| Background and objectiveBone marrow derived Mesenchymal stem cells (Bone marrow MSCs) is another group ofstem cells in bone marrow besides hematopoietic stem cells(HSCs). Bone marrow MSCs candifferentiate to a variety of mesenchymal tissue, including bone, cartilage, fat, and even muscletissue. Bone marrow MSCs are widely used in tissue engineering and cell transplantation inrecent years. In any stage of many heart diseases can occur fatal arrhythmia such asⅢ°atrioventricular block and sick sinus syndrome. Some researchers used the bone marrow MSCsto create biological pacemakers or biological conduction system to treat the fatal arrhythmia.In our investigation, rabbit bone marrow MSCs were treated with 5-azacytidine, andautologously transplanted to left atrioventricular junction to restore a new electricaltransmission passageway which was detected by cardiac electrophysiology methods. We aimedto explore a new therapy for atrioventricular block using biological method.Materials and methodsZelanian rabbits were used as the experimental animals. Three methods of isolating andculturing the bone marrow MSCs were compared, including the Percoll's discontinued densitygradient solution, the whole blood culture method and the Ficoll-Hypaque method. RabbitMSCs were isolated from the bone marrow, and were purified, then expanded by repeatingpassaging in vitro. The MSCs of passage 0, 2, 4 and 6 were treated with 5-azacytidine. Invertedmicroscope and electronmicroscope were used to observe the differentiated phenotype of MSCs.Immunocytochemistrical method was applied to detecting the expression of myocardial cellrelated antibody after differentiation induced by 5-azacytidine. Differentiated MSCs labeledwith DAPI were autologously transplantated into the left atrioventricular junction of rabbits'hearts to create a new atrioventricular electrical transmission pathway. We used the regular cardiac electrophysiologic method to detect the formation of new atrioventricular pathway andits electrophysiologic characteristics. The electrophysiologic characteristics of the newatrioventricular pathway inⅢ°atrioventricular block model were also detected. Moreover, wealso observed the structural features of the myocardiac cells transplantation region usingpathological and immunohistochemical staining techniques.ResultsAmong the three isolation methods, Percoll's solution is the best and in all parameters andhas the highest successful ratio. Fourteen days after treated with 5-AZA, the passage 2, 4 and 6MSCs formed the spindle-shaped multinucleated cells and myotube-like structures were foundafter 24 days. The generation 2, 4 and 6 MSCs treated with 5-AZA were stained positively forcardiacα-Sarcomeric Actin by immunohistochemistry. Furthermore, there were moreα-Sarcomeric Actin positive cells in passage 4 MSCs than in passage 2 or 6 MSCs. Myofilamentwas observed in all of the three passages under transmission electron microscope. However, wecan't observe any positive outcomes in primary cultured MSCs treated with 5-AZA 72 hoursafter cell isolation. After transplantation of DAPI labeled bone marrow MSCs, no positiveevidence of atrioventricular pathway formation was found by electrophysiologic method.Pathological and immunohistochemical staining techniques showed that there were bone marrowMSCs in the atrioventricular junction.Conclusions(1) The Percoll's discontinued density gradient solution is the most preferable method forthe isolation of the bone marrow MSCs in adult rabbits.(2) The MSCs can be induced to differentiate to cardiac-like cells by 5-AZA. The highamplification potential and the cell seeding density are essential for the MSCs to differentiate tocardiac-like cells in passage culture.(3) The differentiated MSCs can grew at the left atrioventricular junction where they weretransplanted. However, routine cardiac electrophysiology examination shows no evidence tosupport that the new electrical transmission passageway is established.
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