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STEM CELL THERAPY FOR MYOCARDIAL REGENERATION OF HUMAN CELLS USING SPECIAL RECEPTOR MARKERS CD45, CD34

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STEM CELL THERAPY FOR MYOCARDIAL REGENERATION OF HUMAN CELLS USING SPECIAL RECEPTOR MARKERS CD45, CD34 S. SHARANYA 1 and S. SUBHA 2 1 Department of Biotechnology, Sathyabama University, Chennai, Tamilnadu,
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STEM CELL THERAPY FOR MYOCARDIAL REGENERATION OF HUMAN CELLS USING SPECIAL RECEPTOR MARKERS CD45, CD34 S. SHARANYA 1 and S. SUBHA 2 1 Department of Biotechnology, Sathyabama University, Chennai, Tamilnadu, India. 2 Department of Biotechnology, St. Michael College of Engineering and Technology, Kalayarkoil , Tamilnadu, India. Correspondence ABSTRACT Stem cells are undifferentiated and pluripotent cells that have the ability to proliferate and potentially self renew throughout the life. The concept of stem cells therapy, the repair of damaged or diseased tissue by transplantation of healthy cells is a innovative and promising therapeutic approach for heart failure. Since the stem cells are derived from the patient s own body, there is no possibility of the body rejecting its own stem cells and this treatment is said to be Autologous. Small aspiration of bone marrow from the Hipbone is done with minimal discomfort using local anesthesia and this process takes about 15 minutes. The aspirate is processed in the laboratory using the latest cell separator technology using special receptor markers such as CD45, CD34 and this process takes about 3 hours. The purified stem cells solution is confirmed bacteria free by means of a Endotoxin test and the stem cells are then injected either into the coronary arteries via special catheter or directly into the heart muscle. Thus the stem cell therapy has sparked intense hope for myocardial regeneration with cells that are from the patient itself and thereby inherently programmed to reconstitute cardiac tissue. Key Words: Stem cell therapy, myocardial regeneration, receptor markers. 1. INTRODUCTION The heart is a muscular pump that provides the force necessary to circulate the blood to all the tissues in the body. Its function is vital because to survive, the tissues need a continuous supply of oxygen and nutrients and metabolic waste products have to be removed. The normal heart pumps about 5 litres of blood every minute throughout life. Deprived of these necessities, cells soon undergo irreversible changes that lead to death. The pathologic conditions of the heart result in loss of cardiomyocytes, scar formation, ventricular remodeling and eventually heart failure, (Barbash et al., 2006) which continuous to be a major health problem worldwide. Stem cells are undifferentiated and pluripotent cells that have the ability to proliferate and potentially self renew throughout the life. Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body, they can divide without limit to replenish other cells as long as the person is still alive. Stem cells have two important characteristics that distinguish them from other types of cells. First they are unspecialized cells that renew themselves for long periods through cell division. The second is that under certain physiologic or experimental conditions, they be induced 1 to become cells with special function such as the beating cells of heart muscle. The stem cells provide a potential therapy for cardiac disease. The potential use of stem cells to treat cardiac diseases has recently been suggested from preclinical and clinical studies. Numerous experimental studies have suggested that stem cells can exert beneficial effects on the failing heart by transdiffertiating into cardiac cell types. These studies demonstrated the potential clinical effectiveness of stem cells approach to the treatment of cardiac diseases. The stem cells are derived from patients own body there is no possibility of the body rejecting its own stem cells and the treatment is said to be autologous (Menasche et al.,2004), (Touchette et al.,2004). These cells carry a marker protein on their surface, which enables the researchers to identify them. Researchers chose the cells bearing these marker proteins because they are believed to have a high probability of becoming blood vessel cells (Emerson et al., 2004).The stem cells may be delivered by means of surgical injection, inter coronary infusion, retrograde venous infusion, transendocardial injections and through catheter (Perin et al.,2004). The injected stem cells fuse with the cardiac cells to produce new muscle (myocyte) cells an forms new blood vessels, the stem cells differentiate or mature by themselves that provide new endothelial cells(aasahara