Few passages required for ES cells with hyper-long telomeres

We showed previously that ES cells lengthen their telomeres upon in vitro expansion35. Thus, here we first addressed whether there was a limit to telomere lengthening during expansion. To this end, we subjected ES cells to over 60 passages in vitro and analysed telomere length by quantitative fluorescence in situ hybridization (Q-FISH) on metaphase spreads at different passages. Mean telomere length was increased until passage 24 and then, was maintained until passage 60 (Fig. 1a and Supplementary Fig. 1a), suggesting that a few passages are sufficient to reach maximum telomere length in ES cells. This finding was further confirmed by telomere Q-FISH and telomapping techniques (Supplementary Fig. 1b,c). To discard that differences in telomere length were caused by changes in probe accessibility, or ploidy, we performed Q-FISH with a centromeric major satellite probe and found no significant differences in centromeric fluorescence at different passages (Methods, Supplementary Fig. 2). To study whether other changes might occur during in vitro expansion of ES cells, we subjected four independent clones of ES cells at passage 6 and passage 16 to RNA-deep sequencing and found only five genes (out of 19,555 genes analysed) significantly differentially expressed for a false discovery rate (FDR) below 0.05 analysed using DESeq (Methods). These genes were Sox18 (upregulated 20-fold compared with ES cells at passage 6) and Sox17, Zbtb48, Chst15 and Jph4 (downregulated less than twofold compared with ES cells at passage 6) (Supplementary Table 1). Thus, RNA-seq analysis showed very few changes in gene expression between ES cells at passages 4 and 16. Importantly, we did not observe alterations in mRNA expression of telomerase genes, nor in components of shelterin, or in other genes associated to telomere biology. We confirmed similar telomeric repeat amplification protocol (TRAP) telomerase activity in passage 4 and passage 16 ES cells (Supplementary Fig. 3). Finally, none of the genes with altered expression levels have been implicated in longevity36.

Figure 1: Analysis of ES cells bearing hyper-long telomeres. (a) Mean telomere length analysed on metaphase spreads in primary MEF (passage 2) and ES cells at the indicated passages. The ‘M’ in the graph indicates the number of metaphases studied. Underneath, the two graphs show the histograms corresponding to telomere signals in the primary MEFs and ES cells at passage 24. (b) Mean TRF1 intensity in primary MEFs (passage 2), blastocysts and ES cells at the indicated passages. Underneath, the graphs show the histograms for TRF1 intensity in MEFs and ES cells at passage 24. (c) Representative micrographs of TRF1 stain in selected samples described in b. Scale bar, 10 μm. (d) Per cent of cells with DNA damage measured by IF with anti γH2AX antibody in primary MEFs (passage 2) and ES cells at the indicated passages. We show a representative graph of three independent experiments. (e) Per cent of cells with more than two TIFs, analysed by IF with anti γH2AX and TRF1 antibodies in primary MEFs (passage 2) and ES cells at the indicated passages. We show a representative graph of three independent experiments. (f) Representative micrographs of control or damaged cells. Cells were subjected to IF with anti γH2AX and TRF1 antibodies. Co-localization of γH2AX and TRF1 is indicated with yellow arrows. Scale bar, 10 μm. (g) Mean number of MTS in metaphases from ES cells at the indicated passages, analysed by telomere FISH. Representative graph of two independent experiments. (h) Representative images of metaphases at passages 5 and 50. A representative telomere shape is highlighted in each micrograph. Scale bar, 5 μm. n=number of independent primary MEFs or clones of ES cells. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values. MEF, mouse embryonic fibroblasts. Full size image

