VISUAL CONFIRMATION OF ISOLATED BONE MARROW MESENCHYMAL STEM CELLS AND DIFFERENTIATED CARDIOMYOCYTE-LIKE CELLS
M. Noorezatey, K.B. Khairul Fidaa, K. Ramin, A.S. Shamsul Azlin, H.A. Durriyyah Sharifah* and S. Asmat
Abstract
VISUAL CONFIRMATION OF ISOLATED BONE MARROW MESENCHYMAL STEM CELLS AND DIFFERENTIATED CARDIOMYOCYTE-LIKE CELLS
- Noorezatey1, K.B. Khairul Fidaa1, K. Ramin1, A.S. Shamsul Azlin1, H.A. Durriyyah Sharifah1 and S. Asmat2
1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia and
2International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.
ABSTRACT
This project attempted in vitro protocols to isolate, culture, expand and induce bone marrow mesenchymal stem cells (BM-MSCs) into cardiomyocyte-like cells. Mixed hematopoietic and mesenchymal bone marrow cells were isolated from Sprague Dawley rats. They were then cultured for 15 days in complete Dulbecco’s Modified Eagle Medium (DMEM), during which the media was changed every 3 days to remove unattached floating hematopoietic cells. Inverted phase contrast microscope was used for morphological detectection of BM-MSCs. Immunocytochemistry was also done to confirm BM-MSCs characteristics based on specific protein markers (positive markers; CD44 and CD117; negative marker: CD34) and secondary antibody IgG Alexa Fluor, visualized by fluorescence microscope. Subsequently, 10-15 days separate treatments of synthetic demethylating agents, 5-azacytidine and zebularine, were conducted to induce differentiation of BM-MSCs into cardiomyocyte-like cells. Acquired mixed bone marrow cells of hematopoietic and mesenchymal cells were seen morphologically roundish/ spherical in shape at the initial culturing. Progressively, more BM-MSCs displaying spindle-shaped fibroblast morphology were observed. Immunostained cells successfully confirmed these BM-MSCs. The separate treatments by selected inducers resulted in morphologically cardiomyocyte-like cells; each having extended cytoplasmic processes and a myotube-like structure. It can be concluded that the protocols used could successfully develop BM-MSCs to cardiomyocyte-like cells.
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Keywords: Bone Marrow, Stem Cell, Cardiomyocyte-like Cell
