cardiomyocyte precursors.
3. The method and process of claim 1, wherein said mesenchymal stem cells
(sterile aspirate) are obtained from autologous bone marrow.
4. The method and process of claim 2, wherein said mesenchymal stem cells
are enriched on density gradient centrifugation.
5. The method and process of claim 2, wherein said enriched mesenchymal
stem cells are expanded in presence of growth factor.
6. The method and process of claim 2, wherein said mesenchymal stem cells
have been co-cultured with condition medium, in vitro.
7. The method and process of claim 2, wherein said mesenchymal stem cells
have been co-cultured with condition medium that have specific protein.

8. The method and process of claim 1, wherein said condition medium is
prepared by culturing atrial tissues in a petridish containing 5ml of DMEM (5-
10% of autologous serum or umbilical cord blood serum), in vitro.
9. The method and process of claim 1, wherein said condition medium is
prepared by culturing the tissue in a petridish containing 5ml of DMEM in 1: 3
ratio.
10. The method and process of claim 6, wherein said specific tissue are potent
in inducing cardiomyocytes from other stem cells including Embryonic Stem
cells.
11. The method and process of claim 1, wherein said mesenchymal stem cells
are exposed to 5- azacytidine.
12. The method and process of claim 1, wherein said 5- azacytidine is present
at a concentration of 5-10 millimoles 13. The method and process of claim 1,
wherein said mesenchymal stem cells have been cultured for atleast 3-4 weeks.
14. The method and process of claim 2, wherein the said centrifugation of atrial
crush is performed at a speed of 800-1000 rpm.
15. The method and process of claim 1, wherein said mesenchymal stem cells
are administered by injection.

A novel process and method for deriving cardiomyocyte precursors from bone-marrow stem cells
5 FIELD OF INVENTION
This invention relates to the field of deriving cardiomyocyte precursors from bone-marrow stem cells and specifically to the process of co-culturing mesenchymal stem cells, in 10 vitro.
More specifically, this invention provides controlled differentiation of human pluripotent stem cells to form cardiomyocytes precursors, using special culture 15 conditions and selection techniques.
BACKGROUND OF INVENTION
Most cardiac diseases if remain untreated eventually turn
20 into heart failure. In the western world, cardiovascular
diseases account for about 43% of deaths and for about
50% of hospitalizations. Cardiac diseases account for 80%
- 85% of cardiovascular death. Researchers have identified
a number of factors that put human heart at risk. Some of
25 the factors are smoking, high blood cholesterol, high blood
1

Pressure, increasing age, diabetes, obesity, lack of exercise etc.
There are low density and high-density lipoproteins in the blood. They are the fat or lipid content of the blood and especially the low-density lipoprotein is responsible for focal accumulation of lipids in the luminal layer of coronary artery. This process is called atherosclerosis. The most common form of arteriosclerosis is a process that in coronary arteries, as in other blood vessels consists of focal inner layer accumulations of lipids, complex carbohydrates, blood and blood products, fibrous tissue and calcium deposits and associated changes in the middle layer of the coronary artery wall, thus producing thickening of the inner layer and middle layer compromising the lumen.
Myocardial infarction in human beings is caused due to the narrowing of the coronary artery and thereby reduction of blood flow to the heart muscle producing ischemia followed by infarction. Ischemic heart disease is caused by an imbalance between the myocardial blood flow and metabolic demand of the myocardium. Reduction in coronary blood flow is related to progressive atherosclerosis with increasing occlusion of coronary arteries. Blood flow can be further decreased by

