Abstract: Objective To investigate the time-dependent effect of embryoid body (EB) adhesion on the cardiac differentiation of mouse embryonic stem cells (ESCs) and its mechanisms. Methods Hanging drop culture was used to induce ESCs differentiating into cardiomyocytes through forming EBs. Suspended EBs were transferred onto tissue culture plates at different differentiation days. The percentage of beating EBs was calculated at different time points. The mRNA expression of Nkx2.5, GATA4 and β-MHC were detected by RT-PCR. The phosphorylation of Src kinase was detected by Western-blot. Results The groups whose EB adhesion times were at differentiation day 3 or 4 had higher percentage of beating EBs and higher gene expression of Nkx2.5, GATA4 and β-MHC compared with those groups whose EB adhesion times were at differentiation day 5, 6 or 7（P<0.05）. EB adhesion triggered the phosphorylation of Src kinase, which was inhibited by PP2（P<0.05）. Furthermore, the percentage of beating EBs and gene expression of β-MHC were enhanced by PP2 at differentiation day 4~6 in the group whose EB adhesion time was at differentiation day 4（P<0.05）. Conclusion Our results indicate that the EB adhesion time is important in regulation of the cardiac differentiation of ESCs. When EBs adhere to gelatin-coated plates at differentiation day 4 or earlier, the cardiac differentiation is significantly inhibited. The mechanism is likely that EB adhesion triggers the phosphorylation of Src kinase.

Key words: embryonic stem cell, cardiomyocyte, adhesion, Src kinase