Competitive interactions eliminate unfit embryonic stem cells at the onset of differentiation

Abstract

A fundamental question in developmental biology is whether there are mechanisms to detect stem cells with mutations that, although not adversely affecting viability, would compromise their ability to contribute to further development. Here, we show that cell competition is a mechanism regulating the fitness of embryonic stem cells (ESCs). We find that ESCs displaying defective bone morphogenetic protein signaling or defective autophagy or that are tetraploid are eliminated at the onset of differentiation by wild-type cells. This elimination occurs in an apoptosis-dependent manner and is mediated by secreted factors. Furthermore, during this process, we find that establishment of differential c-Myc levels is critical and that c-Myc overexpression is sufficient to induce competitive behavior in ESCs. Cell competition is, therefore, a process that allows recognition and elimination of defective cells during the early stages of development and is likely to play important roles in tissue homeostasis and stem cell maintenance.

Graphical abstract

Figure 1

Cells with Defective BMP Signaling Are Eliminated in the Presence of Wild-Type Cells (A) High levels of apoptosis in Bmpr1a^−/− cells in Bmpr1a^−/fx;Mox2Cre^+/− embryos (n = 5/7). act-Casp3, cleaved (activated) Caspase3. (B) Phosphorylation of Smad1/5/8 in the described media (left) and Id gene activation in BMP4 + Lif (right) are decreased in Bmpr1a^−/− ESCs. PCNA, proliferating cell nuclear antigen. (C) Time-lapse imaging of cocultured control and Bmpr1a^−/−-GFP ESCs. (D) Growth curves (left) and ratio (right) of control to Bmpr1a^−/− ESCs show that Bmpr1a^−/−-GFP ESCs are outcompeted when cultured with control cells in N2B27. A minimum of three independent experiments were performed, and the average ± SEM was plotted. t, time. ^∗∗p < 0.005, Student’s paired t test. See also Figure S1 and Table S1.

Figure 2

ESCs with Defective BMP Signaling Are Eliminated by Apoptosis upon Exit of the Ground State of Pluripotency (A) Growth rates (in cell doublings per day) of control and Bmpr1a^−/− ESCs between days 3 and 4 of culture indicate that, while Bmpr1a^−/− ESCs are eliminated, control cells undergo compensatory proliferation. Sep., separate culture; Co-Cul., coculture. (B) Ratio obtained by flow cytometry and confocal images of Bmpr1a^−/− and control cells indicates that addition between days 2 and 4 of coculture of the pancaspase inhibitor ZVAD-FMK blocks the elimination of Bmpr1a^−/− ESCs. DMSO, dimethyl sulfoxide. (C) Growth curves (left) and ratios (right) of control and Bmpr1a^−/− ESCs showing that the elimination of mutant cells occurs under EpiSC culture conditions. (D) Ratio of control to Bmpr1a^−/− ESCs showing that the outcompetition of Bmpr1a^−/−-GFP ESCs is prevented by FCS + Lif, BMP4 + Lif, 2i, CHIR99021, or PD0325901. A minimum of three independent experiments were performed, and the average ± SEM was plotted. ^∗p < 0.05, and ^∗∗p < 0.01; a one-way analysis of variance (ANOVA) followed by Tukey’s test. See also Figures S2 and S3 and Table S1.

Figure 3

Elimination of Bmpr1a^−/− ESCs Depends on Secreted Factors (A) Assays using Corning Transwell inserts indicate that the growth of Bmpr1a^−/− ESCs is inhibited when these cells are grown with overlaying control cells in N2B27. Cell numbers refer to the cells growing underneath the insert; the genotype of the cells growing on the insert is indicated in brackets. (B) Plot of the ratio of control to Bmpr1a^−/− ESCs cultured as separate populations or cocultured in the presence of Noggin (p = 0.15), Bmpr1a-Fc (p = 0.48), BMP4 (p = 0.97), BMP7 (p = 0.51), FGF4 (p = 0.33), FGF5 (p = 0.93), and Lif from day 0 (p = 0.0001) or from day 2 (p = 0.06). The plot shows that only when Lif is added from day 0 of coculture is the outcompetition of Bmpr1a^−/− ESCs significantly blocked. (C) Western blot showing that Lif is less efficient in triggering Stat3 phosphorylation once differentiation is initiated. A minimum of three independent experiments were performed, and the average ± SEM was plotted. pStat3, phosphorylated Stat3. ^∗∗p < 0.005, Student’s paired t test. See also Figure S4 and Table S1.

Figure 4

Autophagy-Deficient and Tetraploid ESCs Are Eliminated by Cell Competition (A) Growth curves and plots of the ratio of control to Atg5^−/− ESCs in separate culture or coculture. (B) Growth curves and plots of the ratio of control to tetraploid ESCs in separate culture or coculture. Both (A) and (B) indicate that both autophagy deficiency and tetraploidy induce cell competition. (C) Histograms for Annexin V levels in control and tetraploid ESCs in separate culture or coculture in EpiSC media. (D) Immunostaining for cleaved-caspase 3 in chimeras generated with tetraploid-GFP ESCs (n = 10). Both (C) and (D) indicate that the tetraploid ESCs are eliminated by apoptosis both in vitro and in vivo. A minimum of three independent experiments were performed, and the average ± SEM was plotted. ^∗∗p < 0.005, Student’s paired t test. See also Figure S5 and Table S1.

Figure 5

Establishment of Differential c-Myc Levels Is Critical for the Elimination of Defective ESCs (A and B) In (A), western blot analysis and quantification of relative c-Myc/PCNA intensity indicate that c-Myc protein expression is significantly downregulated in Bmpr1a^−/− ESCs at day 3 of coculture with control cells, but (B) shows that this difference is abolished by Lif and MEK inhibition using PD0325901. (C and D) c-Myc levels are also lower in (C) Atg5^−/− ESCs and in (D) tetraploid ESCs when cocultured with control ESCs. Cells in coculture were sorted by FACS based on GFP expression prior to analysis. A minimum of three independent experiments were performed, and the average ± SEM was plotted. ^∗p < 0.05, and ^∗∗p < 0.01; a one-way ANOVA was followed by Tukey’s test. See also Figure S5.

Figure 6

c-Myc Is a Key Mediator of the Elimination of Defective ESCs (A and B) WISH in (A) and immunostaining in (B) show that c-Myc is heterogeneously expressed in embryos at 6.5 dpc but that this expression is downregulated from the embryonic region by 7.5 dpc. The lower image is a magnification of the region highlighted by the square. (C) TUNEL staining indicates that cell death peaks at 6.5 dpc in the embryo. (D) Double staining for TUNEL and c-Myc shows that those cells that are dying (white arrows) show low levels of c-Myc expression. (E) Growth curves and plot of the ratio of c-MycER and control ESCs when grown for 3 days in N2B27 and then treated with tamoxifen for 3 days in separate and coculture conditions, showing how c-Myc overexpression induces the elimination of control cells. A minimum of three independent experiments were performed, and the average ± SEM was plotted. ^∗∗p < 0.005, Student’s paired t test. See also Figure S6 and Table S1.

Figure 7

Model for How Cell Competition Eliminates Defective Stem Cells during Early Mammalian Development A first step during cell competition is the mutual sensing that allows identification of those stem cells that present a lower “fitness” level. In response to the mutual sensing, differential levels of c-Myc are established between “fit” and “unfit” cells. Next, a second “mutual sensing” event occurs that monitors c-Myc levels, leading to activation of the apoptotic pathway and elimination of the “weaker,” low c-Myc-expressing stem cells.