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Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor, with 5-year survival rates > 70%. Cranial radiotherapy (CRT) to the whole brain, with posterior fossa boost (PFB), underpins treatment for non-infants; however, radiotherapeutic insult to the normal brain has deleterious consequences to neurocognitive and physical functioning, and causes accelerated aging/frailty. Approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development., Methods: We have developed a clinically relevant in vivo model system that recapitulates the radiotherapy dose, targeting, and developmental stage of childhood medulloblastoma. Consistent with human regimens, age-equivalent (postnatal days 35-37) male C57Bl/6J mice received computerized tomography image-guided CRT (human-equivalent 37.5 Gy EQD2, n  = 12) ± PFB (human-equivalent 48.7 Gy EQD2, n  = 12), via the small animal radiation research platform and were longitudinally assessed for > 12 months., Results: CRT was well tolerated, independent of PFB receipt. Compared to a sham-irradiated group ( n  = 12), irradiated mice were significantly frailer following irradiation (frailty index; P  = .0002) and had reduced physical functioning; time to fall from a rotating rod (rotarod; P  = .026) and grip strength ( P  = .006) were significantly lower. Neurocognitive deficits were consistent with childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; P  = .009) and exhibited spatial memory deficits (Barnes maze; P  = .029). Receipt of PFB did not induce a more severe late-effect profile., Conclusions: Our in vivo model mirrored childhood MB radiotherapy and recapitulated features observed in the late-effect profile of MB survivors. Our clinically relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late effects and the development of novel interventions for their amelioration., Competing Interests: The authors have no conflicts of interest to declare., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Telomere length and DNA methylation have been proposed as biological clock measures that track chronological age. Whether they change in tandem, or contribute independently to the prediction of chronological age, is not known., Methods: We address these points using data from two Scottish cohorts: the Lothian Birth Cohorts of 1921 (LBC1921) and 1936 (LBC1936). Telomere length and epigenetic clock estimates from DNA methylation were measured in 920 LBC1936 participants (ages 70, 73 and 76 years) and in 414 LBC1921 participants (ages 79, 87 and 90 years)., Results: The epigenetic clock changed over time at roughly the same rate as chronological age in both cohorts. Telomere length decreased at 48-67 base pairs per year on average. Weak, non-significant correlations were found between epigenetic clock estimates and telomere length. Telomere length explained 6.6% of the variance in age in LBC1921, the epigenetic clock explained 10.0%, and combined they explained 17.3% (allP< 1 × 10 -7 ). Corresponding figures for the LBC1936 cohort were 14.3%, 11.7% and 19.5% (allP< 1 × 10 -12 ). In a combined cohorts analysis, the respective estimates were 2.8%, 28.5% and 29.5%. Also in a combined cohorts analysis, a one standard deviation increase in baseline epigenetic age was linked to a 22% increased mortality risk (P= 2.6 × 10 -4 ) whereas, in the same model, a one standard deviation increase in baseline telomere length was independently linked to an 11% decreased mortality risk (P= 0.06)., Conclusions: These results suggest that telomere length and epigenetic clock estimates are independent predictors of chronological age and mortality risk., (© The Author 2016. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background: weak grip strength (GS) and chronic inflammation have been implicated in the aetiology of sarcopenia in older adults. Given the interrelationships between inflammatory biomarkers, a summary variable may provide better insight into the relationship between inflammation and muscle strength. This approach has not been investigated in very old adults (aged ≥85) who are at highest risk of muscle weakness., Methods: we used mixed models to explore the prospective association between GS over 5 years in 845 participants in the Newcastle 85+ Study, and inflammatory components identified by principal component analysis (PCA). Cut-offs of ≤27 kg (men) and ≤16 (women) were used to define sub-cohorts with weak and normal GS at each assessment., Results: PCA identified three components, which explained 70% of the total variance in seven baseline biomarkers. Basal interleukin-6 (IL-6) and tumour necrosis factor (TNF-α) had the highest loadings on Component 1; stimulated IL-6 and TNF-α and homocysteine the highest on Component 2; high-sensitivity C-reactive protein (hsCRP) loaded positively and albumin negatively to Component 3. In adjusted mixed models, only Component 3 was associated with GS. One SD increase of Component 3 was associated with a 0.41 kg lower GS initially (P = 0.03) in all participants, but not with GS decline over time. Similar conclusions held for those in the weak and normal GS sub-cohorts., Conclusion: an inflammatory profile including hsCRP and albumin was independently associated with baseline GS. Future studies linking inflammatory profiles and muscle strength are needed to corroborate these findings in older adults., (© The Author 2017. Published by Oxford University Press on behalf of the British Geriatrics Society.)

Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories., Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques., Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy., Conclusions: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories., (© The Author 2014. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Aim: Cross-sectional studies reported associations between short leucocyte telomere length (LTL) and measures of vascular and cardiac damage. However, the contribution of LTL dynamics to the age-related process of cardiovascular (CV) remodelling remains unknown. In this study, we explored whether the rate of LTL shortening can predict CV phenotypes over 10-year follow-up and the influence of established CV risk factors on this relationship., Methods and Results: All the participants from the MRC National Survey of Health and Development (NSHD) with measures of LTL and traditional CV risk factors at 53 and 60-64 years and common carotid intima-media thickness (cIMT), cardiac mass and left ventricular function at 60-64 years were included. LTL was measured by real-time polymerase chain reaction and available at both time points in 1033 individuals. While LTL at 53 years was not linked with any CV phenotype at 60-64 years, a negative association was found between LTL and cIMT at 60-64 years (β = -0.017, P = 0.015). However, the strongest association was found between rate of telomere shortening between 53 and 60-64 years and values of cIMT at 60-64 years (β = -0.020, P = 0.006). This association was not affected by adjustment for traditional CV risk factors. Cardiac measurements were not associated with cross-sectional or longitudinal measures of LTL., Conclusion: These findings suggest that the rate of progression of cellular ageing in late midlife (reflected by the rate of LTL attrition) relates to vascular damage, independently from contribution of CV risk factor exposure., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objectives: to examine the association between subjective and objective measures of sleep and wake and other health parameters in a cohort of the very old., Design: a population-based cohort study., Setting: primary care, North East England., Participants: four hundred and twenty-one men and women, aged 87-89, recruited to the Newcastle 85+ Study cohort., Methods: sleep questionnaires were administered and sleep-wake patterns were assessed over 5-7 days with a novel wrist triaxial accelerometer. Associations between sleep measures and various health parameters, including mortality at 24 months, were examined., Results: only 16% of participants perceived their sleep as severely disturbed as assessed with questionnaire responses. Wrist accelerometry showed marked variation between normal and abnormal sleep-wake cycles that did not correlate with the participants' perception of sleep. Impaired sleep-wake cycles were significantly associated with cognitive impairment, disability, depression, increased falls, body mass index and arthritis but not with any other specific disease markers and with decreased survival., Conclusions: commonly used sleep questionnaires do not differentiate well between those with objectively determined disturbance of sleep-wake cycles and those with normal cycles. Abnormal sleep-wake patterns are associated with institutionalisation, cognitive impairment, disability, depression and arthritis but not with other diseases; there is also an association with reduced survival.

DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.

