Isolation, expansion and characterization of bone marrow derived mesenchymal stem cells and their differentiation into skeletal myocytes in canines
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Date
2017-06
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
The adult stem cell therapy is a blooming area of clinical research and relevance. The therapeutic benefits of mesenchymal stem cells in veterinary regenerative medicine are numerous as they are in human medicine. The characterization of canine derived MSCs is poorly mentioned in literature. The mesenchymal stem cells in canines can be potentially used for cell based therapy to regenerate damaged or lost muscle cells. The motive behind this study was fixed on the differentiation potential of canine mesenchymal stem cells into skeletal
myocytes in vitro. An adult male mongrel dog of 3 years of age and 24kg weight was used for this study. The bone marrow aspirate was aseptically collected from ileac crest of the dog after administration of general anaesthesia by 2.5% thiopentone sodium till effect and atropine
sulphate and diazepam as pre-anaesthetics. The bone marrow aspirate was processed in laboratory within 4 hours of its collection. Mesenchymal stem cells were isolated by density gradient centrifugation technique and cells were seeded in T-25 tissue culture flasks. After
attainment of 80-90% confluence, first passage was performed by using Trypin-EDTA to expand the cell population. Cells were passaged till 4 passages and then grown in skeletal myogenic differentiation media for 21 days in CO2 incubator at 37 ° C and 5% CO2. The morphology of cells was observed at regular intervals and the cells were characterized by use of Hoechst 33342 stain after 21 days with positive result which coloured the nuclei blue. The RT-PCR for the genes Myogenin and Myo-D revealed bands on days 7 and 14 confirming the differentiation process as early differentiation factors. The striations were observed when the sample on gelatin scaffold was subjected to scanning electron microscopy and close association of parallel arranged cells with striations when the pellet was subjected to electron microscopy. On the basis of this study it was concluded that MSCs possess potential to trans differentiate to skeletal myocytes when cultured in skeletal myogenic media in vitro and the identification techniques are successful. The meticulous exploitation of MSCs in tissue engineering and regenerative therapy in veterinary medicine is the need of the hour.
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