Characterization of iPS cells for their pluripotency. Number 1 represents the cell line UTA.04602.WT and 2 the line UTA.04607.WT. A: iPS cells formed typical colonies for pluripotent stem cells that are rather compact and round in shape. B: The iPS cell colonies typically had well defined edges and distinct cell borders, and the iPS cells had a high nucleus to cytoplasm -ratio and a large nucleoli characteristic for stem cells. C: Endogenous pluripotency gene expression was studied using RT-PCR. Nanog, Oct4, Sox2 and Rex1 were all expressed at mRNA level in the iPS cells. β-actin and GAPDH were used as housekeeping control genes for both endogenous and exogenous markers. D: The expression of pluripotency genes was also studied at the protein level by immunocytochemical staining. The iPS cell expressed several markers for the pluripotent state: Nanog, Oct4, Sox2, SSEA4, TRA-1-60, and TRA-1-81 (all in red). Pictures in the left panel are from the line UTA.04602.WT and the ones on the right side are from UTA.04607.WT. Blue in all pictures indicates the DAPI staining of nuclei. E: Using RT-PCR, it was shown that all the transgenes were silenced in the iPS cells. Negative control is marked with “-” and positive control with “+”. F: Embryoid body (EB) -assay was used to define the pluripotency of the iPS cells in vitro. Markers for all three germ layers were detected from the EBs formed from both cell lines. Alpha- fetoprotein (AFP) was used as a marker for endoderm, kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR-2) was used as a marker for mesoderm and nestin was used as an ectoderm marker. GAPDH was used as an endogenous control gene.