Superimposed events such as vasospasm, thrombosis or circulatory changes leading to hypoperfusion. The myocardial infarct signifies death of cardiac muscles in the areas of ischemia due to non-availability of blood and oxygen.
The cardiomyocyte responsible for mechanical pump function are post mitotical terminally differentiated cells that can no more divide and thus react to increased workload with hypertrophy, if they are alive to compensate for the dead cardiac muscle.
The cardiomyocytes are post mitotic and do not have any power of regeneration. Therefore, they are replaced by the scar tissue composed of fibroblast if there is myocardial infarction and necrosis of the cardiac muscle around the site of compromised blood flow. The fibroblasts are connective tissues and do not have the ability to polarization, depolarization and contractility. As a result of multiple infarcts the heart becomes very weak, especially the left ventricle which is required to pump blood into the aorta. There is a possibility for this scar tissue to balloon out and form aneurysm where the blood get sequestered and does not effectively reach the aorta; as a consequence all the organs of the body gets very less blood supply for them to function adequately including the

Heart itself. Heart gradually dilates because of scar tissue and aneurysm formation as it loses its elasticity and contractility. Laplace's law does not hold good anymore and leads to heart failure or pump failure that subsequently leads to death.
Conventionally for treating myocardial injuries cell transplantation techniques were used. Since heart dysfunction after myocardial injury is due to the loss of cardiac cells and scar expansion, replacing lost cardiac cells with muscle cells by cell transplantation may limit scar expansion and prevent heart failure. The transplanted cardiomyocyte may proliferate in vivo and grow to form myocardial tissue. At present there is no permanent solution to prevent heart from failure, however with the advent of the stem cell regenerative therapy it is possible to regenerate the lost heart muscle and thus treat these conditions.
Stem cells have the ability to divide indefinitely in culture and give rise to specialized cells constituting a tissue upon stimulation by a specific differentiation stimulus.
Stem cells are two types: embryonic stem cells (ES cells) and adult stem cells. ES cells are isolated from the inner cell mass (ICM) of embryos at the blastocyst stage and are totipotent, i.e., they are capable of differentiating into

Virtually every type of cells found in an organism, but once they start producing a specific cell type their cell division can become uncontrolled giving rise to tumour formation. Though there are methods for induction of differentiation of embryonic stem cells into cardiomyocytes, the transplanted ES cells can potentially form teratomas is some undifferentiated totipotent cells are still present. In addition, recipients must receive immunosuppressants because ES are allogenic. In contrast, mesenchymal stem cells do not carry any inherent risk of tumour formation and are syngeneic.
In contrast, tissue-specific adult stem cells are generally committed to give rise to cells constituting a specific tissue line. These tissue-specific stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically. Representative tissue-specific stem cells include hematopoietic stem cells and mesenchymal stem cells present in bone marrow. Hematopoietic stem cells give rise to various blood cells such as erythrocytes and leukocytes; and mesenchymal stem cells, to the cells of connective tissues, e.g., osteoblasts, chondroblasts, adipocytes and myoblasts.
Recently, clinical applications of the stem cells have drawn an increasing interest since the successful isolation of

Human embryonic stem cell. But due to uncontrolled cell division and to other ethical issues embryonic stem cell is not being pursued much in regenerative therapy.
The most noticeable potential application of the adult stem cells is their use as a perfect source for a cell replacement therapy, if they can be directed properly to grow the specific tissue. Hardly curable diseases, e.g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction and liver cirrhosis, are caused by the disruption and permanent functional disorder of the cells constituting an organ and the cell replacement therapy, wherein the loss of cells is replenished from the outside, has been presented as an effective remedy.
However, notwithstanding the obvious benefit of the cell replacement therapy, there exist many limitations in its clinical applications. Specifically, the conventional method, wherein fully differentiated cells isolated from the tissues of a donor are transplanted into a patient, has the problem of antigenicity. It requires life long immunosuppressive therapy and very often rejection of the transplanted tissue becomes the ultimate outcome. Over and above all these surgical and medical therapies carry a very high expenditure and requires continuous monitoring; of course

