Abstract
In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.
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Data availability
RNA-seq, scRNA-seq, ATAC-seq and RRBS data from this study have been deposited at the NCBI Gene Expression Omnibus (GEO) under accession number GSE185005. Source data are provided with this paper.
Code availability
The code for the analyses can be found at https://github.com/pengchengtan/Hu-et-al.-2022-ciTotiSC.
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Acknowledgements
We thank Tsinghua University Center of Pharmaceutical Technology for assistance with chemical screening, the Imaging Core Facility and the Center of Biomedical Analysis for assistance in confocal microscopy and flow cytometry analysis, and the Laboratory Animal Research Center for assistance in mouse embryo microinjection and transplantation. We thank J. Yong for technical assistance of Smart seq2 RNA-seq and Y. Li for early exploration of RNA-seq data analysis. This work is supported by the National Key R&D Program of China (2017YFA0104001 to S.D.), the National Natural Science Foundation of China (31771530 to T.M. and 32030031 and 31530025 to S.D.), Center for Life Sciences (to S.D.) and Tsinghua University Initiative Scientific Research Program (to T.M.).
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Contributions
Y.H., Y.Y., T.M., K.L. and S.D. designed the study, Y.H. and Y.Y. performed most of the experiments, and Y.H., Y.Y., T.M., K.L. and S.D. interpreted the data. P.T. performed the bioinformatics analyses of RNA-seq, scRNA-seq and RRBS data. Y.Z. performed the mouse embryo microinjection experiments and mouse breeding. M.H. assisted with parts of the experiments. J.Y., X.Z. and Z.J. assisted in ATAC-seq library preparation and data analysis, Y.L. supervised this part of the work. K.Y. and H.P. assisted in metabolomics assay and data analysis, Z.H. supervised this part of the work. D.W. assisted with the reagents preparation. Y.H., Y.Y., S.D., K.L. and T.M. conceived this project. T.M., K.L. and S.D. supervised the study. Y.H., Y.Y., T.M., K.L. and S.D. wrote the manuscript with input from all authors. S.D. is the lead contact.
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Competing interests
S.D., K.L., Y.H., Y.Y., P.T. and T.M. are listed as inventors on the priority patent application CN202110989429.8 (Induced Totipotent Stem Cell and the Preparation Method Thereof) filed by Tsinghua University, Beijing, on 26 August 2021. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Screening of chemical compounds enabling induction and maintenance of mouse totipotent stem cells.
a, Schematic diagram of MERVL-tdTomato reporter. b, Immunostaining of MERVL-Gag in mouse ES cells with MERVL-tdTomato & OCT4-GFP dual-reporter mouse ES cells under basal 2i/LIF medium. c, Bar graph showing the percentage of MERVL-tdTomato+ cells generated by treatment with indicated RAR agonists. d, Detailed list of compound combinations. e, Bar graph showing the percentage of MERVL-tdTomato+ cells generated by treatment with different compound combinations. f, The number of DEGs (differentially expressed genes, p-value < 0.001) between each of the conditions to mouse 2-cell embryos. L2C: Late 2C embryo; EM2C: Early-Middle 2C embryo. g, OCT4-GFP changes in cells under sustained TTNPB, 1-Azakenpaullone and WS6/TAW treatment over 4 passages. Up: imaging of colony morphology. Middle: imaging of OCT4-GFP reporter. Low: FACS analysis of OCT4-GFP+ cells. h, Immunostaining of pluripotency markers OCT4 and NANOG in mouse ES cells treated with or without TAW. Scale bar: 20 μm. i, The karyotype analysis of mouse ES cells treated with or without TAW. j, Volcano plots showing up- (red) and down- (blue) regulated genes (left, log2 (FC) > 1, FDR < 0.1) and transposons (right, log2 (FC) > 1, FDR < 0.15) in TAW_P1 versus mouse 2C embryo. Benjamini-Hochberg method was used to control the false discovery rate. Some totipotency genes/ transposons and pluripotency genes were labeled. k, Transcriptional changes of pluripotency (blue) and totipotency (red) specific genes among mouse ES cells, mouse ES cells treated with TAW for 1/2/4/8 passages, D-EPSC and L-EPSC. l, Transcriptional changes of pluripotency (blue) and totipotency (red) specific genes in mouse ES cells treated with TAW for 8 passages. m, GSEA analysis of bulk RNA-seq data of mouse ES cells treated with TAW for 8 passages by the indicated embryonic stage-specific gene sets. All datasets in bioinformatic analyses were summarized in Supplementary Table 1.
