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stem cellsadvantagestherapy to mycardial in farction
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ISOLATION AND CHARACTERISATION OF CARDIAC STEM CELLS
ISOLATION AND CHARACTERISATION OF CARDIAC STEM CELLSKHALIDHA NOORDEEN (2011305020)SHAMANTHIKA.T(2011305026)
PROJECT ADVISOR : Dr. C.D.ANURADHAOUR BODYS PUMP--HEARTPost mitotic organCoronary arteriesCells of the heartDISEASES BY HEART !!CARDIOVASCULAR DISEASES are silent diseases. In 2014By 2030 17.1 million23.6 million
RECENT THERAPIES AND THEIR CONTRAINDICATIONSThrombolytic agentsVentriculoplastyAngioplastyAllogenic transplant
MOST PROMISING TREATMENT THERAPY FOR THE PRESENT
STEM CELL TRANSPLANTATION THERAPY
RECENT THERAPIES< XX>Heparin, streptokinaseCONTRAINDICATIONShemorrhagic stroke or cerebro vascular events within 1 year intracranial neoplasmActive internal bleeding
Removal of damaged heart tissues CONTRAINDICATIONS results in the loss of a substantial portion of non diseased myocardium
THROMBOLYTIC AGENTSVENTRICULOPLASTY
reestablishes blood flow to ischemic myocardiumCONTRAINDICATIONSAllergic reaction ArrhythmiasBleeding,infeaction at the insertion siteStroke
PROBLEMS ASSOCIATEDTransplant availablityGraft Vs Host reactionANGIOPLASTYALLOGENIC TRANSPLANTREGENERATIVE THERAPY USING STEM CELLSform teratomas upon transplantation in vivo Ethical issues
Mesenchymal stem Cells
differentiation into fibroblastic scar tissue, which could impair recovery of hearts function
Limited or no true differentiation
Umbilical Cord Blood Stem Cells can form new blod vessels when infarcted int the heart
Embryonic stem cells
Human Adult Bone-Marrow Derived Cells
CARDIAC STEM CELLS in 2003, Beltrami, Barlucchi et al SELF RENEWING, CLONOGENIC, MULTIPOTENT; able to undergo differentiation into cardiomyocytes, smooth muscle, and endothelial cellspositive for various stem cell markers ckit, Sca1, Oct 3 / 4, SOX 2, von Willebrand factor, a-sarcomeric actin
WHY CARDIAC STEM CELLS??
ISOLATION OF CARDIAC STEM CELLSModel organism : Wistar Kyoto RatsMale/Female1-2 months old100g weightExplant method : Messina et al; 2004Higher yield of cells; Shorter time period; Cost effective
STEP 1 : Explant CultureSTEP 2 : Cardiosphere cultureSTEP 3 : Culture of CDCsExcision of rat hearts
Cut into small pieces
Place in fibronectin coated plates in CEM
Cells migrate from explants
* Process takes around 7-14 daysHarvest explant derived cells using trypsin
Place in poly D Lysine coated plates in CGM
Cardiospheres form in suspension
* Process takes around 4-5 days
Harvest CSs from suspension
Plate on fibronectin coated flasks
CDCs form monolayers
* Process takes around 3-4 days
Explant culture Fibroblast like cells being shed from explant 2 days post culture (10X magnification )Migration of phase bright round cells on fibroblast cells 12 days post culture(10X magnification )
Migration of phase bright round cells on fibroblast cells 12 days post culture(40X magnification )White arrow indicates explant border
CARDIOSPHERE CULTURE to enrich stemness xxCells begin to divide & form groups
Microenvironment resembling in vivo niche conditions.
Cardiosphere core with stem cell properties periphery with differentiated cell properties
Electrostatic repulsion between Poly-D-Lysine & CSCs CSCs in suspension
Fig 2: Sphere shaped clusters (cardiospheres) formed 14 days post culture ( 20X magnification)CARDIOSPHERE CULTURE
CARDIOSPHERE DERIVED CELL (CDC) CULTURE xxCells from spheres detach and adhere to fibronectin coated T-25 flasks
Lose spherical shape and become spindle shaped
Monolayer formed can be maintained for many passages and subjected for characterization.