Fig. 6: Modulating miRNA function and P-body assembly alters cell fate.

a, Schematic of the strategy for miR-300 inhibition in mouse naive ES cells. b, Quantitative PCR with reverse transcription (RT–qPCR) analysis of expression of miR-300 targets in P-bodies of mouse naive ES cells after miR-300 inhibition and for the control; n = 3 biological independent samples per group, unpaired two-sided Student’s t-test, mean ± s.d.; *P < 0.05, ***P < 0.001, ****P < 0.0001; P = 0.0009, P = 0.0133, P = 0.0237, P = 0.0195, P = 0.0009, P = 0.0004, P = 0.0002. c, Heatmap showing expression levels of differentially enriched 2C-related genes in mouse naive ES cells after miR-300 inhibition and for the control. d, Representative IF imaging of EDC4 puncta (green) and Nanog-MS2 (red) in Nanog-let-7^WT (top) and Nanog-let-7^Mut cells (bottom). Nuclei were counterstained with DAPI (blue) (scale bar, 10 μm). n = 3 independent experiments. e, RT–qPCR analysis of Nanog expression in Nanog-let-7^WT and Nanog-let-7^Mut cells compared to control cells (Nanog KO). n = 3 biologically independent samples per group, mean ± s.d. f, Representative western blot showing NANOG protein levels in Nanog-let-7^WT and Nanog-let-7^Mut cells compared to control cells (Nanog KO). g–i, Representative pictures (g) and quantification of alkaline phosphatase staining of cell colony number (h) and size (i) of Nanog-let-7^WT and Nanog-let-7^Mut cells cultured with fetal bovine serum and leukemia inhibitory factor (LIF). Unpaired two-sided Student’s t-test, n = 3 biologically independent samples per group, mean ± s.d.; *P < 0.05, ***P < 0.001; P = 0.0002, P = 0.0135. j, Heatmap showing expression levels of differentially enriched 8C-related mRNAs between purified P-body and cytoplasmic fractions in human naive ES cells (n = 2 biological independent samples per group). k, Schematic of the strategy for P-body dissolution in naive human ES cells carrying a blastomere-specific reporter (TPRX1-GFP). Flow cytometric analysis of TPRX1-GFP⁺ expression after 4 d of DDX6 KD. k, 1,000. SSC, side scatter. l, Quantification of TPRX1-GFP⁺ cells upon DDX6 KD. Unpaired two-sided Student’s t-test, n = 4 biologically independent samples per group, mean ± s.d., ****P < 0.0001. m, Representative IF imaging of H3Y1 (green)-positive cells in naive human ES cells upon DDX6 KO compared to control cells. Nuclei were counterstained with DAPI (blue) (scale bar, 50 μm) (left). Quantification of H3Y1⁺ cells upon DDX6 suppression. Unpaired two-sided Student’s t-test, single-guide (sg)Control (n = 5 fields), sgDDX6 (n = 8 fields), mean ± s.d., ****P < 0.0001 (right). hESC, human ES cell. n,o, RNA-seq data from primed, naive and TPRX1-positive human ES cells after DDX6 KD, showing expression levels of pluripotency- and totipotency-related transcripts (n) and transposable elements (o). p, GSEA analysis for the human PGCLC-related gene expression signature⁹⁶ in P-body-versus-cytoplasmic differential expression in human primed ES cells. Enrichment significance was calculated using the permutation test (two sided), with multiple-testing correction using the Benjamini–Hochberg method. q, Schematic of human iPS cell (hiPSC)-to-PGCLC differentiation. hPGCLC, human PGCLC. r, Left: flow cytometric analysis of TFAP2C-GFP and BLIMP1-Tomato expression after 4 d of PGCLC differentiation in three-dimensional aggregates. Right: quantification of TFAP2C-GFP⁺ and BLIMP1-Tomato⁺ cells by flow cytometry with three experiments. Error bars indicate mean ± s.d., n = 3 biologically independent samples per group; statistical significance was determined using a two-sided unpaired Student’s t-test; ***P < 0.001, P = 0.0003. s, RNA-seq analysis of hiPSCs (BTAG cell line⁹⁶), intermediate mesenchymal-like cells (iMeLCs) and TFAP2C-and-BLIMP1-negative and -positive cells after DDX6 suppression, showing expression levels of pluripotency, mesenchymal and PGC-related genes.

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