Quiescence enables unrestricted cell fate in naive embryonic stem cells - PubMed (original) (raw)

. 2024 Feb 26;15(1):1721.

doi: 10.1038/s41467-024-46121-1.

Wentao Yang 1, Mengrou Shan 2, Li Zhang 2, Fengbiao Mao 3, Bo Zhou 4, Qiang Li 5, Rebecca Malcore 4, Clair Harris 4, Lili Zhao 6, Rajesh C Rao 5, Shigeki Iwase 4, Sundeep Kalantry 4, Stephanie L Bielas 4, Costas A Lyssiotis 2, Yali Dou 7

Affiliations

Quiescence enables unrestricted cell fate in naive embryonic stem cells

Le Tran Phuc Khoa et al. Nat Commun. 2024.

Erratum in

Abstract

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

© 2024. The Author(s).

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Conflict of interest statement

C.A.L. is a paid consultant for Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics. C.A.L. received no specific funding for this work. The remaining authors declare no competing interests.

Figures

Fig. 1

Fig. 1. The spontaneous qESCs possess a unique transcriptional landscape.

a Representative images of AP staining for naive ESCs with top (high) and bottom (low) 5% of TMRM signals at day 4 post FACS sorting. Scale bar, 200 µm. AP Alkaline phosphatase. Images were representatives from three independent experiments. b Left, MA plots showing up- (red) and down-regulated (blue) genes in low vs. high ΔΨm ESCs. Selected 2C and cell cycle genes were highlighted. RNA-seq data from biological duplicates were used to determine differential gene expression ([FDR] < 0.05). Right, heatmap showing expression levels of 145 2C genes ([FDR] < 0.05) in high and low ΔΨm ESCs. See Supplementary Data 1 for the 2C gene list. 2C, 2-cell. **c** MA plots from RNA-seq data showing up- (red) and down-regulated (blue) repetitive elements ([FDR] < 0.05) in low vs. high ΔΨm ESCs. Selected repetitive elements were highlighted. **d** RT-qPCR validation for upregulation of _MERVL_ and 2C genes in low ΔΨm ESCs. Relative mRNA expression was normalized against that in high ΔΨm ESCs, which was arbitrarily set 1. Data were presented as mean ± SEM from four independent FACS sorting. _P_ values were calculated using the two-sided Student’s _t_ test. **e** A representative FACS plot from three biological replicates showing TMRM staining in naive ESCs treated with DMSO or CCCP. **f** RT-qPCR analysis for _MERVL_ and 2C genes in naive ESCs treated with DMSO or CCCP. Relative mRNA expression was normalized against that in the CCCP-treated naive ESCs, which was arbitrarily set 1. Data were presented as mean ± SEM from three biological replicates. _P_ values were calculated from the two-sided Student’s _t_ test. **g** Unsupervised clustering using transcriptome of high and low ΔΨm ESCs, reported 2-cell-like cells and developmental stages of mouse embryogenesis. Green-shaded area denotes the close transcriptome correlation of low ΔΨm qESCs with other datasets. **h** Gene ontology (GO) analysis for genes significantly upregulated in low ΔΨm qESCs. GO terms with fold enrichment > 2 and the P value < 0.05 (defined by the Benjamini-Hochberg method) were considered as significant enrichment. Source data are provided as a Source Data file.

Fig. 2

Fig. 2. The spontaneous qESCs give rise to extraembryonic trophoblast stem cells.