Proper telomere function requires binding of the shelterin complex to telomeric repeats1,5. The shelterin protein TRF1 (telomere repeat binding factor 1) is involved in telomere capping and telomere length regulation37,38,39,40. Interestingly, TRF1 is highly expressed in ES cells as well as in induced pluripotent stem (iPS) cells35,41 and this enrichment is also maintained in adult stem cell compartments compared with more differentiated compartments in the context of the organism41. Thus, we next asked whether in vitro expansion of ES cells affected TRF1 levels. To this end, we quantified TRF1 levels at a single-cell level by immunofluorescence (IF) with anti-TRF1 antibodies using confocal microscopy (Fig. 1b,c). We found higher TRF1 levels in the ICM of the blastocyst compared with the trophectoderm or to mouse embryonic fibroblasts (Fig. 1b,c and Supplementary Fig. 4a,b). Interestingly, we found that early passage ES cells (up to passage 24) retained similarly high TRF1 levels to those present in the ICM but TRF1 levels decreased at higher passages, which may reflect a change in ES cell properties at later passages. These results indicate that maximal telomere length is achieved and high TRF1 levels are maintained after moderate in vitro passaging of ES cells (up to passage 24).

ES cells with hyper-long telomeres do not show increased DNA damage

We next asked whether continuous telomere lengthening associated to in vitro ES cell expansion would cause DNA damage, particularly at regions difficult to replicate such as telomeres. γH2AX is a marker of double-strand breaks and dysfunctional telomeres, the latter also known as telomere damage-induced foci or telomere-induced foci (TIFs)42,43. To asses DNA damage specifically at telomeric chromatin, we performed double IF with anti-γH2AX and TRF1 antibodies to quantify TIFs (Fig. 1d–f). We found very few ES cells with DNA damage (>3 γH2AX foci per cell) up to passage 24, but this percentage significantly increased at later passages with more than 40% of the cells showing DNA damage (Fig. 1d). In the case of telomere-specific damage, we also observed low numbers of TIF-positive cells until passage 24, and this was significantly increased at later passages (Fig. 1e,f). We found similar results when we analysed telomere damage by telomere FISH combined with IF using anti 53BP1 antibody (Supplementary Fig. 4c,d). To study the specific type of DNA damage present at later passages in cells with hyper-long telomeres, we arrested cells with colcemid and performed telomere Q-FISH on metaphase spreads. We found that the only telomere aberration increased with ES cell passaging was the presence of multitelomeric signals (MTS) (Fig. 1g,h), a type of aberration previously associated to increased telomere fragility as the result of replication stress at telomeres13,44. Interestingly, TRF1, which is also decreased at later passages (Fig. 1b), has been previously shown to protect from telomere fragility38,44.

Taken together our results demonstrate that hyper-long telomeres after moderate ES cell passaging (up to 24 passages) are well capped and do not show increased DNA damage.

Hyper-long telomeres contribute to healthy chimaeric mice

We next addressed the capability of ES cells bearing hyper-long telomeres to contribute to chimaera formation in vivo and to retain hyper-long telomeres in the adult organism (Fig. 2a–f). To this end, we aggregated GFP-positive ES cells with hyper-long telomeres (passage 16) with eight-cell morulae, and derived from them chimaeric mice (see Fig. 3a; note longer telomeres in GFP-positive ICM cells compared with the GFP-negative ICM of the unmodified species). Gross phenotypic analysis of the resulting chimaeric mice showed that they were normal compared with the unmodified species. To track cells derived from GFP-positive ES cells with hyper-long telomeres, we performed immunohistochemistry of different tissues with an anti-GFP antibody. In particular, we focused our analyses in the intestine and the skin as an example of two highly proliferative tissues, as well as in the brain, as an example of a low proliferative tissue. Tissues were analysed at four time points during the mouse lifespan: 0; 1; 6; and 12 months of life. We observed the presence of both GFP-positive and -negative cells in all tissues analysed (Fig. 2a,b). The percentage of GFP-positive cells was ∼20–50 per cent in all tissues studied, and this was maintained when chimaeric mice were analysed at different time points indicating that cells bearing long telomeres are functional and maintained with ageing (Fig. 2a,b). Importantly, these chimaeric tissues showed a normal histology and we did not observe any pathological finding (Fig. 2a and Supplementary Fig. 5a). To study dynamically the fate of cells with hyper-long telomeres in vivo, we performed a longitudinal analysis of the percentage of cells expressing GFP in peripheral blood samples from chimaeric mice bearing either normal or hyper-long telomeres (Fig. 2d) from 4 until 8 months of age. We found that GFP-positive cells with hyper-long telomeres are maintained, or even increased in some cases, over time with respect to the starting point (Fig. 2d). As control, while GFP-positive cells containing normal telomere length were also maintained with time (Supplementary Fig. 5b). We further confirmed telomere length in GFP-positive or -negative cells by Q-FISH in blood samples from these chimaeric mice (Supplementary Fig. 6a). Note that in chimaeric mice bearing GFP-positive cells with normal telomere length both the GFP-positive or -negative cells display similar telomere intensities (upper graph and pictures), while in chimaeric mice bearing GFP-positive cells with hyper-long telomeres, GFP-positive cells display brighter telomeres than the GFP-negative cells (lower graph and pictures; Supplementary Fig. 6a,b).

