Twelve-day-old embryoid bodies (EBs) differentiated from
-OFF human embryonic stem cells (hESCs) displayed abnormal morphologies and expression levels of developmentally regulated genes. (A) Day 12 EBs that were differentiated from MEG3-OFF NTU1 hESCs were smaller and not well bordered in structure than EBs differentiated from MEG3-ON NTU1 hESCs. Scale bars, 1,000 μm. The bar chart illustrated the differences in diameters of the 12-day-old EBs derived from MEG3-ON and MEG3-OFF hESC sublines. Error bars represent the standard error of the mean (SEM) generated from three biological samples with 20 to 40 EBs in each group. **P <0.01 with respective MEG3-ON groups by Student’s t test.
(B) Day 12 EBs differentiated from MEG3-OFF hESCs displayed unusual expression levels of developmentally regulated genes, including higher expression levels of endoderm- and mesoderm-related genes (SOX17 and HAND1, respectively) and low but detectable expression levels of an ectoderm-related gene (PAX6). ‘d’ represents the day of EB formation. GAPDH was used as an internal control for mRNA expression analysis. The quantitation of mRNA expression was performed by using the 2−ΔΔCp method. Error bars represent the SEM generated from three biological samples with three technical repeats each. **P <0.01 compared with the corresponding MEG3-ON groups by Student’s t test. MEG3, maternally expressed gene 3.