a Left, immunofluorescence of OCT4 (red), CDX2 (green) and nuclei (DAPI blue) at day 7 post TSC differentiation. Scale bar, 200 µm. Right, quantification of CDX2+/OCT4− cells. Data were presented as mean ± SEM from three independent experiments. P values were calculated using the two-sided Student’s t test. TSC trophoblast stem cell. b Scheme for TSC derivation. c Left, representative images of typical TSC-like colonies at day 14. The TSC-like colonies were only observed from low ΔΨm ESCs, but not from high ΔΨm ESCs. Scale bar, 200 µm. Right, efficiency of TSC derivation. The success rate was calculated by dividing the number of successfully derived iTSC lines from total number of picked colonies. d RT-qPCR analysis for indicated genes in ESCs, TSCs and iTSCs. Relative mRNA expression was normalized against that in naive ESCs. Data from two independent cell lines were presented as mean ± SEM in the form of Log10 (fold change). e Immunofluorescence of indicated proteins in indicated cell types. Nuclei were co-stained with DAPI (blue). Scale bar, 200 µm. Images were representatives from two independent experiments. f Top, heatmap showing DNA methylation level at the upstream region of Elf5 promoter. The heatmap score (Log2) was shown on the right. Each dot indicates a methylated CpG site. Dark arrowheads denote five CpG sites that exhibit substantial differences in methylation levels between ESCs and TSCs. Bottom, quantification of methylation level for five CpG sites shown from the top. Violin plots show the median, 1st and 3rd quartiles. Data from two independent cell lines were presented. P values were determined by a two-tailed, nonparametric Mann-Whitney test. g Representative images from two independent iTSC lines at day 6 of differentiation. The white arrow indicates the trophoblast giant cells. Scale bar, 200 µm. h RT-qPCR analysis for indicated genes in the iTSCs at day 0 and 6 of differentiation. Relative mRNA expression was normalized against that of β-actin. Data from two independent iTSC lines were presented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 3

Fig. 3. Low one-carbon metabolism confers unrestricted cell fate of the spontaneous qESCs.

a Heatmap (Z-score) showing significantly changed metabolites in high and low ΔΨm ESCs. Metabolites with P < 0.05 were selected. Data from four biological replicates were presented as mean ± SEM. P values were determined by the two-sided Student’s t test. Metabolites, except Cystine, involved in one-carbon (1C) metabolism were highlighted on the right. b Pathway analysis for metabolites significantly decreased in the low ΔΨm qESCs. P < 0.05, determined by the Fisher’s Exact Test, was used as the cutoff for significantly enriched pathways. Selected pathways were highlighted. c An overview of 1C metabolism. 3-PG 3-phosphoglycerate, Homo homocysteine, Met methionine, SAM _S_-adenosylmethionine, SAH _S_-adenosylhomocysteine. d Levels of 1C-related metabolites as indicated at the bottom. Data were presented as mean ± SEM from four biological replicates. a.u. arbitrary unit. P values were determined by the two-sided Student’s t test. e Heatmap (Z-score) showing expression levels of 1C-related genes in high and low ΔΨm ESCs. RNA-seq data from biological duplicates with [FDR] < 0.05 were shown. f Scheme for TSC induction in the low ΔΨm qESCs treated with H2O or SAM. g Left, immunofluorescence of CDX2 (red), GATA6 (green) and nuclei (DAPI blue) at day 7 post TSC differentiation. Scale bar, 200 µm. Right, quantification of CDX2+/GATA6− cells for data shown from the left. P values were calculated using the two-sided Student’s t test from three independent experiments. Source data are provided as a Source Data file.

Fig. 4

Fig. 4. Global reduction of H3K27me3 enables unrestricted cell fate of the spontaneous qESCs.