Figure 2: Tissues bearing cells with hyper-long telomeres are healthy. (a) Micrographs show histology of intestine, skin and brain from chimaeric mice bearing cells with normal telomere length and other cells with hyper-long telomeres (GFP-positive). Note that tissues containing GFP-positive cells are healthy. Scale bar, 50 μm. (b) The graphs show the percentage of GFP-positive cells found in intestine, brain and skin of chimaeric mice bearing hyper-long telomeres, over the period of time indicated. (c) The scheme shows how chimaeric mice were generated. (d) Per cent of GFP-positive cells in blood from chimaeric mice bearing hyper-long telomeres during the period of time is indicated. (e) Scheme shows that the chimaeric mice analysed were constituted by two different backgrounds, 129S1 with hyper-long telomeres and CD1 cells with normal length telomeres. (f) Graphs show mean telomere length in intestine, skin and brain in newborns and 6 months chimaeric mice compared with aged-matched animals from the 129S1 and CD1 backgrounds. Representative graph of two independent experiments. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values. E&H, eosin and haematoxylin. Full size image

Figure 3: Both hyper-long and normal length telomeres shorten with age in vivo. (a) The scheme shows how the chimaeric mice were generated. Underneath, The graphs show mean telomere length in ES cells used for microinjection, The ICM of the blastocyst, intestine, skin and brain from chimaeric mice bearing hyper-long (GFP-positive) and normal length (GFP-negative) telomeres, at the indicated time points. Samples were subjected to IF with anti-GFP antibody combined with telomere FISH. Telomere length was analysed by telomapping. Representative graph of two independent experiments. (b) Representative images showing GFP-positive cells (green), GFP-negative cells and telomere FISH in blastocyst and in 1-month-old skin. Scale bar, 10 μm. (c) Graphs present the rate of mean telomere length shortening per month in intestine, skin and brain for the indicated periods. n=number of blastocyst, or independent clones of ES cell (passage 16) or independent chimaeric mice. Representative graph of two independent experiments. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values. Full size image

Because the chimaeric mice contained two different genetic backgrounds (Fig. 2e), 129S1 cells (with hyper-long telomeres) and CD1 cells (with normal length telomeres) from receptor morulae, we compared telomere length in the chimaeric mice with age-matched mice of the 129S1 and CD1 backgrounds in newborns and 6-months-old chimaeric mice. As shown in Fig. 2f and Supplementary Figs 6c and 7, GFP-positive cells, bearing hyper-long telomeres had a higher mean telomere length than cells of both the 129S1 and CD1 backgrounds with normal length telomeres. Indeed, the mean telomere length was similar in the chimaera eGFP-negative cells and in cells from non-chimaeric mice from either background (Fig. 2f).

These results demonstrate the contribution of GFP-positive ES cells with hyper-long telomeres to the formation of healthy tissues and mice which contain GFP-positive cells with longer telomeres than those of the unmodified species (GFP-negative cells). These findings suggest that cells with hyper-long telomeres are maintained during both embryonic and adult mouse development.

