Daily qRT-PCR analysis in aggregate differentiation of hESCs.

<p>Undifferentiated aggregates of hESCs were treated by Activin A for 1 day and then for 4 days by BMP4. At day 5, the aggregates were plated without cytokines. The data show the maximum expression of the mesoendodermal marker, <i>Brachyury</i>, one day after Activin A treatment (day 2 after differentiation initiation). By continuing differentiation with BMP4 for the next 4 days <i>Isl1</i>, a marker of precardiac mesoderm, and <i>Actinin</i> were reached to their highest expression level. <i>Isl1</i> expression was remained at high level for the next 3 days and by decreasing its expression, <i>Mef2c</i>, a cardiac progenitor marker showed its maximum expression and after that, other cardiac progenitor genes, <i>Gata4 Nkx2.5</i> and <i>Tbx5</i> reached to their highest expression level respectively. Finally, the expression of <i>MHC</i> and <i>cTnT</i>, which are structural cardiomyocytes markers, got to maximum level (Fig. 1). These data shows that 3-dimentional structures of the cells are very important for cardiac differentiation and aggregated differentiation method enhances cardiac differentiation and functionality.Target genes were normalized by the reference gene <i>Gapdh</i>. The relative expression was calculated by dividing the normalized target gene expression of the treated sample with that of the undifferentiated state (day 0). All data represented as log2-linear plots. All data are statistically significant otherwise marked with “ns” (P>0.05).