a Left, heatmaps of H3K27me3 CUT&RUN signals from biological duplicates. The number of differential peaks was shown on top. Right, line plots showing H3K27me3 peak signals from the left. b Left, immunofluorescence of H3K27me3 (green) and nuclei (DAPI blue) in high and low ΔΨm ESCs. Scale bar, 10 µm. Right, quantified H3K27me3 fluorescence signals of polycomb bodies (foci) relative to DAPI. Violin plots show the median, 1st and 3rd quartiles. Data from three biological replicates were presented. P values were determined by a two-tailed, nonparametric Mann-Whitney test. n number of foci. c GO analysis for H3K27me3 peaks that were decreased in the low ΔΨm qESCs. “Count” denotes the number of genes found in each GO term. d Top, line plots showing changes of H3K27me3, H3K4me3, and ATAC-seq signals at 662 MERVL elements in low and high ΔΨm ESCs. Bottom, violin box plots representing the peak values of the top plots show the median, 1st, 3rd quartiles, lower adjacent value, upper adjacent value, and outside points. P values were determined by a nonparametric Mann-Whitney test. e Levels of intracellular SAM in naive ESCs treated with DMSO or CCCP. Data were presented as mean ± SEM from four biological replicates. a.u. arbitrary unit. P values were determined by the two-sided Student’s t test. f Left, immunofluorescence of H3K27me3 (green) and nuclei (DAPI blue) at indicated conditions. Scale bar, 10 µm. Right, quantification of the left. Violin plots denote quantified H3K27me3 fluorescence signals of foci relative to DAPI. Data were from three biological replicates. P values were determined by a two-tailed, nonparametric Mann-Whitney test. n number of foci. Source data are provided as a Source Data file.

Fig. 5

Fig. 5. Knockdown of Mat2a induces 2C-like features in naive ESCs.

a mRNA expression levels of Mat1a, Mat2a and Mat2b in naive ESCs. FPKM values were from RNA-seq data. Data from three biological replicates were presented as mean ± SEM_. P_ values were calculated using the two-sided Student’s t test. FPKM, fragments per kilobase of transcript per million. b RT-qPCR validation for Mat2a knockdown. Relative mRNA expression was normalized against that in the siControl naive ESCs, which was arbitrarily set 1. Data from two biological replicates were presented as mean ± SEM. c Confocal images of H3K27me3 (green) and nuclei (DAPI blue) immunofluorescence at indicated conditions. Scale bar, 10 µm. Representative images from two independent experiments were shown. d MA plots showing up- (red) and down-regulated (blue) genes in si_Mat2a_ versus siControl naive ESCs. Selected 2C genes, Mat2a, and MERVL were highlighted. RNA-seq data from biological duplicates were used to determine differential gene expression ([FDR] < 0.05). e RT-qPCR validation for upregulated 2C gene signature in si_Mat2a_ naive ESCs. Relative mRNA expression was normalized against that in siControl naive ESCs, which was arbitrarily set 1. Data were presented as mean ± SEM of two biological replicates. f Left, representative live and GFP images of MERVL-2C::EGFP naive ESCs at indicated conditions. Scale bar, 200 µm. Right, quantification of GFP + cells (%) for data shown from the left. Data from three independent experiments were presented as mean ± SEM. P values were calculated using the two-sided Student’s t test. g Immunofluorescence of OCT4 (red), MERVL (green) and nuclei (DAPI blue). Scale bar, 10 µm. Asterisks indicate MERVL+ cells that lose chromocenters and OCT4 protein. Note that chromocenters were marked by DAPI-dense regions in the MERVL− cells, which become spread in the MERVL+ cells. Images were representatives from two independent experiments. h Unsupervised clustering using transcriptome data of indicated samples (see method). Green-shaded area denotes the close transcriptome correlation of samples highlighted in red with other datasets. Source data are provided as a Source Data file.

Fig. 6

Fig. 6. Induction of quiescence in naive ESCs is sufficient to unlock the unrestricted developmental potential via downregulating H3K27me3 and metabolic modulation.

a RT-qPCR analysis for MERVL in naive ESCs treated with FAO, MYC or mTOR inhibitor (see method). Relative mRNA expression was normalized against that of the control cells, which was arbitrarily set 1. Data were presented as mean ± SEM from six biological replicates. P values were calculated using the two-sided Student’s t test. b Scheme for TSC induction in FAOi or MYCi induced quiescent naive ESCs. c Left, immunofluorescence of CDX2 (red), GATA6 (green) and nuclei (DAPI blue) at day 7 post TSC differentiation at indicated conditions. Scale bar, 200 µm for FAOi and 400 µm for MYCi. Right, quantification of CDX2+/GATA6− cells for data shown from the left. Data were presented as mean ± SEM from three independent experiments. P values were determined by the two-sided Student’s t test from three independent experiments. d Venn diagram comparing downregulated (left) and upregulated (right) pathways for metabolites that showed significant changes in low ΔΨm, FAOi and MYCi (see Supplementary Data 6 for more details). e Levels of intracellular SAM in naive ESCs treated with FAOi or MYCi as compared to control. Data were presented as mean ± SEM from four biological replicates. a.u. arbitrary unit. P values were determined by the two-sided Student’s t test. f Heatmap (Z-score) showing expression levels of the PRC2 core subunits in indicated conditions. RNA-seq data from biological duplicates were shown. g Confocal images of H3K27me3 (green) and nuclei (DAPI blue) immunofluorescence at indicated conditions. Scale bar, 10 µm. Representative images from three independent experiments were shown. Source data are provided as a Source Data file.

