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.