Both long and normal length telomeres shorten with age

Next, we sought to investigate the length dynamics of hyper-long telomeres in the context of the organism with increasing age. To this end, we performed Q-FISH using a telomere probe to measure telomere length combined with IF using anti-GFP antibody to track the cells derived from ES cell with hyper-long telomeres in sections from the intestine, skin and brain, from chimaeric mice at 0, 1, 6 and 12 months of age (Methods). We confirmed longer telomeres in the GFP-positive ES cells used for morulae aggregation and in the derived GFP-positive ICM cells compared with the GFP-negative ICM cells within the same blastocyst (Fig. 3a,b and Supplementary Figs 8 and 9)35. After birth, we observed telomere shortening with age in all mouse tissues analysed, including the brain, in agreement with previous findings (Fig. 3a and Supplementary Figs 8–10)16. At all the different ages studied, we observed that the mean telomere length of GFP-positive cells (derived from ES cells with hyper-long telomeres) was higher than the mean telomere length of the GFP-negative cells (derived from ES cells of the unmodified species with normal length telomeres) in all tissues analysed (Fig. 3a and Supplementary Figs 8 and 9), in spite of a slightly increased rate of telomere shortening in the GFP-positive cells (Fig. 3c), probably due to the starting differences in telomere length in these cells. Note that cells with hyper-long telomeres were never found or were very rare in the GFP-negative tissue compared with the GFP-positive cells (Fig. 3a and Supplementary Figs 7–9). Interestingly, we noticed that telomere shortening with age was not uniform over time, with the highest rate of telomere shortening per month occurring during the first month of life in both the GFP-positive and -negative cells (Fig. 3c), which has also been observed in human blood45. This high rate of telomere shortening also affected to the brain. This fast telomere shortening soon after birth may reflect on the higher proliferation rates in newborn mice to reach the size of adults.

In summary, these findings indicate that telomere shortening occurs associated with ageing in the context of mouse tissues in both cells derived from ICM of the recipient blastocyst (GFP-negative cells) with normal length telomeres and ES cells with hyper-long telomeres (GFP-positive), and that this is exacerbated during the first month of life. Importantly, adult cells derived from GFP-positive ES cells with hyper-long telomeres showed longer telomeres at any time point in all tissues analysed indicating that we achieved generation of adult tissues bearing longer telomeres than those of the unmodified species without any genetic manipulation and in particular without transgenic germ line telomerase overexpression.

To further confirm these results, we studied chimaeric mice showing a 100% of chimaerism. In particular, we determined telomere length, shelterin levels, as well as telomerase levels in blood samples at 20 and 40 weeks after birth. We confirmed longer telomeres in the 100% GFP-positive chimaeric mice with hyper-long telomeres at both time points compared with 100% GFP-positive cells chimaeric mice with normal telomere length, as well as compared with chimaeric mice with normal telomeres and age-matched control mice (Supplementary Fig. 11a,c). In addition, 100% chimaeric mice with hyper-long telomeres showed less percentage of short telomeres compared with 100% chimaeric mice with normal telomere length (Supplementary Fig. 11a–c). We then analysed the levels of different shelterin proteins in 100% chimaeric mice with normal or hyper-long telomeres. By IF, we found similar levels of TRF1 or RAP1 shelterins in blood samples from either 100% chimaeric mice with normal or hyper-long telomeres (Supplementary Fig. 11d–f). By quantitative PCR, we also confirmed similar mRNA levels of shelterin and telomerase, both in blood and skin samples from 100% chimaeric mice with normal or hyper-long telomeres or controls (Supplementary Fig. 12). Finally, we demonstrate similar telomerase TRAP activity in spleen from chimaeric mice with normal length and hyper-long telomeres as well as in the control mice (Supplementary Fig. 13).

These results suggest that mice with hyper-long telomeres do not have globally an altered expression of shelterin or telomerase gene compared with control mice.

