The Study Of Inducing Human Marrow Mesenchymal Stem Cells To Differentiate In Vitro Into Cardiomyocytes

Objective: To probe the condition for cultivation ,expansion of human marrow mesenchymal stem cells (hMSC) from human bone marrow and inducing them to differentiate in vitro into cardiomyocytes. Methods: 1. Human marrow cells were obtained from children sternum of 1-5 year old with congential heart disease when the CPB operation had been performed, collected by Percoll gradient centrifugation. These cells were cultured and expanded in DMEM medium with 20% FBS. At last, human marrow mesenchymal stem cells (hMSC) can be obtained. About several days after cultivation, the cells were treated with 5-azacytidine and observed. The contents of DNA and CD-antigen, such as CD14, CD31, CD34, CD45, CD71, CD90, were tested in the cells of passage 2 and passage 6,7 respectively. The number of cells was counted before and on the 15,24,38 day of culturing. 2. Some heart muscle cells were obtained from the children of 1-5 year old with congential heart disease when the CPB operation had been performed. A part of cells were used to culture in vitro , the other was used to analyse with immunohistochemistry and observe with Electron microscopy. Results: Human marrow cells began to confluent after 3 days when cultivated. About 9 days or so, the cells began to hyperpleasia very quickly. The DNA content observation showed that S-stage cells of passage 2 was accounted for 19.2% .The number of the cultured cells was (4.56 0.14) 106, (3.21 0.35) 107, (5.23 0.19) 108, (7.32 0.56) 109. These cells of passage 6,7 still can not negative to anti-CD14, anti-CD31, anti-CD34, anti-CD45, anti-CD71 and positive toanti-CD90.The cells were treated with 5-azacytidine, but the typical ultrastructure of cardiomyocytes can not be seen with electron microscopy. The developing heart muscle cells for cultivation of passage 5,6 in vitro still can keep the ultrastructure of cardiomyocytes. They also can be stained with anti-Actin , anti-Myosin, anti-Desmin, but the cells began to hyperpleasia slowly after passage 4 and change the shape gradually. The cells of passage 6 almost stopped to grow. Conclusion : Human marrow mesenchymal stem cells(hMSC) were multipotent stem cells. These cells can be collected by Percoll gradient centrifugation , moreover these cells can be cultured in vitro for a long time, subcultured for several times and still maintain the characteristic of stem cells .It was a good experimental basic of using these cells in the future. The developing cardiomyocytes could be proliferated in culturing in vitro. It may be a ideal material of providing cells for the celluar cardiomyoplasty, it may also provide the methods of assay cardiomyocytes.