Patient is also subjected to the adverse effects of immunosuppression. That is why worldwide search has been focused on autologous stem cell therapy in regenerative medicine.
In the past decade, clinical observations have shown that bone marrow derived stem cells can migrate to the site of infarct and repair the damaged heart by transdifferentiating into cardiomyocytes. However this repair is not enough to improve the clinical conditions; the amount of stem cell induced cardiomyocytes produced are very small and as they are cardiomyocytes they can no longer multiply, and if the blood supply is not adequate they do not survive long. Therefore there is need for a process to differentiate bone marrow derived mesenchymal stem cells into cardiomyocyte precursor cells and implant these into the infracted area of the heart to derive healthy cardiomyocytes and thus treat the clinical conditions. Existing methods for deriving cardiomyocytes from bone marrow stem cells use a combination of fibroblast growth factor and 5-azacytidine. This method is very expensive due to the use of recombinant growth factors and also does not yield optimum numbers of cardiomyocyte precursors. Mesenchymal stem cells are capable of differentiating into more than one type of mesenchymal cell lineage. Mesenchymal stem cells have been identified and

Another objective of the invention is to identify, isolate and express as recombinant product, novel factors which can induce stem cells to differentiate cardiomyocyte precursors.
Yet another objective of this invention is to provide an inexpensive process of differentiating stem cells to cardiomyocytes precursor.
Mesenchymal stem cells are used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration. The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. The stem cells are co-cultured with atrial tissues.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the drawing, which respectively show: Fig. 1: a photomicrograph; (hereinafter, same magnification is applied) shows spindle shaped cells with intercalated disc like structure, resembling cardiomyocyte.

Fig 2 is a block diagram explaining the various steps involved in the process of co-culturing bone marrow stem cells and atrial condition media
Fig 3 shows the picture of human long-term bone marrow stroma (21 days old), from which the stem cells are obtained.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides method for differentiating mesenchymal stem cells isolated from bone - marrow into cardiomyocytes precursor.
The autologous mesenchymal stem cells differentiate into cardiac muscle cells and integrate with the healthy tissue of the recipient to replace the function of the dead or damaged cells, thereby regenerating the cardiac muscle as a whole. Cardiac muscle does not normally have reparative potential. The mesenchymal stem cells are used, for example, in cardiac muscle regeneration for a number of principal indications (i) - ischemic cardiomyopathyIk(ii) therapy for heart failure patients and (iii) reduction of scar tissue area in aneurysmal heart wall undergoing coronary artery bypass graft.

The proper environmental stimuli convert mesenchymal stem cells into cardiac myocytes. Differentiation of mesenchymal stem cells to the cardiac lineage is controlled by factors present in the cardiac environment. Exposure of mesenchymal stem cells to a simulated cardiac environment directs these cells to cardiac differentiation as detected by expression of specific cardiac muscle lineage markers. Local chemical, electrical and mechanical environmental influences alter pluripotent mesenchymal stem cells and convert the cells grafted into heart into cardiac lineage.
In order to derive cardiomyocyte precursors from mesenchymal stem cell, it is preferred to co-culture the mesenchymal stem cells with a condition medium. The condition medium has specific factors that are potent transdifferentiators of the bone marrow derived stem cells.
Mesenchymal stem cell (MSC) therapy can serve as a means to deliver high densities of repair- competent cells to a defect site when adequate numbers of mesenchymal stem cell and mesenchymal stem cell lineage - specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly producing large numbers of MSC are necessary. While MSC have been exposed to a number of growth factors in

Vitro, only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra), while none have been demonstrated to independently induce differentiation. Methods that increase the ex vivo proliferation and differentiation of MSC will greatly increase the utility of MSC therapy.
Similarly, methods that increase in vivo proliferation and differentiation of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site.
Furthermore, methods that enhance the proliferation of lineage-specific descendants of MSC, including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites.
Since adult stem cell banking has come into existence, specific conditioning media and/or the factors isolated from it along with 5-Azacytidine and angiogenic agents may protect the human heart whenever necessary.
Thus, there exists a need in the art for methods that increase the proliferation and differentiation of