Study Question: Can the basal epithelial compartment of the human endometrium be defined by specific markers?, Summary Answer: Human endometrial epithelial cells from the basalis express nuclear SOX9 and the cell-surface marker SSEA-1, with some cells expressing nuclear β-catenin. In vitro, primary endometrial epithelial cells enriched for SSEA-1+ show some features expected of the basalis epithelium., What Is Known Already: The endometrial glands of the functionalis regenerate from the basalis gland stumps following menstruation. Endometriosis is thought to originate from abnormal dislocation of the basalis endometrium. In the highly regenerative intestinal epithelium, SOX9 and nuclear β-catenin are more highly expressed in the intestinal crypt, the stem/progenitor cell region., Study Design, Size, Duration: A large prospective observational study analysing full-thickness human endometrial hysterectomy samples from 115 premenopausal women, 15 post-menopausal women and ectopic endometriotic lesions from 20 women with endometriosis., Participants/materials, Setting, Methods: Full-thickness endometrium from hysterectomy tissues was analysed by immunohistochemistry for SSEA-1, SOX9 and β-catenin. Primary human endometrial epithelial cells from short-term cultures were sorted into SSEA1+/- fractions with a cell sorter or magnetic beads and analysed for markers of differentiation and pluripotency and telomere lengths (TLs) using qPCR, telomerase activity [telomere repeat amplification protocol (TRAP)] and growth in 3D culture., Main Results and the Role of Chance: Similar to the intestinal crypt epithelium, human endometrial basal glandular epithelial cells expressed nuclear SOX9 and contained a rare subpopulation of cells with nuclear β-catenin suggestive of an activated Wnt pathway. The embryonic stem cell-surface marker, SSEA-1, also marked the human endometrial basal glandular epithelial cells, and isolated SSEA-1(+) epithelial cells grown in monolayer showed significantly higher expression of telomerase activity, longer mean TLs, lower expression of genes for steroid receptors and produced a significantly higher number of endometrial gland-like spheroids in 3D culture compared with SSEA-1(-) epithelial cells (P = 0.009). Cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, suggesting that the basalis contributes to ectopic lesion formation in endometriosis following retrograde menstruation., Limitations, Reasons for Caution: This is a descriptive study with only short-term culture of the primary human epithelial cells in vitro., Wider Implications of the Findings: The surface marker SSEA1 enriches for an endometrial epithelial cell subpopulation from the basalis. Since the functional endometrium originates from these cells, it is now possible to study basalis epithelium for stem/progenitor cell activity to extend our current understanding of endometrial biology in health and diseases., Study Funding/competing Interest(s): The work included in this manuscript was funded by Wellbeing of Women project grant RG1073 (D.K.H. and C.G.). We also acknowledge the support by National Health and Medical Research Council, RD Wright Career Development Award 465121 and Senior Research Fellowship 1042298, and the Victorian Government's Operation Infrastructure Support Program to C.G. and MRC G0601333 to T.V.Z. All authors have no conflict of interest to declare., Trial Registration Number: N/A.

Background: reduced telomere length in blood cells has been associated with increased risk of multiple age-related diseases and is widely regarded as a general biomarker of ageing. Therefore, it is important to know both the extent and limitations of this association. We investigated the relation between telomere length and anaemia in two independent cohorts, with the prior expectation of adding anaemia to the list of conditions for which telomere reduction is a risk factor., Participants and Methods: the present study is embedded in the Newcastle 85-plus Study and Leiden 85-plus Study, two population-based studies of inhabitants of Newcastle and North Tyneside, UK (n = 749) and Leiden, the Netherlands (n = 658) aged 85 and over. High-molecular-weight DNA was isolated from full fresh blood (Newcastle) and peripheral blood mononuclear cells samples (Leiden). Telomere length was measured as abundance of telomeric template versus a single gene by quantitative real-time polymerase chain reaction. Anaemia was defined according to World Health Organization criteria., Results: in both studies, no differences in median telomere length were observed between participants with anaemia and participants without anaemia (Newcastle: 2,846 bp (interquartile range (IQR) 2,433-3,630) versus 2,920 bp (IQR 2,425-3,570), P = 0.63; Leiden: 4,136 bp (IQR 3,879-4,428) versus 4,167 bp (IQR 3,893-4,501), P = 0.41). Telomere length also did not correlate with any other haematological parameter in both men and women., Conclusions: in contrast to other age-related diseases, telomere length is not associated with anaemia or any other haematological parameter in older individuals in the general population.

During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many 'real-life' studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate 'tissues' such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 μM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.

A precise understanding of mechanisms used by human embryonic stem cells (hESCs) to maintain genomic integrity is very important for their potential clinical applications. The G1 checkpoint serves to protect genomic integrity and prevents cells with damaged DNA from entering S-phase. Previously, we have shown that downregulation of cyclin-dependent kinase 2 (CDK2) in hESC causes G1 arrest, loss of pluripotency, upregulation of cell cycle inhibitors p21 and p27 and differentiation toward extraembryonic lineages. In this study, we investigate in detail the role of CDK2 in cellular processes, which are crucial to the maintenance of genomic stability in hESC such as G1 checkpoint activation, DNA repair, and apoptosis. Our results suggest that downregulation of CDK2 triggers the G1 checkpoint through the activation of the ATM-CHK2-p53-p21 pathway. Downregulation of CDK2 is able to induce sustained DNA damage and to elicit the DNA damage response (DDR) as evidenced by the formation of distinct γ-H2.AX and RAD52-BRCA1 foci in hESC nuclei. CDK2 downregulation causes high apoptosis at the early time points; however, this is gradually decreased overtime as the DDR is initiated. Our mass spectrometry analysis suggest that CDK2 does interact with a large number of proteins that are involved in key cellular processes such as DNA replication, cell cycle progression, DNA repair, chromatin modeling, thus, suggesting a crucial role for CDK2 in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in hESC., (Copyright © 2011 AlphaMed Press.)

