Abstract
Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-l-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.
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Data availability
Sequencing raw data are deposited as sequence read archives and available under accession number PRJNA891955. The scRNA-seq data reported in this paper are deposited in the Genome Sequence Archive of the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA010829) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa. All original images are deposited with Mendeley (https://doi.org/10.17632/2xt47pcfwb.3). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
B.B.G. is a recipient of the ‘Vernadski bourse’ scholarship given by the French government and a postdoctoral fellowship from Ulysseus European University. Research in the laboratory of D.V.B. is supported by the Foundation FRM, ANR and INSERM programme ‘Agemed’. Research in the laboratory of S.S. is supported by the Agence Nationale de la Recherche, research in the laboratory of F.L. was supported by grants from the National Natural Science Foundation of China (31830061, 32030032) and research in the laboratory of O.N.D. was supported by RSF grant 19-75-20128. We thank M. Serrano and H. Li for providing i4F mouse strains19. We thank W. Li (Institute of Zoology, Institute for Stem Cell and Regeneration, Beijing) and L. Chao (Institute of Zoology, Institute for Stem Cell and Regeneration, Beijing) for their assistance in morula aggregation and transplantation. We thank S. R. Jaffrey (Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, USA) for providing SAM sensor plasmids35. We acknowledge the ImagingCore Facility (PICMI), histology, genomic and animal housing facilities of IRCAN supported by le Cancéropole PACA, la Région PACA, le Conseil Départementale 06, l’INSERM, ARC, IBiSA and the Conseil Départemental 06 de la Région PACA.
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B.B.G., D.E., G.L., L.G., A.E., Z.K., A.K., B.K., C.M., E.L., F.L. and S.S. designed, conducted and analysed the experiments. D.E. and S.S. conducted the ChIP–seq and RNA-seq analyses, and G.L., Z.K. and F.L. conducted the scRNA-seq analysis. O.N.D. and C.G. provided methodological assistance, and D.V.B. secured funding, designed and supervised all the experiments, analysed the data and wrote the final version of the paper with input from all authors.
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B.B.G. and D.V.B. filed a patent application (PCT/EP2023/059341) related to this study. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Molecular aspects of p16 activation during in vitro and in vivo reprogramming.
a) Colocalization of p16High (EGFP +) cells with markers of DNA damage response (pATM, pKu80, pRad51) and cell cycle inhibitors p27 and p57. Positive controls for pATM, pKu80, pRad51, p27 derived by treatment DF with doxorubicin (500 nM, 24 h). Mouse placenta serves as a positive control for p57. Scale bars – 50 μm. b) Teratomas emerged during in vivo reprogramming in p16Cre;i4F;rtTA;mTmG mice produce all three germ layers. H&E staining shows the presence of Car, cartilage (mesoderm); ET, endodermal tube (endoderm); NR, neural rosette (ectoderm). Scale bar represents 100 μm. c) Immunofluorescent analysis of teratomas, obtained by in vivo reprogramming of p16Cre;i4F;rtTA;mTmG mouse co-stained for p16High and mesenchymal (Desmin, a-SMA), endothelial (Cd31), macrophagic (F4/80) and ectodermal (GFAP) markers. Scale bars – 50 μm. d) Immunofluorescent analysis of teratomas derived by grafting feeder-free p16Cre;i4F;rtTA;mTmg iPS cells into NSG mouse for p16High cells and mesenchymal markers Desmin and α-SMA, vascular endothelial marker Cd31, macrophage marker F4/80 and neuroectodermal marker Gfap. Scale bar – 50 μm.
Extended Data Fig. 2 Ablation of p16High senescent cells during in vivo reprogramming and distribution of Line1 and Mervl retroelements in mouse ES cells.
a) p16High cells depletion during in vivo reprogramming produce teratomas with all three germ layers. H&E staining of teratomas obtained from young (2-3-month-old) mice consists of derivatives of three germ layers: endoderm (endodermal tubes, ET); ectoderm (squamos epithelium, SE); mesoderm (bone nidus, BN). Scale bars – 100 μm. b) Immunofluorescent analysis of IOUD2 mouse ES cells for Mervl and Line-1 expression. Arrows indicate double positive cells. Scale bars – 20 μm.
