Expression of pluripotency factors in 2D and 3D cultured cells. Expression of embryonic markers (green) in 2D and 3D cultured cells, as determined by immunocytochemistry. Cell nuclei were stained with Hoechst 33342 (blue). Scale bar: 20 μm (A). Flow cytometry analysis of C-MYC, OCT4, NANOG, and SOX2 expression in 2D and 3D cultured cells (B). Percentage of cells positive for the expression of embryonic markers as assayed by flow cytometry (n = 4, P >0.01) (C). Quantitative real-time polymerase chain reaction assay and Western blot assay for the four factors (D to G). (n = 4, P >0.01). 2D, plate cultures; 3D, spinning bottle cultures.