Kandpal, ManjulKhalid Mubarik2018-07-052018-07-052017-07http://krishikosh.egranth.ac.in/handle/1/5810057085Mesenchymal stem cells are valuable source of multipotent cells which have the ability to differentiate into various types of tissue cells and contribute to the regeneration of a variety of mesenchymal tissues including bone, cartilage, muscle, and adipose. Bone marrow-derived mesenchymal stem cell (MSC)-based therapy is an emerging approach for the treatment of both acute and chronic heart failure. The aim the current study was to focus on canine mesenchymal stem cells and to analyse their differentiation capacity into cardiomyocyte phenotype in vitro. An adult mongrel dog of 5 years of age weighing 25 kgs was used in this study. Bone marrow was aspirated from iliac crest after anaesthesia using xylazine and ketamine. Mesenchymal stem cells were harvested using density gradient centrifugation from the mononuclear cells of bone marrow. The cells were seeded in tissue culture flasks and allowed to grow in monolayer till the attainment of 80-90% of confluency. At this confluency passaging was done using trypsin-EDTA. The cells were subcultured upto four passages. These cells were characterized at their baseline for morphology, culture behavior and also by scanning electron microscopy. At passage three, the cells were exposed to medium containing 5-Azacytidine (10µl/ml) to induce differentiation into cardiac myocyte. The media was changed into media without 5-Azacytidine after 24 hours. The cells were cultured for 21 days in an incubator maintained at 5% CO2 and 37oC. The differentiation of these cells was then analysed using cellular morphology, gene expression and scanning electron microscopy. PCR was carried out for expression of genes Nkx 2.5, Cardiac troponin-T (cTnT), β-Cardiac myosin in differentiated cells. SEM was also done for analyzing the morphology of differentiated cells. Canine MSCs demonstrated morphological changes towards cardiomyocyte like phenotype during differentiation. Cells assumed a stick-like morphology and were connecting with adjoining cells forming myotubelike structures. The cells demonstrated increased expression of cardiac specific markers. On the basis of results obtained in this study it was concluded that Mesenchymal stem cells possess potential for differentiating into cardiac myocyte like cells in vitro. The exploitation of these in vitro harvested cardiac myocyte like cells may prove an effective therapy for cardiac repair under various pathological conditions.ennullThesis