Extended Data Fig. 3 Treatment of already established iPS cell line with D + Q does not expand cellular plasticity.
a) In vivo reprograming was induced with DOX in drinking water and 3 weeks later mice were administered with D + Q by gavaging or mock-treated. Analysis of p16High cells in developed teratoma showed a strong reduction in mice treated with D + Q. Scale bar - 200 μm. b) Analysis of macrophage infiltration in teratoma of a control mTmG mouse and mouse received D + Q. Scale bars 50 μm. c) H&E staining of teratoma obtained from mouse treated with D + Q. Mesoderm: smooth muscles, SM; ectoderm: neural rosette, NR; endoderm: endodermal tubes, ET. d) qPCR analysis of line-1 retroelement (total and two subclasses: md_a and md_t) and the 2C gene, zfp352, in control iPS cells and control iPS cells treated with D + Q after iPS line was established. Error bars represent SD, p-value evaluated by unpaired, two-tailed Student’s t-test with Welch’s correction, independent repeats, n = 3.
Extended Data Fig. 4 Deep sequencing reveals methylation pattern during iPS reprogramming in absence of p16High senescent cells.
a) Example genome browser view of H3K4me3 levels in fibroblasts on d12 of reprogramming. b) Unsupervised hierarchical clustering of H3K4me3 levels at peaks with differential coverage between samples (relative standard deviation between samples ≥0.5; clustering based on Ward’s minimum variance). c) Distributions of promoter H3K4me3 levels at fibroblast-specific genes, in fibroblasts before reprogramming (‘mTmG’ & ‘DTA’) and at d9 of reprogramming. Decrease in high-H3K4me3 promoters associated with reprogramming (‘mTmG’ vs ‘mTmG+dox’): p = 8.4 × 10−5, (‘DTA’ vs ‘DTA+dox’): p = 8.4 × 10—9; enhancement from DQ treatment (‘mTmG+DQ+dox’ vs ‘mTmG+dox’): p = 7.1 × 10−8; enhancement in DTA cells (‘DTA+dox’ vs ‘mTmG+dox’): p = 3.5 × 10−2 (Binomial test)). d) Example genome browser view of H3K27me3 levels in fibroblasts on d12 of reprogramming. e) Genome-wide extent of changes to H3K27me3 levels. Areas of pies correspond to the fraction of the genome exhibiting the indicated changes; blue/white slices depict the fraction among these that is unchanged (blue) or augmented (white) by depletion of senescent cells by DQ treatment. f) Genome-wide distribution of relative H3K27me3 levels in 10 kb bins, in normal fibroblasts (mTmG; x-axis) or in fibroblasts on d12 of reprogramming (mTmG+dox; y-axis). The black dotted line indictes the mean H3K27me3 level in during reprogramming corresponding to each level of coverage before reprogramming. g) Heatmap indicating the augmentation of H3K37me3 hypermethylation imparted by D + Q treatment during reprogramming (concordant results were obtained using DTA fibroblasts), in genomic regions as defined in (F): note that removal of senescent cells selectively promotes augmented H3K27me3 methylation at regions with low coverage in normal fibroblasts, and that already undergo increases in H3K27me3 during normal reprogramming. h) Example genome browser view of H3K27me3 (top tracks) and H3K4me3 (bottom tracks) levels in iPS cells. Red arrow highlights the H3K4me3 peak at the promoter of the Dppa3 (Stella) gene. For A-H the integration of all examined independent biological replicates (n = 3) examined. The accession number for genome-wide date is PRJNA891955. i) qPCR analysis of xkr9 and ripply2 mRNA in control and D + Q-treated iPS cells. p-value evaluated by unpaired, two-tailed Student’s t-test with Welch’s correction, independent repeats, n = 3.
Extended Data Fig. 5 iPS cells derived in senescence-free conditions exhibit totipotent-like features.
Analysis of Nanog and Gata6 protein content in blastoids, derived from a single cell of respective iPS cell line (Ctrl (mTmG), D + Q or DTA). Scale bar – 50 μm. b) Uncropped images of immunofluorescent analysis of 12 dpc placentas of chimeric embryo derived by aggregation of D + Q iPS with 8-cell morulas (related to Fig. 5d). Yellow boxes represent magnified part of images presented in Fig. 5d. Scale bars – 50 μm. c) Immunofluorescent analysis of 12 dpc placenta of chimeric embryo derived by aggregation of GFP−positive DTA iPS cells with 8-cell morulas. Scale bars - 50 μm. d) Violin plots showing the quality control results for single RNA-Seq analysis. e) Cell clusters in the 12.5dpc placental cells visualized by UMAP (left). Feature plots showing the expression of GFP for each cell cluster (right). f) Heatmap showing the DEGs (top 5 genes) for each cell cluster of GFP+ D + Q iPS-derived cells. Data plotted in graphs D-F is integration of all examined independent biological replicates (n = 3) examined. The accession number for scRNA-Seq data is CRA010829.
