Figure 4.

Dido3 is dispensable for ES cell self-renewal and pluripotency. (a) Colcemid-arrested metaphase spreads from Wt/Wt and ΔCT/ΔCT ES cells were karyotyped. Cells with more or less than 40 chromosomes (2N) were scored as aneuploid. The number of aneuploid metaphases relative to the total number of metaphases analyzed is shown above each bar. The Fisher exact test was used for statistical comparison. (b) Two representative examples for the presence of multipolar spindles (γ-tubulin; red dots) in interphase and mitotic nuclei of ΔCT/ΔCT ES cells. (c) Cell cycle profiles of propidium iodide (Pi)-stained Wt/Wt and ΔCT/ΔCT ES cells maintained as undifferentiated ES cells (top) or differentiated in vitro by aggregation into hanging drops and subsequent adhesion of the EB obtained to gelatinized cell culture dishes (bottom). Apoptotic cells (Ap) and cell ploidy (1N, 2N and 4N) are indicated; the inset shows representative photomicrographs for Wt/Wt and ΔCT/ΔCT EB. The proliferative capacity of ES cells and EB was assayed by BrdU incorporation and quantified on scatter-plots of BrdU versus Pi signal. The percentage of proliferating cells is indicated. (d) Pluripotency of Wt/Wt and ΔCT/ΔCT ES cells was assessed by ectopic transplant into immunodeficient recombination activating gene 2 knockout mice (10⁶ cells/injection). Representative examples are shown of teratoma formation after injection of Wt/Wt and ΔCT/ΔCT ES cells. (e) Bar graph quantifies the efficiency of teratoma formation from Wt/Wt and ΔCT/ΔCT ES cells. The number of injections that yielded overt teratomas within 6 weeks relative to total number of injections is given above each bar. The Fisher exact test was used for statistical comparisons. (f) Mice were killed at 6 weeks post-injection to isolate teratomas. Histology of HE-stained ΔCT/ΔCT teratoma sections confirms that ΔCT/ΔCT ES cells retain the capacity to differentiate into all three germ layers (endoderm, mesoderm, ectoderm)