et al., 1997; Assmus et al., 2002) that patch vessel damages that improve blood flow to the heart that repopulate the ailing organ (Anderson et al.,2007). Adult stem cell therapy is used by cardiologists treat heart diseases such as coronary artery diseases, cardiomyopathy congestive heart failure. The stem cells are harvested from the patient itself and this makes the procedure very safe and non controversial. This therapy creates new blood vessels that prove blood flow to the heart as well as generate new tissue in the heart muscle itself (Therative s, Times magazine). The present investigation was aimed to repair the damaged heart by transplantation of healthy cells by stem cell therapy. Small aspiration of Bone Marrow from the hip area is done with minimal discomfort using latest painless anesthesia and this process takes place about 15 minutes. The sample is processed in the lab using the latest cell separator technology using special reporter markers and this process takes about 3 hours. The stem cell solution is confirmed bacteria free by means of a endotoxin test and stem cells are than injected either into the coronary arteries via special catheter directly into the great muscle along angioplasty angiogram, keyhole with some growth factors which enhances the differentiation of stem cells into cardiomyocytes. 2. MATERIALS AND METHODS BLOOD SAMPLE COLLECTION AND STORAGE The collection of blood sample was done in a totally sterile fashion from the Hipbone (Bone Marrow). Aspiration was done by a specialist and done with minimal discomfort using latest painless anesthesia and this process takes about 15 minutes. Blood samples should be processed as soon as possible after collection to ensure optimal results. Storage for 24 hours at room temperature has been reported to result in reduced lymphocyte yield. For lymphocyte separation, defibrinated or anticoagulant treated blood was diluted with an equal volume of Phosphate Buffered Saline and layered over ficoll paque PLUS (without intermixing) in a centrifuge tube. After a short centrifugation at room temperature (typically at 400 g for min) lymphocytes, together with monocles and platelets, are harvested from the interface 2 between the Ficoll paque PLUS and sample layers. This material was then centrifuged twice to wash the lymphocytes and to remove the platelets. To a 5 ml test tube add 15 ml of defibrinated or anticoagulant treated blood and diluted with equal volume of phosphate buffered saline (Final volume 30 ml). Mix gently by drawing the blood and the buffer in and out of Pasteur pipette without formation f any air bubbles. Viability count was performed with the help of haemocytometer. The % viability of the cell suspension was calculated. LAL TEST (ENDOTOXIN TEST) The LAL test was the most sensitive and specific means to detects and measure endotoxin, which was a fever producing by product of gram negative bacteria commonly known as pyrogen enzymatic test is that endotoxin produces an opacity and gelatin in reaction with LAL that is readily recognized FOLLOW UP Follow up period of a patient was very important in determining the success of the stem cell therapy performed. Numerous techniques can be employed for determining the success of stem cell therapy which includes chest X ray, Electrocardiogram, Echocardiography, cardiac magnetic resonance imaging and nuclear cardiology. Cardiac MRI Advanced technique with high resolution image quality accurately measure cardiac volumes, wall thickness and left ventricular mass, also reliably detects thickened pericasdwim and quantiates mycocardial necrosis, perfusion and function. Nuclear cardiology provides accurate measurements of left and to a lesser extent, right ventricular ejection fractions cardiac volume and regional wall motion, diastolic function can also be analysed.echocardiography establish the presence of systolic and/ or diastolic impairment of the left or right ventricle. They also reveal actiology (value disease, regional wall motion abnormalities in ischaemic heart disease, cardiomyopathy). Chest X ray reliable technique used for detecting cardiac size and evidence of pulmonary congestion. 