Objectives this study is to explore the effect of differentiation of BMSCs into myocardial cells in different inducing conditions In vitro .
Methods BMSCs were isolated with the density gradient centrifugation combined with adherent method and cultured. The growth curve, cell cycle, phenotype of BMSCs were determined. BMSCs were induced to differentiate into osteoblasts and adipocytes. In vitro BMSCs were cultured in the medium supplemented with 5-azacytidine (5-aza) or serum of rat with DCM combined with 5-aza or cadiocyte lysate of rat with DCM. Then the morphological changes were observed and the expression of cardiac troponin was detected using the methods of immunocytochemistry and RT-PCR.
Results Most of BMSCs isolated with the density gradient centrifugation combined adherent method and cultured adhered to the wall with the colony growth. Passaged cells quickly adhered, proliferated rapidly, and almost no colonies were formed. About 6 - 7d later, the cells crowded the bottom of the flask with the spindle-shaped uniform distribution. After many passages, cells have a more uniform and orderly distribution of fibroblast-like. After passaged for consecutive 9 times, cells had no significant changes in morphology, no signs of ageing. The passage cycle is 6–7d, but after the 9 passage, the proliferation of cells was slow and the passage cycle was extended. The Passage1, 3, and 5 cells were high proliferation, the growth curve shape of which were similar. The majority of the cells were relatively quiescent. BMSCs could differentiate into osteoblasts and to fat cells under particular induced conditions. After the phenotypes of BMSCs were determined by flow cytometry, they were CD44-positive andCD34-negative. All the three methods could induce BMSCs to differentiate into cardiocytes-like cells with the expression of troponin, among which the growth state of cells in 5-aza group was poor; the cells in the other two groups grew better. The positive-expression rate of cardiac troponin of differentiated cells in the serum of rat with DCM combined 5-aza group was highest.
Conclusions In vitro the three methods can induce BMSCs to differentiate into cardiocyte-like cells. The induced effects were better in the medium supplemented with the serum of rat with DCM combined with 5-aza, the myocardium lysate of rat with DCM than that with 5-aza. The cells growth state and differentiation into cardiocyte-like cells were the best in the medium supplemented with serum of rat with DCM combined with 5-aza (5μmol / L).