Extended Data Fig. 6 Maintenance of high SAM during iPS reprogramming level is crucial for the expansion of cellular plasticity to totipotent-like stage.
a) Analysis of SAM levels in control, D + Q, NNMTi and DTA iPS cells by immunofluorescent approach. Scale bar represents 50 μm. b) Quantification of SAM levels by ELISA during HEK293 cells after transfecting control and 2 different SAM sensor plasmids. c) Efficiency (mean ± SD) of mTmG iPS reprogramming in the presence of 5 mM cycloleucine, independent repeats, n = 3. d) Analysis of p16High cells (mean ± SD) on d12 and d15 of reprogramming in the population of control and SAM sensor 5.1-infected DF, n = 3. e) qPCR analysis of pluripotency genes oct4 and nanog in mTmG and DTA iPS lined established in the presence of cycloleucine, independent repeats, n = 3. f) Reprogramming efficiency of control (untreated) and treated with 10 μm SAM. Data represent as mean ± SD, independent repeats, n = 5. g) qPCR analysis of mervl retroelement activity and inducated 2 C markers in control iPS cells and iPS cells derived in presence of 10 μm SAM. Data represent as mean ± SD, independent repeats, n = 3. h) Reprogramming efficiency of control (untreated) and treated with Nnmt inhibitor (1 μm) JBSNF-000088 DF. Data plotted as mean ± SD, independent repeats, n = 3. i) Analysis of mervl, line-1 retroelement activity and 2 C marker tcstv3 of WT iPS cells, treated with NNMTi for 2 passages. Data plotted as mean ± SD, independent repeats, n = 3. j, l) Heatmaps of relative H3K27me3 levels at 10 kb genomic intervals (J) or at peaks (L) (rows) with differential coverage between samples (columns), with the major clusters (C1- C4) of similarly-behaving peaks indicated in each case. Heatmaps are normalized by rows. k) Unsupervised hierarchical clustering of H3K27me3 levels at 10 kb genomic intervals with differential coverage between samples. m) Distributions of H3K4me3 levels at gene promoters marked by H3K4me3 in fibroblasts before reprogramming. Decrease in high-H3K4me3 promoters associated with repgrogramming (‘mTmG’ vs ‘mTmG+dox’): p = 7.6 × 10−6; enhancement from DQ treatment (‘mTmG+DQ+dox’ vs ‘mTmG+dox’): p = 8.4 × 10−4; enhancement from NNMTi-treatment (‘mTmG+DQ+dox’ vs ‘mTmG+dox’): p = 3.2 × 10−4 (Binomial test)). For J-M the integration of all examined independent biological replicates (n = 3) examined.
Extended Data Fig. 7 Removal of p16High cells improves liver plasticity during partial 4F reprogramming in aging mice.
a) A scheme for 4F-induced rejuvenation experiments with different mouse strains as indicated. b) Immunofluorescent analysis of DNA damage marker 53bp1 in the liver of 2-month-old mouse (mTmG) and 18-month-old mice: p16Cre;mTmG (mTmG), p16Cre;DTA (DTA), p16Cre;i4F;mTmG (i4F), p16Cre;i4F;DTA (DTA+i4F). Quantification is shown in the graph ( ± SD). Scale bar – 20 μm. c) Immunofluorescent staining for endothelial marker Cd146 in liver samples as in (B). Percentage of the area covered by Cd146+ cells is shown in the graph. Scale bar – 100 μm. d) Immunofluorescent analysis for fibrosis marker Desmin in liver samples as in (B). Scale bar – 100 μm. For panels B-D p values estimated by Brown-Forsythe and Welch ANOVA test with Dunnett multiple comparison test and only difference between i4F and DTA+i4F is shown. Data presented as mean ± SD, independent repeats, n = 3.
Extended Data Fig. 8 A graphical abstract for the role of p16High senescent cells in reducing SAM availability during iPS reprogramming in vitro and in vivo.
p16High senescent cells induce expression of a SAM-consuming enzymes such as NNMT, lowering the level of SAM. This in turn reduces cellular plasticity during iPS reprogramming as cells no longer can achieve a totipotent-like state. Similarly, in vivo partial 4F reprogramming in mice significantly rejuvenates old livers only in combination with simultaneous removal of p16High senescent cells.
Extended Data Fig. 9 Example of gating strategy for the FACS of GFP+ cells isolated from mTmG mouse.
The general strategy for FACS-sorting of p16High cells is shown.
Supplementary information
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An Excel table with primer sequences.
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Grigorash, B.B., van Essen, D., Liang, G. et al. p16High senescence restricts cellular plasticity during somatic cell reprogramming. Nat Cell Biol 25, 1265–1278 (2023). https://doi.org/10.1038/s41556-023-01214-9
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DOI: https://doi.org/10.1038/s41556-023-01214-9