Hematopoietic and mesenchymal pluripotent and lineage-specific cells that are useful in rapidly providing a large population of such cells for use in cell therapy and for making a large population of transfected cells for use in gene therapy.
The present invention involves a novel process of differentiating bone marrow derived mesenchymal stem cells, into cardiomyocyte precursor. Bone-marrow aspirates are collected in sterile containers containing preservative free heparin. The bone marrow is diluted with sterile tissue culture medium containing autologous serum and plated into sterile tissue culture petri dishes under a laminar airflow hood. These plates are incubated in 5%-7% carbon dioxide incubator at 33 - 37 degree centigrade for two hours. This procedure removes the adherent neutrophils and monocytes which inhibits the growth of other cells.
The cell suspension is again replated into fresh dishes and incubated as before for 24 hours. The non-adherent cells are transferred to fresh dishes and incubated as before. The adherent cells are again fed with fresh medium containing 10% v/v of atrial-conditioned medium. This procedure is repeated till three plates are made from the starting plate. Incubation with conditioned medium is for 7 days, with a weekly feeding with fresh medium. The plates

Are incubated for a total of three weeks. At the end of three weeks all the plates are treated with 5-Azacytidine for 24 hours before termination of cultures. The cells are fixed for immunocytochemistry and electron microscopy. The condition media is made by mincing autologous atrial tissue in tissue culture medium and incubating it for three days at 5% carbon dioxide and at 33 - 37 degree centigrade temperature. The cell free supernatant fluid is collected by centrifuging the culture at 800g - 1000g for 20 - 30 minutes, at room temperature. Atrium has more precursors similar to that of cardiac muscle. As the atrial crush is autologous this avoids the occurrence of harm to the patient. The cells used in the process are autologous hence it is safe for clinical use, no antigenic reaction, no immunosuppression and thereby ethical issues do not arise. The atrial tissues have specific protein factors which lead to deriving of cardiomyocyte by co-culturing. with mesenchymal stem cells.
The present invention is potent in inducing cardiomyocytes precursors from other methods of deriving cardiomyocytes.
The present invention along with angiogenic therapy may create a fool-proof treatment for ischemic myocardial failure in "no option" ischemic heart diseases.

Isolation and identification of the atrial conditioning media may be helpful for intravenous delivery of the media, thereby enhancing the in vivo process of cardiomyocyte precursor to differentiate into cardiomyocyte whenever such situation warrants.
The mesenchymal therapy of the invention can be provided by several routes of administration such as Intracardiac muscle injection, which avoids the need for an open surgical procedure, can be used where the mesenchymal stem cells along with the condition medium are in an injectable liquid suspension preparation or when they are in a biocompatible medium which is injectable in liquid form and becomes semi-solid at the site of damaged myocardium. A conventional intracardiac syringe or a controllable arthroscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter that shear forces will not damage the mesenchymal stem cells and the condition media. The injectable liquid suspension preparations can also be administered intravenously, either by continuous drip or as a bolus.
Culturing Mesenchymal Stem Cells: A needle was punctured to the bone and used to aspirate the bone marrow and approximately 1gm of specific tissue that has factors, which are potent transdifferentiators of the bone

Marrow derived stem cells. Different cells present at different portions of the bone marrow sample are obtained. The sediment cells constitute majorly RBCs and the supernatant especially that of its central region posses the desired stem cells.
The particular part of the bone marrow aspirate was collected carefully in a sterile container containing preservative free heparin. The collected bone marrow aspirates were added into a petridish in which 5 ml of DMEM media with autologous serum in ratio of 1:3 were already added. The cells were incubated with 95% air and 5% CO2 at 33-37°C for 24 hours.
The procedure was repeated for two or more times and all the petridish were incubated for 7 days in the incubator. After every 7th day the cells grown in petridishes were passaged.
To test the effect of cell passaging on cardiomyocyte differentiation of mesenchymal stem cells, samples of MSCs, prepared as described above were passaged once prior to preparing of cell cultures described below. In this case, the term passaging refers to the distribution of cultured cells growing in the culture dish into new culture dishes after the cells reach confluence i.e. when the cells