Background: To test our hypothesis that eutopic secretory phase endometrium from women with endometriosis is similar to proliferative phase endometrium from fertile women without endometriosis, we explored the expression of regulators of cell fate across the menstrual cycle., Methods: Endometrial biopsies were taken from 73 women, comprising 38 women with surgically diagnosed active peritoneal endometriosis (Group 1) and 35 fertile women without endometriosis (Group 2). Nucleolin, proliferating cell nuclear antigen (PCNA), telomerase and histone gamma-H2AX expression was evaluated by immunohistochemistry and mean telomere length (TL) by quantitative PCR., Results: We have immunolocalized nucleolin and gamma-H2AX in the benign premenopausal endometrium for the first time. All markers were present in the proliferative phase endometrium of all women. In Group 2, during the secretory phase, proliferative markers declined with a paradoxical increase in stromal gamma-H2AX. Women in Group 1, however, showed a persistent immunoreactivity for the proliferative markers, while the staining for gamma-H2AX decreased in secretory endometrium (P < 0.05). This difference between groups was significant in both stroma and glands for nucleolin (P < 0.0001), PCNA (P < 0.01) and gamma-H2AX (P < 0.05) in the secretory phase. We showed a positive correlation between mean TL and nucleolin expression (glandular r = 0.37, P = 0.002; stromal r = 0.4, P = 0.001), telomerase immunoreactivity (glandular r = 0.33, P = 0.009; stromal r = 0.4, P = 0.001) and glandular PCNA (r = 0.35, P = 0.004), whereas a negative correlation was seen between mean TL and gamma-H2AX (r = -0.28, P = 0.04)., Conclusions: These findings demonstrate that the state of replication seen in secretory phase endometrium from women with active peritoneal endometriosis is not a simple extension of the proliferative phase.

Background: In order to test our hypothesis that endometriosis is associated with abnormal expression of telomerase and telomere lengthening in endometrium, we assessed endometrial expression of the human telomerase enzyme and telomere length (TL)., Methods: This prospective pilot study, included 29 women with symptomatic, surgically diagnosed endometriosis (Group 1) and 27 healthy, fertile, symptom-free women without endometriosis (Group 2, confirmed by laparoscopy). Seventeen women in Group 1 and 15 women in Group 2 had endometrial biopsies taken on Day 21 +/- 2 of the cycle. A further 12 women in each group were biopsied on Day 26 +/- 2. Telomerase and estrogen receptor beta (ERbeta) expression was evaluated by immunohistochemistry. Mean TL was determined by quantitative PCR., Results: The endometria of fertile healthy women showed either weak or no telomerase immunoreactivity throughout the luteal phase. Immunostaining for telomerase was significantly increased during the implantation window and the premenstrual endometria of women with endometriosis (P < 0.0001). This was associated with a loss of stromal and vascular ERbeta immunostaining (P < 0.05). The mean TL were significantly longer in endometria of women with endometriosis during the implantation window (P = 0.005), indicating the biological relevance of our novel finding of telomerase in benign endometrium. There was positive correlation of the circulating estradiol with peripheral blood TL in women., Conclusions: We speculate that aberrant endometrial expression of telomerase mediates alterations in cell fate that enhance proliferation, contributing to the pathogenesis of endometriosis.

