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. Author manuscript; available in PMC: 2009 Jun 8.
Published in final edited form as: Stem Cells. 2008 May 8;26(8):2019–2031. doi: 10.1634/stemcells.2007-1115

Figure 1.

Figure 1

Knockdown of Tcf3 in ESCs upregulates Oct4 and limits differentiation potential. (A): LIF was removed from culture media 24 h after control shRNA, vector, and Tcf3 shRNA transfection. Puromycin (1 μgml-1) was added to select for transfected cells. By d 7, Tcf3 shRNA-treated cells formed ESC colonies that could be propagated in the absence of LIF. Immunostaining showed that Tcf3 shRNA treatment in the absence of LIF maintained strong SSEA1 and AP expression. At least two different shRNA designs were used, and results were comparable (data not shown). Scale bars = 200 μm. (B): Control shRNA, vector, and Tcf3 shRNA-transfected stable ESC clones were generated, and one representative clone for each condition is presented. Tcf3 shRNA clones maintained spherical and intact EBs that stained positive for AP after 21 d, whereas control shRNA and vector-treated clones showed signs of disintegration with the appearance of cavities indicative of differentiation and negative for AP. Scale bars = 200 μm (left column) and 150 μm (right column). (C): Retinoic acid (RA) treatment of control shRNA ESCs showed extensive differentiation to flattened cells, with the loss of colony forming ability by d 5. Tcf3 shRNA ESCs maintained colony structures along with differentiated cells after 5 d of RA treatment. These cells could be propagated at least five times in the presence of RA, whereas control shRNA cells could not. During RA treatment, Tcf3 shRNA ESCs were able to maintain higher levels of Oct4 and Sox2 transcripts compared with control shRNA cells. Scale bar = 100 μm; error bars represent SEM. (D): Knockdown of Tcf3-upregulated Oct4, Utf1, Sox2, and Hist1h1b transcript levels after 3 d of transfection. *, significantly different from control shRNA control; p < .01; n = 3. Error bars represent SEM. (E): In both the presence and the absence of LIF, transient treatment of ESCs with Tcf3 shRNA upregulated the levels of Nanog and Oct4 proteins by 48 h. (F): Knockdown of Tcf3, but not Tcf2, Tcf4, or Lef1, upregulated Oct4 promoter activity in mouse ESCs, relative to control shRNA. Oct4 RNAi served as a positive control, whereas vector transfection served as a negative control. Four shRNAs were constructed for each gene to ensure specificity. *, significantly different from control shRNA control; p < .01; n = 3. Error bars represent SEM. Abbreviations: AP, alkaline phosphatase; d, day(s); h, hour(s); LIF, leukemia inhibitory factor; RNAi, RNA interference; shRNA, short hairpin; Tcf3, T-cell factor 3.