Contact each other and cover the surface of the dish. The passaging process was repeated every 3 weeks.
Culturing condition media: The 1gm of specific tissue which has factors which are potent transdifferentiators of the bone marrow derived stem cells, were cut into 1cm3 and washed with gentamycin containing the medium which is obtained from the plasma extracted from autologous blood.
The tissue condition media taken from transport medium are chopped into thin pieces of 1mm3 size. Prior to the process of chopping, any additional fat present is separated from tissue. The chopped tissues are transferred to the petridishes containing 5ml of DMEM (5-10% of autologous serum or umbilical cord blood serum) and left for incubation for 3 days at 33-37'C in CO2 incubation (5% CO2).
Preparation of condition media: The atrial tissues, which were grown in the petridishes after incubation period of 3 days, were used for preparation of condition media. The media along with the cells in it were mixed thoroughly for 3-4 times and then supernatant was collected in a centrifuge tube. The centrifugation process is performed to separate bone marrow cells and red blood cells. The

Collected media was centrifuged at 800-1OOOrpm for 5mins. After the centrifugation process the residue was left and the supernatant was collected and used as condition media. These petridishes were subjected to centrifugation.
After the condition media preparation was completed, it was .stored at 0°C until the stem cells were ready for treatment. At the end of 3 weeks, the stem cells, which were grown in final passaging, were used. These petridishes were mixed thoroughly for 2-3 times and the supernatant was discarded. To .this fresh medium of about 5ml and 100µL of the above- prepared condition media was added. These plates were left for incubation at 37°C for three days in 5% CO2 incubator
Two major types of cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells, were isolated from percoll gradients of bone marrow cells. The MSCs were spindle-shaped, attached to the culture dish tightly and proliferated in the culture medium. The hematopoietic stem cells were round-shaped, did not attach to culture dish, and were washed away with the culture medium changes.
The Atrial condition medium promoted the growth of bone marrow mesenchymal cells. The morphology of

Mesenchymal cells grown in the presence of atrial tissue condition media resembled the cardiomyocytes.
Qualitative analysis of the cell samples from the co-culturing experiments indicated that mesenchymal stem cells co-cultured with atrial tissues and 5-azacytidine formed more small striped, spindle shaped intercalated disc (cardiomyocyte precursors) as shown in Fig. 1 than those mesenchymal stem cells which were cultured with 5-azacytidine and ventricular cardiomyocytes. Mesenchymal stem cells did not form any cardiomyocyte precursors when cultured alone.
The present invention successfully proves that mesenchymal stem cells can be directed to differentiate into cardiomyocyte precursors by co culturing with atrial tissues. The present method that result into formation of cardiomyocyte precursors is more effective as compared to the existing prior methods that use ventricular tissues for co-culturing as it leads to an increased amount of cardiomyocyte precursor.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the

Invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

We Claim:
1. A method and process of deriving cardiomyocyte
from bone-marrow stem cells, by co-culturing bone
- marrow derived mesenchymal stem cells with
atrial condition media, in vitro wherein the stem
cells are treated with 5-azacytidine.
2. A method and process of deriving cardiomyocytes
from bone marrow stem cells of claim 1, wherein
the said co-culturing comprises of stem cell
culturing and tissue culturing. The steps involved in
culturing are as follows:
a. The atrial crush containing the tissue
condition medium is centrifuged at
. predetermined speed and temperature, to obtain atrial condition medium.
b. The bone marrow derived mesenchymal
stem.cells are incubated and co-cultured
with atrial condition medium.
c. The cultured condition medium is incubated
with 5% of CO2 and then treated with 5-
azacytidine at predetermined concentration
to derive the cardiomyocyte precursors

