ABSTRACT
Increasing attention is being paid to the use of mesenchymal stem cells [MSCs] for treatment of human diseases such as myocardial infarction. To study the differentiation of the bone marrow mesenchymal stem cells [BM-MSCs] into cardiomyogenic cells using 5-azacytidine. Forty adult male albino rats were used in this study. BM-MSCs were isolated and cultured in a complete Dulbecco's modified Eagle's medium containing 1% antibiotics and 10% fetal bovine serum [the control group]. Second passaged cells were treated with 10micro moI/I 5-azacytidine for 72h. Then, the medium was removed and kept in a 5-azacytidine-free medium for 4 weeks [the 5-azacytidine-treated group]. The adherent cells of both groups were examined using a phase-contrast microscope and a transmission electron microscope. Expressions of cytoskeleton protein desmin and cardiac muscle-specific cardiac troponin T were assessed by immunohistochemistry. BM-MSCs of the control group were spindled and star shaped with multiple processes and vesicular nuclei. After adding 5-azacytidine for 1 week, the cells showed multinucleation. On the second week, the cells formed stick-like structures. The cells showed extensive cytoplasmic striations in the third week. Finally, in the fourth week, the cells formed myotube-like structures. Immunohistochemical staining of cells of the 5-azacytidine-treated group revealed a positive immune reaction for desmin and cardiac troponin-T. Ultrastructural examination of the 5-azacytidine-treated group revealed that the cells were elongated with central oval large nuclei. The mitochondria were elongated with well developed cristae. There were abundant free ribosomes and extensive dilated rough endoplasmic reticulum. Myofibrils started to appear in the peripheral part of the cytoplasm and T-tubules appeared. MSCs can be differentiated in vitro by 5-azacytidine into cardiomyogenic cells, which are important for repairing infracted myocardium
Subject(s)
Azacitidine , Cell Differentiation , Myocytes, Cardiac/physiology , Mesenchymal Stem Cells/ultrastructure , Microscopy, ElectronABSTRACT
Mesenchymal stem cells [MSCs] have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. To isolate, increase the expansion rate and define rat bone marrow MSCs by immunohistochemical and electron microscopic studies. Twenty adult male albino rats were used and divided equally into two groups. The bone marrow was harvested and flushed out from the long bones of each group. In group 1, the cells were cultured using complete medium containing Dulbecco's modified Eagle's medium, 1% antibiotics, and 10% fetal bovine serum. In group 2, the cells were cultured in complete medium supplemented with 8% fetal bovine serum and 2% autologous rat serum. When the primary culture became nearly confluent, the adherent cells were subcultured. After 12 days from the primary culture, aliquots of the cells were prepared for Giemsa stain, transmission electron microscopy, and immunohistochemical staining for CD44, CD105, and CD34. The adherent cells in both groups were heterogeneous in appearance, and most of them were spindle or star-shaped with vesicular nuclei. Some cells were binucleated. The MSCs in group 2 reached confluency more rapidly than those in group 1. After passaging of group 2, the adherent cells became more homogeneously fibroblast-like in appearance. Immunostaining of MSCs revealed a positive reaction for CD44 and CD1 05 and a negative reaction for CD34. Ultrastructural examination revealed that the native MSCs appeared with many pseudopodia, the nucleus was eccentric, and the inner zone of the cytoplasm was rich in free ribosomes, many mitochondria, few rough endoplasmic reticulum, with obvious Golgi complex, and some lysosomes. Some MSCs showed no pseudopodia with central nucleus. Others appeared with two euchromatic elliptical nuclei and a nucleolus in one of them. MSCs can be easily purified, cultured, and expanded more rapidly using autologous rat serum