Longer telomeres in adult stem and differentiated cells

Stem cell compartments are enriched in cells with the longest telomeres compared with the more differentiated compartments within the same tissue both in mice and humans16,46. Furthermore, long telomeres are advantageous for stem cell function in vivo17, as critical telomere shortening in stem cells impairs their ability to mobilize and regenerate tissues17 and short telomeres impair self renewal and repopulation capacity in blood and intestine47,48. Thus, we set to address whether stem cells derived from GFP-positive ES cells with hyper-long telomeres retained longer telomeres in the adult organism. In particular, we compared telomere length in GFP-positive and negative cells located at known stem cells compartments in both the skin and small intestine. Briefly, we combined IF with anti-GFP antibody with telomere Q-FISH on tissue sections to generate maps of telomere length at a single-cell level within tissues (immuno-telomapping; Methods). In the case of the intestine and skin from 6-month-old chimaeric mice (Fig. 4), GFP-positive cells located at known stem cell compartments (the intestinal crypts in the case of the small intestine and hair bulge in the case of the skin) had longer telomeres than the GFP-negative counterparts at the same compartments (Fig. 4a–d). In the case of the differentiated compartments (villi and basal layer), we also observed longer telomeres in the GFP-positive cells compared with the GFP-negative neighbouring cells (Fig. 4a–d).

Figure 4: Hyper-long telomeres remain long in stem cell niches and differentiated compartments despite ageing. (a) Representative image of a 6 months chimaeric intestine after IF with anti-GFP antibody combined with telomere FISH (left). The map shows telomere intensity of each cell, analysed by telomapping. The colour-intensity code is specified over the image (right). (b) Representative image of 6 months chimaeric skin after IF with anti-GFP antibody combined with telomere FISH (left). The map shows telomere intensity for each cell, analysed by telomapping. The colour-intensity code is specified over the image (right). Below, mean telomere-length quantification of the cells contained in the stem cell niches (crypt or hair bulge) and differentiated compartments (villi and basal layer), differentiating the GFP-positive versus GFP-negative patches. Note that longer telomeres coincide with the GFP-positive patches. Representative images of five independent analysis. n=number of chimaeric mice. (c) Magnification of the square area in picture a separating the telomere FISH and the GFP signals. (d) Magnification of the square area in b separating the telomere FISH and the GFP signals. Bar, 20 μm. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values. Full size image

Together, these results indicate that aggregation of ES cell with hyper-long telomeres results in both adult stem cell compartments and differentiated compartments containing cells with longer telomeres compared with the corresponding adult compartments in the unmodified species. This finding is in agreement with the fact that the percentage of GFP-positive cells in the tissues studied is similar in chimaeric mice of different ages.

Lower accumulation of short telomeres with aging

Next, we addressed whether the accumulation of cells with short telomeres with ageing was also lower in GFP-positive cells. In M. musculus, the 10% percentile in the reference population (in this case, the GFP-negative newborns, whose telomere length is similar to the 129S1 unmodified species in age-matched animals) is used to quantify short telomeres. Percentile 10% corresponded to a telomere length of approximately <15 kb (ref. 31). The percentage of cells with short telomeres was zero in the case of the ICM of the blastocyst for both genetic backgrounds (Fig. 5). Strikingly, the percentage of cells with short telomeres was lower for the GFP-positive cells at the different ages and tissues studied (Fig. 5a). Interestingly, tissues with a higher rate of proliferation accumulated more cells with short telomeres in both positive and negative GFP cells at the 12 months time point (Fig. 5a). In both positive and negative GFP cells, the biggest increase in the percentage of cells with short telomeres occurred during the first month of life (Fig. 5b), in agreement with a faster rate of telomere shortening early in life, until the adult organism is formed. Of note, after the first month of life, and for the rest of the time points analysed, the biggest accumulation of cells with short telomeres continued to occur in the GFP-negative cells.

Figure 5: The amount of cells containing short telomeres is reduced in chimaeric mice containing hyper-long telomeres. (a) The graphs show the per cent of cells with short telomeres in the ICM of the blastocyst, intestine, skin and brain at the indicated time points. Samples were subjected to IF with anti-GFP antibody combined with FISH and analysed by telomapping. (b) Graphs show the increase in the per cent of cells with short telomeres per month in intestine, skin and brain at the indicated time points. Samples came from chimaeric mice and were analysed as described in a. n=number of blastocysts or chimaeric mice. Representative graphs of two independent experiments. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values. Full size image

Our results suggest that adult cells derived from ES cell with hyper-long telomeres preserve longer telomeres and accumulate lower numbers of short telomeres with age.