Fig. 7

Fig. 7. Deletion of Eed promotes unrestricted fate potential in naive ESCs.

a RT-qPCR analysis for Eed and Cdx2 in Eed fl/− (heterozygous) and Eed −/− (null) naive ESCs. Relative mRNA expression was normalized against that of the Eed fl/- naive ESCs, which was arbitrarily set 1. Data were presented as mean ± SEM from three biological replicates. P values were calculated using the two-sided Student’s t test. b Left, confocal images of H3K27me3 (green), CDX2 (red) and nuclei (DAPI blue) immunofluorescence in the Eed fl/− and Eed −/− naive ESCs. Scale bar,10 µm. Right, scatter plot showing per-nucleus intensity of CDX2 and H3K27me3 shown from the left. A total of 145 nuclei (69 nuclei for Eed fl/- and 76 nuclei for Eed −/−) from one representative experiment were shown. c MA plots showing up- (red) and down-regulated (blue) genes in the Eed −/− naive ESCs relative to Eed fl/− naive ESCs. 2C genes were selectively highlighted. RNA-seq data from biological duplicates were used to determine differential gene expression ([FDR] < 0.05). **d** RT-qPCR analysis showing elevated expression of _MERVL_ and 2C genes in the _Eed_ _−/−_ naive ESCs. Relative mRNA expression was normalized against that in _Eed_ _fl/-_ naive ESCs, which was arbitrarily set 1. Data were presented as mean ± SEM of three biological replicates. _P_ values were calculated by the two-sided Student’s _t_ test. **e** Heatmap (Z-score) showing expression levels of trophectoderm genes in the _Eed_ _fl/−_ and _Eed_ _−/−_ naive ESCs. RNA-seq data from biological duplicates were shown. **f** GO analysis for genes significantly upregulated in the _Eed_ _−/−_ naive ESCs. GO terms with fold enrichment > 2 and the [FDR] < 0.05 (defined by the Fisher’s Exact Test) were considered as significant enrichment. g Heatmap (Z-score) showing expression of genes associated with ectoderm, mesoderm and endoderm in indicated cell types. RNA-seq data from biological duplicates were shown. Source data are provided as a Source Data file.

Fig. 8

Fig. 8. Quiescence preserves innate unrestricted cell fate in naive ESCs.

a Top, scheme for generating new naive ESC lines from the low ΔΨm single qESC. Bottom, TMRM staining showing the similarity of ΔΨm heterogeneity between parental cells and three newly established naive ESC lines. b RT-qPCR analysis for genes related to 2C (left) and pluripotency (right) in high and low ΔΨm ESCs isolated from three single cell derived naive ESC lines. Relative mRNA expression was normalized against that in the high ΔΨm ESCs, which was arbitrarily set 1. Average values were presented from three single cell derived naive ESC lines. c Left, immunofluorescence of OCT4 (red), CDX2 (green) and nuclei (DAPI blue) for TSC induction at day 7 from three single cell derived naive ESC lines. Scale bar, 200 µm. Representative images from three independent experiments for each cell lines were shown**. d** Quantification of CDX2+/OCT4− cells for data shown in (c). Average values were presented from three single cell derived naive ESC lines. Source data are provided as a Source Data file.

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