Figure 7. Dax1 is indispensable for self-renewal of Nanog^low ESC.

(a) Luc KD- and Dax1 KD-Nanog:GFP cells were sorted into Nanog:GFP^low and Nanog:GFP^high populations (day 0). Cells were cultured in the presence of LIF for 6 days and FACS analyses were repeated. Number given is the percentage of cells in each of the indicated gates. Immunoblot analyses confirmed that GFP fluorescence reflected Nanog expression and Dax1 was efficiently silenced in both sorted Dax1 KD-Nanog:GFP populations. (b) qRT–PCR to measure expression of ExEn markers in the indicated FACS-purified cells. All data are normalized to Gapdh and shown relative to the mean of Luc KD-Nanog:GFP^high cells (set at 1.0). Data are means±s.d.; n=3. (c) The indicated FACS-sorted cells (1 × 10³ cells per cm² in 12-well plates) were cultured for 6 days with LIF and stained for AP. (d) Percentage of colony types formed by cells shown in c. Data are means±s.d.; n=3. NA, not available. (e) Total colony number counts after plating the indicated FACS-sorted cells at a density of 1 × 10³ cells per cm² in 12-well plates and culturing for 6 days with LIF. Data are means±s.d.; n=3. (f) Morphology and GFP fluorescence of the indicated FACS-sorted cells cultured with LIF. Scale bar, 100 μm. (g) Growth curves of the indicated FACS-sorted cells cultured with LIF. Diff., differentiated; Undiff., undifferentiated.