Immunolabeling of primary cilia in undifferentiated hESCs. (A) Characterization of undifferentiated colonies of H1 hESCs grown on matrigel for 5 d in DME:F12 with serum replacement. Undifferentiated cells are identified by nuclear colocalization of anti–OCT-4 (OCT-4, green) and DAPI (dark blue) in the merged image (light blue). More than 97% of cells on average expressed OCT-4 in a nuclear pattern indicating their undifferentiated state. Primary cilia stained with anti-AcTb (tb, red) are indicated by arrows. (B) H1 hESCs grown on matrigel for 5 d in DME:F12 with serum replacement (i.e., unstarved), labeled with anti-AcTb (tb, green) to show primary cilia (arrows). (C) H1 hESCs grown on matrigel for the same period of time in DME:F12 without serum replacement (i.e., starved) for 24 h and labeled with anti-AcTb (tb, green). Primary cilia are indicated by arrows. (D) Characterization of undifferentiated colonies of LRB003 hESC grown on 0.1% gelatin with conditioned medium. Undifferentiated cells are identified by nuclear colocalization of anti–OCT-4 (OCT-4, green) and DAPI (dark blue) in the merged image (light blue). Anti-AcTb (tb, red) marks the primary cilia (arrows) as well as the microtubular network in the cytoplasm. Less than or equal to 95% of the cells were ciliated and positive for OCT-4. Primary cilia are not easily visualized at the low resolution images (as shown in the insets). (E) A primary cilium (tb, red, arrow) in undifferentiated LRB003 hESCs emerges from one of the centrioles (asterisks) marked with anti-centrin (centrin, green). Nuclear localization of anti–OCT-4 (OCT-4, blue) denotes that the cell has not differentiated. The inset shows anti-pericentrin (Pctn, green) marking the centrosome (¤) at the base of the primary cilium (arrow).