3. RESULT AND DISSCUSIONS ISOLATION OF LYMPHOCYTES Lymphocyte isolation preceded normally using Ficoll-Paque PLUS and resulted, in combination with purification of granulocytes from the red blood cell pellet by dextran sedimentation.the upper layer, which contains the plasma. Lymphocytes which were present next to plasma were creamy white in colour were isolated successfully. RBC settles at bottom of the conical bottom centrifuge tube due to higher density. Ficoll-Paque PLUS seen above RBC. 3 Table 1. TOTAL VIABILITY COUNT OF BONEMARROW BMMNC s S.NO SAMPLE NO QUANTITY OF SPECIMEN PROCESSED TOTAL COUNT OF BMMNC S ml million cell suspended in ml 537 million cell suspended in ml 210 million cell suspended in ml 670 million cell suspended in ml million cell suspended in ml 530 million cell suspended in ml 540 million cell suspended in ml million cell suspended in ml 220 million cell suspended in ml 840 million cell suspended in 40 REPORTED AVERAGE 4 TOTAL VIABILITY COUNT S AM P LE NUM BE R TOTAL COUNT OF BMMNC'S Fig 1. The Total viability count of samples taken for BMMNCS x FLOW CYTOMETRY REPORT POSITIVE % OF LEUKOCYTES FOR CD34 SAMPLE NUMBER Fig 2. The flow cytometry report of positive leukocytes of CD34marker. 5 X FLOW CYTOMETRY REPORT POSITIVE % OF LEUKOCYTES FOR CD45 SAMPLE NUMBER Fig 3. The flow cytometry report of positive leukocytes of CD45marker. DISCUSSIONS The collection of sample was done in a totally sterile fashion from the Hip bone (Bone marrow).bone marrow aspiration was done by a specialist and done with minimal discomfort using latest painless Anaesthesia and this process takes about 15 minutes. The stem cells were injected into the Heart vessel. The injection was done painlessly by specialists. The follow up was done at three months, six months after the stem cell therepy. The various techniques employed are Chest x ray, Electrocardiogram, Echocardiography; Magentic Resonance Imaging and Nuclear imaging.the follow up report suggested that stem cells have beneficial effects such as infract size reduction, improvement in ejection fraction, myocardial perfusion and wall motion.patel AN et al., (2005) performed adult stem cell transplantation in patients with performed adult stem cell transplantation in patients with ischemic cardiomyopathy and an ejection fraction of less than 35% who were scheduled for primary off pump coronary artery bypass grafting, angiographic follow up was done, the ejection fraction of the off pump coronary artery bypass grafting plus stem cell transplantation group were as follows: preoperative, 30.7% +/- 2.5% versus 29.4% +/- 3.6%; 1 month, 36.4% +/-3.0% versus 42.1% +/- 3.5%; 3 months 37.2% +/-3.0 versus 45.5% +/-2.2% and 6 months 37.2% +/- 3.4% versus 46.1% +/-1.9%. The autologous stem cell transplantation led significant improvement in cardiac function in patients undergoing off pump coronary artery bypass grafting for ischemic cardiomyopathy, since the stem cells are harvested from the patient itself there is no problem of rejection and also there is no need for expensive immunosuppressive drugs. 6 REFERENCE 1. Mensache P Autologous skeletal myoblast transplantation for cardiac insufficiency. Nature (2002).420: Mensache P Myoblast transplantation for heart failure. Lancet (2002).352: Drexler H, Meyer Gand Wollert k.bone marrow derived cell trans after ST elevation myocardial infraction.cardiovascu Med (2006). 1: Medvinsky A and Smith SStem cell fusion brings down barriers.nature (2003).42: Touchette F and Nancy G Bone marrow stem cell transplantation for heart disease.genome news network (2004).45: Perin E, Geffner L and Vina RF Transen-docardial autologous bone marrow transplantation for severe ischemic heart Failure. Circulation (2001).130: regeneration enhancement in acute myocardial infraction. Circulation (2002).106: Grigoropolous N and Mathur A Stem cells in cardiac repair. Curr Pharmol (2002).6 (2): Orlic D, Kajstura J and Chimentic S.Bone marrow cells regenerate infracted myocardium. Nature (2001).410: Kalka C, Masuda H, Takahashi M Transplantation of exvivo expanded progenitor neovascularization. Science (2002).97: Kocher A,Schustec M and Szabolics M.Neovascularization of ischemic myocardium By human bone marrow derived angioblasts prevents cardiomyocyte apoptosis, reducesremodeling and improves cardiac function. Nature (2002).7: Fraser J, Schreiber R, Zuk, and Hedrick P.Adult stem cell therapy for heart. Inti J.Biochem.Cell Biol (2004).36: Dawn B, Zuba surma E and Tiwari S Cardiac stem cell therapy for myocardial regeneration. Minnerva cardioangol (2005).53 (6) Perin E, Silva G and donk H.Stem cell therapy for cardiac diseases. Nature (2004).11 (6): Aasahara T, Murohara Tand Sullivan A.Isolation of putative Progenitor Endothelial cells for angiogenesis. Science (1997).275: Assmus B, Schachinger Vand Teupe C.Transplantation of progenitor cells and 16. Anderson M, Edward T and Yeh Sticky proteins fuse adult stem cells to cardiac muscle repairing hearts.circulation (2002).109: Patel AN, Dohmann HF and Borojevic R Surgical treatment for congestive heart failure with autologous bone marrow celll transplantation for severe ischemic heart failure. Circulation (2003).106: Segev H, Kenyagin B and Fishman B.Molecular analysis of cardiomyocytes derived from human stem cells. Nature ((2006).47:
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