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. 2013 Feb;23(2):248-59.
doi: 10.1101/gr.141945.112. Epub 2012 Oct 18.

Pluripotent stem cells escape from senescence-associated DNA methylation changes

Carmen M Koch  1 , Kristina Reck, Kaifeng Shao, Qiong Lin, Sylvia Joussen, Patrick Ziegler, Gudrun Walenda, Wolf Drescher, Bertram Opalka, Tobias May, Tim Brümmendorf, Martin Zenke, Tomo Saric, Wolfgang Wagner
Affiliations

Pluripotent stem cells escape from senescence-associated DNA methylation changes

Carmen M Koch et al. Genome Res. 2013 Feb.

Abstract

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.

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Figures

Figure 1.
Figure 1.
Long-term culture-associated changes in MSC. (A) Long-term growth curves demonstrate that MSC reach a senescent state after 2–3 mo of culture expansion. (B) Cells at higher passage acquire morphological changes, senescence-associated beta-galactosidase staining, and loss of adipogenic differentiation potential (green: BODIPY-staining of lipid droplets; blue: DAPI) and decline in osteogenic differentiation potential (Alizarin Red staining of calcium phosphate precipitates; size bars 100 μm). (C) Comparison of DNAm between early (P2–P3) and late (P7–P16) passages. CpG sites with relevant SA-hypermethylation (red: 1702 CpG sites) and SA-hypomethylation (green: 2116 CpG sites) are depicted (adjusted P-value < 0.05 and DNAm change >20%). (D) SA-DNAm changes were classified according to gene regions, and (E) in relation to CpG islands. (F) Signal intensity of gene expression microarrays was used as an indicator for gene expression level. In particular, genes with SA-hypomethylated CpG sites were significantly less-expressed than the average. (G) Genes with SA-DNAm changes were subsequently mapped to genes with H3K9me3, H3K27me3 (Delbarre et al. 2010), and PRC2 targets (Wei et al. 2011) in human MSC. Enrichment analysis revealed that particularly genes with SA-hypomethylated CpG sites have been associated with these repressive histone marks. As an alternative, we focused on the subset of promoter-associated CpG sites (TSS1500, TSS200, or 5′UTR; striped bars), and this analysis also revealed association of genes with SA-hypomethylated CpG sites with those genes that have repressive histone marks (H3K9, P < 10−28; H3K27, P < 10−13; EZH2, P < 10−2).
Figure 2.
Figure 2.
Irradiation-induced senescence does not result in SA-DNAm changes. (A) Representative growth curves of MSC after irradiation with different doses at passage 2 (0–25 Gy; black arrow). (B) Upon irradiation with 15 Gy, MSC acquire the typical senescent morphology with proliferation arrest and moderate SA-β-gal staining. Furthermore, adipogenic (green: BODIPY; blue: DAPI) and osteogenic differentiation potential (red: Alizarin Red; size bars 100 μm) is impaired after irradiation with 15 Gy. (C) Scatterplot analysis of DNAm of nonirradiated versus irradiated MSC. Long-term culture-associated hypermethylated (red) and hypomethylated CpG sites (green) are depicted. Irradiation did not result in any significant DNAm changes.
Figure 3.
Figure 3.
Immortalization does not affect SA-DNAm. (A) MSC at passage 2 were either retrovirally transfected with an TERT-IRES-GFP construct or with IRES-GFP (control; n = 5). Flow cytometic analysis of one sample reveals a growth advantage of TERT-transduced cells at late passages. (B) The DNAm profile of this TERT-transfected sample was compared with the corresponding control (both passage 14). CpG sites that were hypermethylated (red) or hypomethylated upon long-term culture (green) are indicated. Overall, TERT expression does not affect SA-DNAm changes. (C) MSC were immortalized by tetracycline-inducible expression of TERT and SV40-TAg. Upon Dox removal from the culture medium, these cells acquire a typical senescent morphology within 10 d, express SA-β-gal, and lose osteogenic differentiation potential. (D) The Epigenetic-Senescence-Signature was used to estimate cellular aging of all samples (Koch et al. 2012). DNAm was determined at six SA-CpG sites by pyrosequencing and used for the linear regression model to predict the number of passages. The predictions correlate with the real passage number for all MSC samples, including irradiated, TERT-transfected, and TERT/SV40-TAg immortalized cells. In contrast, senescence-predictions for iP-MSC and ESC rather correspond to noncultured cells. (E) Relative telomere length was determined by the telomere to single-copy gene (T/S) ratio with quantitative multiplex PCR (each sample measured in triplicate). Telomere length is significantly reduced in MSC of later passage, whereas it is not affected by irradiation. TERT expression, TERT/SV40-TAg immortalization (particularly at passage 62), and generation of iP-MSC result in telomere extension.
Figure 4.
Figure 4.
Reprogramming into iP-MSC prevents SA-DNAm changes. (A) Induced pluripotent MSC reveal typical ESC-like morphology and express the pluripotency markers POU5F1, NANOG, and TRA-1-80. (B) Unsupervised hierarchical clustering of global DNAm profiles clearly separates pluripotent cells (iP-MSC and ESC) and it discerns MSC of early and late passage. (C) Scatter plot of DNAm profiles of iP-MSC in comparison to MSC of late passage (P7–P16). In particular, CpG-sites that are hypomethylated upon replicative senescence (green) are hypermethylated in iP-MSC. In contrast, SA-hypermethylated CpG sites (red) are rather hypomethylated in iP-MSC. (D) Differential DNAm upon long-term culture is plotted against methylation changes upon reprogramming into iP-MSC. Overall, SA-DNAm changes are prevented in iP-MSC. (E,F) Venn diagrams demonstrating the overlap of SA-DNAm changes with differential methylation between MSC of late passage with either iP-MSC or ESC.
Figure 5.
Figure 5.
Schematic overview of how SA-DNAm changes may influence cellular senescence. Senescence is associated with a continuous gain of SA-DNAm changes that are enriched in genes with repressive histone marks. They may be associated with permanent gene silencing, but this link is yet unproven and therefore labeled with a question mark. SA-DNAm changes are not affected by ionizing radiation or telomere length. However, these processes evoke chromosomal aberrations and may ultimately activate the same downstream signal cascades of cellular senescence (including activation of CDKN1A, CDKN2A, and TP53). Pluripotent cells hardly reveal SA-DNAm changes; avoidance from these epigenetic modifications might contribute to the escape of pluripotent cells from cellular senescence.
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