Zscan4 restores the developmental potency of embryonic stem cells

Abstract

The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo, is known to deteriorate during long-term cell culture. Previously, we have shown that ES cells oscillate between Zscan4(-) and Zscan4(+) states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency.

Figure 1. Constitutive expression of a Zscan4c-ERT2 fusion protein increases developmental potency

a, The structure of a Zscan4c-ERT2 fusion protein. Zscan4c contains one SCAN domain and four C2H2 zinc finger domains. b, Fluorescence microscopy of MC1-ZE3 cells, in which a Zscan4 promoter drives the expression of Emerald marker (left), MC1-ZE3-ZERT2 clone #15 cells, in which the Zscan4c-ERT2 fusion protein is constitutively expressed, cultured in the absence of Tmx (middle), MC1-ZE3-ZERT2 clone #15 cells cultured in the presence of Tmx for 3 days (right). Scale bar, 50 μm. c, Flow-cytometry analysis of MC1-ZE3 ES cells (left, control) and MC1-ZE3-ZERT2 #15 ES cells (right) in the absence or presence of 1 μM Tmx. Em fluorescence levels (average ± S.E.M.; n=6) are shown. Note 3-fold increase of Em⁺ cells by the constitutive expression of a Zscan4c-ERT2 fusion protein even without Tmx. d, Quantitative RT-PCR analysis of endogenous Zscan4 expression measured by using PCR primer pairs specific for 3’-UTR of Zscan4 in MC1-ZE3 ES cells (left, control) and MC1-ZE3-ZERT2 #15 ES cells (right) in the absence or presence of 1 μM Tmx. The fold-induction of endogenous Zscan4 expression levels (average ± S.E.M.; n=6) compared to that of control MC1-ZE3 is shown. Note the 6-fold increase of endogenous Zscan4 at the RNA level by the constitutive expression of a Zscan4c-ERT2 fusion protein even without Tmx. e, V6.5 parental ES cells (passage number 14), V6.5 ZERT2 #2 (p.20), V6.5 ZERT2 #18 (p.22) ES cell colonies after whole-mount RNA in situ hybridization of a Zscan4 full-length probe, which detects both endogenous and exogenous Zscan4 RNAs: upper panel) or a Zscan4 3’-UTR probe, which detects only endogenous Zscan4 RNAs. Scale bar, 100 μm. f, Representative coat colors of chimeric mice generated by injecting various ES cells into blastocysts. Higher chimerism represents higher contribution of injected ES cells to mice, indicating higher developmental potency of the ES cells. g, Percent distribution of chimerism levels among “n” number of mice born from various ES cell lines.

Figure 2. Forced expression of Zscan4 enhances developmental potency of ES cells

a, Schematic presentation of experimental procedure. b, Expression levels of Zscan4 (endogenous and ORF) in Dox⁺ and Dox^- conditions in G418-selected clones. Error bars indicate S.E.M., n=3 c, Forced expression of βgeo in Dox⁺ and Dox^- conditions. d, Induction of Stra8 and Tmem92 (Zscan4-downstream genes) in Dox⁺ condition. Error bars indicate S.E.M., n=3 e, Forced expression of Zscan4 protein detected by immunohistochemistry. Scale bar, 100 μm. f, Assessment of developmental potency of ES cells by 4N complementation assays.

Figure 3. Molecular features of ES cells expressing Zscan4-ERT2 and a proposed mode of Zscan4 actions

a, Constitutive presence of Zscan4-ERT2 and forced expression of Zscan4 made telomeres longer than controls in both F1-hybrid (129/C57BL/6)-derived ES cells (V6.5) and C57BL/6-derived ES cells (MC2). Telomere lengths (average ± S.E.M.; n=3) are shown. b, Constitutive presence of Zscan4-ERT2 and forced expression of Zscan4 maintained better karyotype in long-term-culture of ES cells. c, Comparisons of global expression profiles between V6.5 ZERT2 #18 ES cells and V6.5 #2 control ES cells (left scatter plot) and between MC1 Em⁺ (Zscan4⁺) ES cells and Em^- (Zscan4^-) ES cells (right scatter plot). Red and green dots represent genes that show expression differences with statistical significance (False discovery rate < 0.05 and fold-change > 2). Venn diagram showing the overlap between genes that are more highly expressed in V6.5 ZERT2 #18 cells than V6.5 #2 cells and genes that are more highly expressed in Em⁺ (Zscan4⁺) cells than Em^- (Zscan4^-) cells (center). d, A proposed model. Upper panel: ES cells oscillate between Zscan4^- and Zscan4⁺ states, resulting in the normal ES cells with only 1% - 5% of Zscan4⁺ cells. While in Zscan4^- state, the potency of ES cells gradually decreases, which is restored to some extent by transient activation of Zscan4 (Zscan4⁺ state). However, the potency of ES cells still decreases during the long-term culture. Lower Panel: When the Zscan4 is more frequently activated by expressing an exogenous Zscan4, ES cells are rejuvenated more frequently, resulting in the longer maintenance of the high developmental potency.