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. 2024 Jul 3;19(8):1217–1232. doi: 10.1016/j.stemcr.2024.06.003

Figure 7.

Figure 7

Variant 1q and MDM4-overexpressing cells are less sensitive to DNA double-strand breaks, which are more abundant in hPSCs grown in E8/VTN compared to KOSR/MEF

(A) Western blot analysis of MDM4 in MIFF3 and MIFF3 v1q cells under different conditions. MDM4 abundance is reduced in KOSR/MEF. Also, the addition of KOSR to E8/VTN reduces MDM4 expression. β-actin was used as a loading control.

(B) The addition of KOSR to E8/VTN reduces growth rates of v1q to levels like those of wild-type cells. Data shown are the mean ± SD of three independent experiments. ns, non-significant; ∗∗p < 0.01; two-way ANOVA with Holm-Sidak’s multiple comparison test.

(C) MDM4 has a more nuclear and punctate localization in hPSCs grown in E8/VTN compared to KOSR/MEF. Representative images of MIFF3 hPSCs grown in E8/VTN (upper panels) or KOSR/MEF (lower panels) stained with antibodies against MDM4 and OCT4 (POU5F1). The nuclei are counterstained with Hoechst 33342. Scale bar: 50 μm.

(D) Quantification of the MDM4 nuclear expression in E8/VTN versus KOSR/MEF condition in MIFF3 line. Data shown are the mean ± SD of three independent experiments. ∗∗p < 0.01; Unpaired t test.

(E) Genome damage is heightened in E8/VTN compared to KOSR/MEF condition. Quantification of γH2AX marker of double-strand breaks in hPSCs grown in E8/VTN compared to KOSR/MEF. Data shown are the mean ± SD of three independent experiments. ∗p < 0.05; paired t test.

(F) Cells with a gain of chromosome 1q or overexpressing MDM4 are more resistant to genome damage-induced apoptosis. Quantification of cell numbers of wild-type, v1q and wild-type-MDM4 cells treated with 10 μM camptothecin (CPT) for 2 h. Data shown are normalized to untreated control and represent the mean ± SD of three independent experiments. ∗∗∗p < 0.001; One-way ANOVA.

(G) A model summarizing a differential competitive advantage of v1q in E8/VTN versus KOSR/MEF conditions. E8/VTN confers high levels of genome damage in hPSCs. Amplification of MDM4 through the gain of chromosome 1q bestows v1q cells with the resistance to genome damage-induced cell death. Consequently, v1q outcompete wild-type hPSCs in E8/VTN. The KOSR/MEF condition does not generate the same selective pressure as the levels of genome damage are reduced compared to E8/VTN. The shift from feeder-based to feeder-free conditions over the last two decades has contributed to an increase in frequency of chromosome 1q gains detected in hPSC cultures. See also Figures S7C–S7E.