3. The method and process of claim 1, wherein said
mesenchymal stem cells (sterile aspirate) are
obtained from autologous bone marrow.
4. The method and process of claim 2, wherein said
mesenchymal stem cells are enriched on density
gradient centrifugation.
5. The method and process of claim 2, wherein said
enriched mesenchymal stem cells are expanded in
presence of growth factor.
6. The method and process of claim 2, wherein said
mesenchymal stem cells have been co-cultured
with condition medium, in vitro.
7. The method and process of claim 2, wherein said
mesenchymal stem cells have been co-cultured
with condition medium that have specific protein.
8. The method and process of claim 1, wherein said
condition medium is prepared by culturing atrial
tissues in a petridish containing 5ml of DMEM (5-
10% of autologous serum or umbilical cord blood
serum), in vitro.
9. The method and process of claim 1, wherein said
condition medium is prepared by culturing the
tissue in a petridish containing 5ml of DMEM in 1:3
ratio.
10. The method and process of claim 6, wherein said
specific tissue are potent in inducing

Cardiomyocytes from other stem cells including
Embryonic Stem cells. 11. The method and process of claim 1, wherein said
mesenchymal stem cells are exposed to 5-
azacytidine. 12The method and process of claim 1, wherein said 5-
azacytidine is present at a concentration of 5 - 10
millimoles
13. The method and process of claim 1, wherein said
mesenchymal stem cells have been cultured for
atleast 3-4 weeks.
14. The method and process of claim 2, wherein the
said centrifugation of atrial crush is performed at a
speed of 800 -1000 rpm.
15. The method and process of claim 1, wherein said
mesenchymal stem cells are administered by
injection.

Documents:

960-CHENP-2006 FORM-3 09-10-2009.pdf

960-chenp-2006 form-3 26-03-2010.pdf

960-CHENP-2006 CORRESPONDENCE OTHERS.pdf

960-CHENP-2006 CORRESPONDENCE PO.pdf

960-CHENP-2006 FORM 18.pdf

960-CHENP-2006 FORM 3.pdf

960-CHENP-2006 FORM 5.pdf

960-CHENP-2006 OTHER DOCUMENT 17-08-2009.pdf

960-chenp-2006-claims.pdf

960-chenp-2006-correspondnece-others.pdf

960-chenp-2006-description(complete).pdf

960-chenp-2006-drawings.pdf

960-chenp-2006-form 1.pdf

960-chenp-2006-form 26.pdf

960-chenp-2006-form 3.pdf

960-chenp-2006-pct.pdf

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Patent Number

241512

Indian Patent Application Number

960/CHENP/2006

PG Journal Number

29/2010

Publication Date

16-Jul-2010

Grant Date

08-Jul-2010

Date of Filing

20-Mar-2006

Name of Patentee

GUHATHAKURTA, Dr. Soma,

Applicant Address

D6 Casagrande, 13 & 14 Ellai Amman Koil Street Adyar, 600020 Chennai

Inventors:

#	Inventor's Name	Inventor's Address
1	GUHATHAKURTA, Dr. Soma,	D6 Casagrande, 13 & 14 Ellai Amman Koil Street Adyar, 600020 Chennai
2	AGARWAL, Dr. Ravi,	194/15 TNHB Apartments, R - 30A Annanagar West Extn, 600101 Chennai
3	KOTTURATHU MAMMEN, Dr. Cherian,	61(New no:18), Taylors Road, Kilpauk, 600010 Chennai
4	RAO, Dr. S G , Anand	72, Anugraha, SBM Colony, Anandnagar, 560024 Bangalore

PCT International Classification Number

C12N 5/06, A61K 35/12

PCT International Application Number

PCT/IN2003/000382

PCT International Filing date

2003-12-04

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	PCT/IN2003/000382	2003-12-04	India