Characterization and differentiation potential of rat bone marrow Mesenchymal stem cells into cardiac-like cells / Ramin Khanabdali

Khanabdali, Ramin (2014) Characterization and differentiation potential of rat bone marrow Mesenchymal stem cells into cardiac-like cells / Ramin Khanabdali. Masters thesis, University of Malaya.

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Abstract

Heart diseases are the leading cause of death worldwide. Despite the development of a broad array of treatment options, current therapies only delay progression of the disease and failed to prevent myocardial scar formation and replace the lost cardiomyocytes (cardiac muscle cells). Over the past decade the use of adult stem cells, particularly bone marrow derived mesenchymal stem cells, to safely facilitate recovery of cardiac function after myocardial infarction has received a lot of interest. Mesenchymal stem cells (MSCs), which are adherent stromal cells of a non-hematopoietic origin, have great differentiation potential and under appropriate in vitro culture conditions can trans-differentiate into cardiomyocyte cells. This study investigated the characterization of rat bone marrow derived-mesenchymal stem cells (BM-MSCs) and in vitro differentiation potential of them into cardiomyocyte-like cells by two DNA-demethylating agents, 5-azacytidine and zebularine. MSCs were isolated from Sprague Dawley’s bone marrow and cultured in complete Dulbecco’s Modified Eagle Medium (DMEM). Morphological characteristics of MSCs were analyzed by phase contrast microscopy. Selected surface antigens CD44, CD117, known MSCs markers, and CD34, a hematopoietic marker (negative marker), were analyzed by immunocytochemistry. In addition, CD45, known hematopoietic marker (negative marker) and CD44 were analyzed by flow cytometry for the MSC cell population count. Passage 1 (P1) cultured MSCs were then treated in separate culture flasks for 24 hours with a 3μM optimized concentration of 5-azacytidine and zebularine. After 20 days, treated cells were analyzed for the expression of rat cardiac specific genes; namely, alpha-myosin heavy chain (CAMHC), cardiac troponin-T (cTnT), and cardiac transcription factor (GATA-4) by reverse transcriptase polymerase chain reaction (RT-PCR). The endogenous housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard gene for normalization of mRNA. The isolation of MSCs from rat bone marrow was successfully completed. Isolated MSCs exhibited spindle-shaped morphology with adherence ability to the surface of flasks and proliferated in the culture medium. Immunocytochemistry results showed that cell surface antigen expression was observed to be positive for CD44 and CD117. However, MSCs were negative for CD34 (hematopoietic marker); hence, confirming the absence of hematopoietic cells. Furthermore, CD44 was found to be >85% positive, while CD45 was more than 60% negative in MSCs after flow cytometry cell population analysis. Upon induction with 5-azacytidine and zebularine, the morphology of the MSCs changed and the cells showed extended cytoplasmic processes with ball-like appearance. After 20 days, they were connected with adjoining cells forming myotube-like structures. The mRNAs of CAMHC, cTnT and GATA-4 were detected in both treated and untreated cells. However, RT-PCR analysis for the expression of cardiac specific genes showed that treated MSC cells expressed cTnT, CAMHC and GATA-4 significantly higher compared to untreated cells. While there were no significant differences between 5-azacytdine and zebularine treated cells, zebularine could be a good replacement for 5-azacytidine as it is more stable and less toxic to biological system. These results showed that bone marrow mesenchymal stem cells (BM-MSCs) could differentiate in vitro towards a cardiomyogenic lineage.

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