Extended Data Fig. 2 ciTotiSCs exhibit characteristic transcriptome features close to totipotent blastomeres at the single-cell level.
a, Representative images and flow cytometry analysis of MERVL-tdTomato of ciTotiSCs. Scale bars, 500 μm. b, UMAP plot from scRNA-seq displaying three clusters (A-C) identified in ciTotiSCs culture. The expression of representative 51 totipotency genes, 47 pluripotency genes and 6 primitive endoderm genes were shown in UMAP plot. c, Violin plots showing the expression distribution of specific marker genes in each cluster shown in (b). d, Transcriptome PCA analysis of ciTotiSCs, mouse ES cells, L-EPSC, D-EPSC, TBLCs and mouse embryos from zygote to E6.75 at the single-cell level. e, FeaturePlots projecting expression of representative pluripotency and totipotency genes, overlaying Fig. 2c UMAP. All datasets in bioinformatic analyses were summarized in Supplementary Table 1.
Extended Data Fig. 3 The epigenomic and metabolic features of ciTotiSCs are similar to totipotent 2C-embryo blastomeres.
a, Comparison of the chromatin accessibility among mouse ES cells, ciTotiSCs and mouse embryos at the indicated developmental stages by ATAC-seq. b, Chromatin accessibility changes after chemical induction of TotiSC. Totipotency and pluripotency genes located in top open (red) and closed (blue) peaks were indicated. c, Chromatin accessibility (log10 (RPKM+1) transformed value) of 2C specific retrotransposon elements in ciTotiSCs and mouse ES cells. The central line corresponds to the median, the boxes indicate the lower and upper quartiles. P values were determined using two-sided student’s t-test, and then adjusted using Holm’s method. d, Boxplots of DNA methylation levels on 2C specific retrotransposon elements in mouse ES cells, ciTotiSCs and mouse embryos at the indicated stages. The central line is the median, the boxes indicate the lower and upper quartiles. e, Different CpG methylation pattern of Zscan4 clusters in mouse ES cells and ciTotiSCs. f, Different CpG methylation pattern of X chromosome in mouse ES cells and ciTotiSCs. g, Abundance of metabolites involved in the TCA cycle, purine metabolism pathway, one-carbon metabolism and redox metabolism-related pathway. Mean relative fold-change and error bar were calculated from n = 5 or 8 biological experiments. P values determined by two-sided Student’s t-test. All datasets in bioinformatic analyses were summarized in Supplementary Table 1.
Extended Data Fig. 4 Characterization of ciTotiSCs’ chimerism potential in vivo.
a, Live images of chimeras at E4.5, which were developed from 8-cell embryos injected with tdTomato+ ciTotiSCs or mouse ES cells. Embryos with tdTomato+ cells integrated trophectoderm were pointed by white arrows. Scale bars: 100 μm. b, Representative images showing expression of CDX2 in chimeric blastocysts at E4.5 in vitro, developed from 8-cell embryos injected with multiple tdTomato+ ciTotiSCs or mouse ES cells. Scale bars: 20 μm (left), 10 μm (right). c, Representative images showing expression of ELF5 and OCT4 in chimeras at E7.5 in vivo, developed from 8-cell embryos injected with single tdTomato+ ciTotiSC or mouse ES cells. Scale bars: 100 μm (left), 25 μm (right). d, Representative images of extraembryonic tissues from E13.5 chimeric conceptuses derived from uninjected control 8-cell embryos or 8-cell embryos injected with tdTomato-labeled ciTotiSCs or mouse ES cells. JZ: junctional zone; Lab: labyrinth; CP: chorionic plate. e, Representative images of the multiple ciTotiSCs-derived E13.5 chimeric embryo sections, derived from 8-cell embryos injected with tdTomato-labeled ciTotiSCs or mouse ES cells. f, A representative image of E13.5 gonads from chimera contributed by ciTotiSCs and mouse ES cells. g, ciTotiSCs or mouse ES cells-derived chimeric mice. h, FACS analysis of developmental contribution of tdTomato+ cells in fetus, yolk sac and placenta of E13.5 chimeric conceptuses, derived from uninjected control 8-cell embryos or 8-cell embryos injected with tdTomato-labeled ciTotiSCs or mouse ES cells. i, Representative images of placenta sections from E13.5 chimera, derived from uninjected control 8-cell embryos or 8-cell embryos injected with tdTomato-labeled ciTotiSCs or mouse ES cells co-immunostained with trophoblast cell marker PROLIFERIN. The insets showed enlarged images of single cells. Scale bars: 500 μm. j, Violin plots showing relative expression distribution of specific marker genes for each cluster shown in (Fig. 5f).