Mice with longer telomeres show less DNA damage and tumours

First, as an indication of proper telomere capping, we examined TRF1 levels in both GFP-positive and -negative cells. TRF1 is an essential shelterin component that plays a role in telomere protection by preventing telomere fragility and fusions, which in turn are associated to premature tissue ageing and increased cancer susceptibility5,13,44,49. To this end, we analysed TRF1 abundance at telomeres in both ES cells used for aggregation experiments as well as in 6- and 12-moths-old chimaeric mice. A tendency to find the mean TRF1 intensity higher in the GFP-positive cells than in the GFP-negative was observed in all the tissues analysed (Fig. 6a and Supplementary Fig. 14), reflecting on adequate telomere protection.

Figure 6: Analysis of TRF1 and DNA damage in chimaeric tissues. (a) Mean TRF1 intensity in cells bearing normal (GFP-negative) or hyper-long telomeres (GFP-positive) in ES cells used for the generation of chimaeric mice, and in brain, intestine and skin from the chimaeric mice at 6 and 12 months. Tissues were subjected to IF with anti-GFP and anti-TRF1 antibodies. Representative graphs of two independent experiments. (b) Per cent of cells positive for γH2AX in brain, intestine and skin tissue from chimaeric mice at 6 and 12 months. Tissues were subjected to IF with anti-GFP and anti-γH2AX antibodies. Representative graphs of two independent experiments. (c) Representative images of brain tissues as described in b. Scale bar, 10 μm. (d) The graph shows the per cent of P53-positive cells in skin from 1-year-old chimaeric mice bearing hyper-long telomeres for both GFP-positive and -negative cells. Underneath, a representative image of intestine stained for GFP and P53 and in 1-year-old chimaeric mice. The yellow arrowhead indicates GFP stain and the red arrow indicates p53 stain. Scale bar, 50 μm. (e) The graph shows the per cent survival of three different cohorts of mice, chimaeric mice with normal telomere length, chimaeric mice bearing cells with hyper-long telomeres and control mice. (f) The graphs show the percentage of spontaneous tumour incidence in the three cohorts of mice described in e. (g) Chemical carcinogenesis experiment. The graph shows the total number of papillomas in chimaeric mice bearing hyper-long telomres, normal length telomeres or in age-matched mice of the 129S1 and C57Bl6 backgrounds. Representative graph of two independent experiments. (h) The graph shows the percentage of area affected by hyperkeratosis in the mice described in g. Representative graph of two independent experiments. (i) Representative micrograph of back skin of mice affected with hyperkeratosis. (j) E&H images on skin affected with hyperkeratosis. The lesion was diagnosed parakeratotic hyperkeratosis, as no signs of inflammation were observed. n=number of independent clones of ES cells or chimaeric or control mice. Scale bar, 50 μm. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values, except for the graph of e, where a log rank test was used. E&H, eosin and haematoxylin. Full size image

Next, we studied accumulation of DNA damage. Long telomeres could be a target of DNA damage owing to more difficulties to replicate repetitive DNA, although DNA damage can occur independently of telomere length due to genotoxic stress50,51. We determined DNA damage by % of cells showing >2 γ-H2AX foci in tissues at 6- and 12-months-old animals (Fig. 6b,c and Supplementary Fig. 15a). We observed a moderate number of cells showing DNA damage in all the tissues analysed and this increased with ageing, but there was a tendency to accumulate more of these cells in the GFP-negative cells at both time points, in agreement with shorter telomeres in these cells. Although in this assay we cannot distinguish damage at telomeres from other regions, these findings are in line with the notion that longer telomeres are more efficiently protected from damage by maintaining a functional telomere capping structure41,46,49. In agreement with the lower DNA damage, we also detected increased numbers of cells positive for p53 staining in the GFP-negative population compared with the GFP-positive cells both in intestine and skin (Fig. 6d and Supplementary Fig. 15b). These results suggest that hyper-long telomeres are well capped in the context of the organism as indicated by high levels of the TRF1 protein, which in turn ensures a lower accumulation of DNA damage and of p53 in tissues with ageing.