Evolutionary theory predicts that cellular maintenance, stress defense, and DNA repair mechanisms should be most active in germ line cells, including embryonic stem cells that can differentiate into germ line cells, whereas it would be energetically unfavorable to keep these up in mortal somatic cells. We tested this hypothesis by examining telomere maintenance, oxidative stress generation, and genes involved in antioxidant defense and DNA repair during spontaneous differentiation of two human embryonic stem cell lines. Telomerase activity was quickly downregulated during differentiation, probably due to deacetylation of histones H3 and H4 at the hTERT promoter and deacetylation of histone H3 at hTR promoter. Telomere length decreased accordingly. Mitochondrial superoxide production and cellular levels of reactive oxygen species increased as result of increased mitochondrial biogenesis. The expression of major antioxidant genes was downregulated despite this increased oxidative stress. DNA damage levels increased during differentiation, whereas expression of genes involved in different types of DNA repair decreased. These results confirm earlier data obtained during mouse embryonic stem cell differentiation and are in accordance with evolutionary predictions.

Aims: Heart failure is a condition increasingly prevalent at older ages; however, mechanisms by which the ageing process affects cardiac function are largely unknown. Telomere length is a biomarker of ageing that has been suggested to be associated with a variety of diseases of late onset, but its relationship with cardiac function has not previously been studied. We measured telomere length in peripheral blood mononuclear cells and carried out echocardiography in a group of 85-year old subjects recruited from the community as part of the Newcastle 85+ Study., Methods and Results: Eighty-nine subjects were recruited through local family practitioners. They were visited in their homes for clinical assessment and echocardiography, which was performed using a handheld device. Telomere length was measured by a real-time PCR method. High sensitivity C-reactive protein was measured using ELISA. Echocardiographic M-mode ejection fraction (EF) was strongly associated with telomere length (P=0.006) in subjects without evidence of previous MI. Sex and telomere length were significant predictors of EF while current smoking, blood pressure, plasma high sensitivity C-reactive protein, and use of cardiovascular medications were not. One standard deviation longer telomeres were associated with a 5% higher EF. Telomere length accounted for 12% of the observed variability in EF., Conclusion: These data show influences of the ageing process on myocardial function in the oldest old, apparently independent of other specific disease processes. This may be of importance in the aetiology of heart failure in this age group.

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence.

Embryonic stem cells (ESCs) are capable of extended self-renewal and maintenance of pluripotency even after many population doublings. This is supported by high levels of telomerase activity and enhanced antioxidant protection in ESCs, both of which are downregulated during differentiation. To examine the role of telomerase for ESC self-renewal and differentiation, we overexpressed the reverse transcriptase subunit (Tert) of murine telomerase in ESCs. Increased telomerase activity enhances the self-renewal ability of the Tert-overexpressing ESCs, improves their resistance to apoptosis, and increases their proliferation. The differentiated progeny of wild-type ESCs express little Tert and show shortening of telomeric overhangs. In contrast, the progeny of Tert-overexpressing ESCs maintain high telomerase activity, as well as the length of G-rich overhangs. In addition, these cells accumulate lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Finally, differentiation toward hematopoietic lineages is more efficient as a result of the continued expression of Tert. Microarray analysis revealed that overexpression of Tert altered expression of a variety of genes required for extended self-renewal and lifespan. Our results suggest that telomerase functions as a "survival enzyme" in ESCs and its differentiated progeny by protecting the telomere cap and by influencing the expression patterns of stress response and defense genes. This results in improved proliferation of ESCs and more efficient differentiation, and these results might have profound consequences for stem cell-replacement therapies.

A very small number of embryonic stem (ES) cells gives rise to all tissues of the embryo proper. This means that ES cells should be equipped with highly efficient mechanisms to defend themselves against various stresses and to prevent or repair DNA damage. One of these mechanisms is a high activity of a verapamil-sensitive multidrug efflux pump. Because reactive oxygen species are a major source of DNA damage, we further tested the idea that murine ES cells might differ from their more differentiated counterparts by high levels of antioxidant defense and good DNA strand break repair capacity. This was confirmed by comparing cellular peroxide levels, total antioxidant capacity, and activity of radiation-induced strand break repair between murine ES cells and embryoid bodies or embryonic fibroblasts. Using microarrays and confirmation by reverse transcription-polymerase chain reaction, we identified several candidate antioxidant and stress-resistance genes that become downregulated during differentiation of ES cells into embryoid bodies.