Extended Data Fig. 5 The derivation of ciTotiSCs is dependent on TAW cocktail.
a, Heatmaps revealing expression changes of totipotency genes, ZGA genes and maternal genes after the removal of individual molecule from the TAW condition. b, Analysis of Gene Ontology (GO) terms enriched in 2C embryo versus blastocyst. P values determined by two-sided Student’s t-test. c, GO analysis of terms enriched in ciTotiSCs versus mouse ES cells. Specific pathways of interest were colored. P values determined by two-sided Student’s t-test. d, GO analysis of cells cultured with AW (-TTNPB) versus ciTotiSCs. P values determined by two-sided Student’s t-test. e, Representative fluorescence images of cells with MERVL-tdTomato reporter, treated with (TTNPB + AW) or (all-trans RA + AW) in the presence or absence of RAR antagonist AGN193109 for 72 h. Scale bars: 100 μm. f, RT-qPCR analysis of representative totipotency genes in mouse ES cells treated with (TTNPB + AW) or (all-trans RA + AW) in the presence or absence of RAR antagonist AGN193109 for 72 h. Expression levels are relative to Gapdh. Data are mean ± s.d. (n = 3). g, The enrichment of RARE binding motif in maternal, totipotency and pluripotency gene regulatory regions. Dot size: -log10 (p value). P values determined by two-sided Student’s t-test. h, GO analysis of cells cultured with TW (-1AKP) versus ciTotiSCs. Upregulated genes are shown for specific Wnt signaling pathway. P values determined by two-sided Student’s t-test. i, Flow cytometry analysis (left) and quantification (right) of cell cycle distribution of mouse ES cells, ciTotiSCs, and ciTotiSCs cultured with TW (-1AKP). j, GO analysis of cells cultured with TA (-WS6) versus ciTotiSCs. Upregulated genes are shown for two pathways of interest. P values determined by two-sided Student’s t-test. k, The expression of genes involved in NF-κB-mediated signaling in mouse ES cells, ciTotiSCs and cells cultured with TA (-WS6).
Extended Data Fig. 6 Dux or p53 knockout impairs ciTotiSC generation.
a, Percentage of endogenously fluctuating MERVL-tdTomato+ cells in WT, Dux and p53 knockout mouse ES cells, analyzed by flow cytometry. n = 2 biological replicates. b, Percentage of MERVL-tdTomato+ cells in WT, Dux and p53 knockout mouse ES cells treated with TAW for 1 passage, analyzed by flow cytometry. n = 2 biological replicates. c, Expression of representative totipotency MERVL repeats and genes in WT and Dux knockout mouse ES cells treated with or without TAW for 1 passage, detected by RT-qPCR. Data are mean ± s.d. (n = 3). P values determined by two-sided Student’s t-test. d, Expression of representative totipotency MERVL repeats and genes in WT and p53 knockout mouse ES cells treated with or without TAW for 1 passage, detected by RT-qPCR. Data are mean ± s.d. (n = 3). P values determined by two-sided Student’s t-test.
Supplementary information
Supplementary Figure 1
Representative gating strategies in flow cytometry analysis. (a) Gating strategy for mouse ES cells and ciTotiSCs, related to Extended Data Fig. 1g, Extended Data Fig. 2a and Extended Data Fig. 6a-b. (b) Gating strategy for analysis of cell cycle distribution identification, related to Extended Data Fig. 5i. (c) An example of the gating strategy to analyse the contribution of tdTomato+ cells to fetus, yolk sac and placenta of E13.5 chimeria, related to Extended Data Fig. 4h.
Supplementary Table 1
Summary of published datasets used in this study. Cited references and accession numbers of all datasets are listed.
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Hu, Y., Yang, Y., Tan, P. et al. Induction of mouse totipotent stem cells by a defined chemical cocktail. Nature 617, 792–797 (2023). https://doi.org/10.1038/s41586-022-04967-9
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DOI: https://doi.org/10.1038/s41586-022-04967-9
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