EBs formed from Tim knockdown cells retain pluripotent cells.

<p>EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. <b>A</b>, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. <b>B</b>, Side profiles from merged images in part <b>A</b>; the location of the cavity in the control EB is indicated with a “c”. <b>C</b>, Side profiles from merged images obtained from 12 day EBs and stained as for 6 day EBs. <b>D</b>, Secondary EB assay. Six-day EBs from part <b>A</b> were trypsinized to single cells, replated in methylcellulose at the cell numbers indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture ± S.E.M. is shown in the bargraph (n = 8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each case (p<0.01).