Importantly, we found that hyper-long telomeres did not result in detectable long-term deleterious effects for mice, as indicated by a similar survival of the chimaeric mice bearing cells with hyper-long telomeres compared with chimaeric mice with normal telomere length (see Fig. 6e). In line with this, we did not find increased incidence of spontaneous tumours in the chimaeric mice with hyper-long telomeres, indeed, none of the chimaeric mice bearing hyper-long telomeres developed any spontaneous tumours in the curse of the survival follow-up (Fig. 6f). To further study whether hyper-long telomeres could be influencing cancer, we performed a 7,12-dimethylbenz[a]anthracene (DMBA) -phorbol 12-myristate 13-acetate (TPA) chemical carcinogenesis protocol on the skin of chimaeric mice with normal or hyper-long telomeres, as well as age-matched controls (Methods). DMBA was applied once on mouse skin, followed by TPA treatment during 15 weeks. The evolution of papillomas was further observed during at least 20 weeks after DMBA treatment. After 20 weeks following DMBA treatment, only 129S1 control mice showed papillomas, whereas C57BL6 control mice and chimaeric mice with normal and hyper-long telomeres did not show papillomas (Fig. 6g and Supplementary Fig. 16). Note that the C57Bl6 background is very resistant to papilloma formation52,53,54. In addition, at 10 weeks after DMBA treatment, we observed the presence of preneoplastic lesions such as parakeratotic hyperkeratosis, produced by external addition of TPA and not due to inflammation processes since neutrophils were not present in a systematic way (Fig. 6h–j) in the skin of control mice and chimaeric mice with normal telomere length. Interestingly, in chimaeric mice with hyper-long telomeres, the patches of skin with hyper-long telomeres (GFP-positive cells) did not show presence of hyperkeratosis while these lesions were readily observed in the patches of skin with normal length telomeres (GFP-negative cells) from the same mice (see Fig. 6h,i).

Cells with hyper-long telomeres show better skin wound healing

Previous studies have shown that wild-type long telomeres have advantageous effects over short telomeres in the context of the telomerase-deficient mouse model, particularly in the ability of skin stem cells to mobilize and maintain skin homeostasis17 and liver regeneration48. Here, we wondered whether hyper-long telomeres would be advantageous compared with normal length telomeres in the context of the organism. To address this, we generated two types of chimaeric mice: chimaeric mice with normal length telomeres by microinjecting ES cells at passage 4 (telomere length being similar to telomere length of the inner cells mass of the blastocyst35) in morulae and expressing GFP (in order to follow these cells in chimaeric tissue), as well as chimaeric mice with hyper-long telomeres by microinjecting ES cells expressing GFP at passage 16 (with hyper-long telomeres) (see scheme in Fig. 7a). In particular, ES cells with normal or hyper-long telomeres were microinjected in recipient morulae of the C57Bl6 background. Adult chimaeric mice bearing either normal or hyper-long telomere between 6 and 12 months and adult non-chimaeric mice of the 129S1 or C57Bl6 backgrounds (used as age-matched controls) between 6 and 12 months were anaesthetized to remove the back hair with depilatory cream. After a period of 3 days, they were anaesthetized again to cause superficial wounds of 4 mm diameter with a circular razor blade. We quantified the surface of the 4 mm wound every day. The area of the wound was reduced with days until its closure. In chimaeric mice with normal telomere length wounds were healed at the same rate than in control non-chimaeric mice (Fig. 7b). In contrast, in chimaeric mice with hyper-long telomeres, we found that rate of wound-healing was higher in the GFP-positive cells with hyper-long telomeres compared with the GFP-negative cells and the in the non-chimaeric age-matched controls (Fig. 7b,c and Supplementary Fig. 17). Telomere length was analysed in the skin extracted from chimaeric mice with either normal or hyper-long telomeres as well as in age-matched controls when wounds were caused, confirming longer telomeres in the GFP-positive cells derived from ES cells with hyper-long telomeres (Supplementary Fig. 18). Histopathology analysis confirmed that GFP-positive cells contributed to wound-healing in the chimaeric mice with hyper-long telomeres (Supplementary Fig. 19). Together, these results indicate that hyper-long telomeres are advantageous for skin regeneration compared with normal length telomeres.