Abstract: [Abstract] Objective To investigate the role of different culture densities of embryoid bodies (EBs) in cardiac dif?ferentiation of mouse embryonic stem cells (ESCs). Methods  The mouse ESCs were cultured in hanging drops for 3 days,followed by another 2 days for suspension culture to form EBs. Suspended EBs of different densities (60 or 120 EBs/60 mm tissue culture dish) were transferred onto tissue culture plates. The cardiac specific troponin T (TnT) was detected by immunocytochemistry. The percentage of beating EBs was calculated at different time points. The mRNA expression of cardiac specific transcriptional factors Nkx2.5, GATA4 and cardiac specific proteins β-MHC and ANF were detected by RT-PCR. The protein expressions of TnT and p38 were detected by Western-blot assay. Results    Spontaneously beating EBs were positively stained with TnT. There were significantly higher percentage of beating EBs, higher gene expression levels of Nkx2.5,GATA4, β-MHC and ANF and higher protein expression of TnT in high density culture group than those of low density culture group (P＜ 0.05). Furthermore, there was significantly higher activity of p38 pathway in high density culture group than
that of low density culture group. And the percentages of beating EBs and TnT protein expression were decreased by p38 pathway inhibitor SB203580. Conclusion   The culture density of EBs is important in regulating the cardiac differentiation of ESCs. The high cell-density density culture of EBs enhances the cardiac differentiation of ESCs, which might be mediated by p38 signaling pathway.

Key words: embryonic stem cell, Cell count, myocytes, cardiac, cell differentiation, cell differentiation, p38 mitogen-activated protein kinases, 拟胚体