BackgroundHuman aging is associated with DNA methylation changes at specific sites in the genome. These epigenetic modifications may be used to track donor age for forensic analysis or to estimate biological age.ResultsWe perform a comprehensive analysis of methylation profiles to narrow down 102 age-related CpG sites in blood. We demonstrate that most of these age-associated methylation changes are reversed in induced pluripotent stem cells (iPSCs). Methylation levels at three age-related CpGs - located in the genes ITGA2B, ASPA and PDE4C - were subsequently analyzed by bisulfite pyrosequencing of 151 blood samples. This epigenetic aging signature facilitates age predictions with a mean absolute deviation from chronological age of less than 5 years. This precision is higher than age predictions based on telomere length. Variation of age predictions correlates moderately with clinical and lifestyle parameters supporting the notion that age-associated methylation changes are associated more with biological age than with chronological age. Furthermore, patients with acquired aplastic anemia or dyskeratosis congenita - two diseases associated with progressive bone marrow failure and severe telomere attrition - are predicted to be prematurely aged.ConclusionsOur epigenetic aging signature provides a simple biomarker to estimate the state of aging in blood. Age-associated DNA methylation changes are counteracted in iPSCs. On the other hand, over-estimation of chronological age in bone marrow failure syndromes is indicative for exhaustion of the hematopoietic cell pool. Thus, epigenetic changes upon aging seem to reflect biological aging of blood.

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 longterm 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 doxycyclineinducible 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 SADNAm in pluripotent cells may play a central role for their escape from cellular senescence.

Aging is associated with highly reproducible DNA methylation (DNAm) changes, which may contribute to higher prevalence of malignant diseases in the elderly. In this study, we analyzed epigenetic aging signatures in 5,621 DNAm profiles of 25 cancer types from The Cancer Genome Atlas (TCGA). Overall, age-associated DNAm patterns hardly reflect chronological age of cancer patients, but they are coherently modified in a non-stochastic manner, particularly at CpGs that become hypermethylated upon aging in non-malignant tissues. This coordinated regulation in epigenetic aging signatures can therefore be used for aberrant epigenetic age-predictions, which facilitate disease stratification. For example, in acute myeloid leukemia (AML) higher epigenetic age-predictions are associated with increased incidence of mutations in RUNX1, WT1, and IDH2, whereas mutations in TET2, TP53, and PML-PARA translocation are more frequent in younger age-predictions. Furthermore, epigenetic aging signatures correlate with overall survival in several types of cancer (such as lower grade glioma, glioblastoma multiforme, esophageal carcinoma, chromophobe renal cell carcinoma, cutaneous melanoma, lung squamous cell carcinoma, and neuroendocrine neoplasms). In conclusion, age-associated DNAm patterns in cancer are not related to chronological age of the patient, but they are coordinately regulated, particularly at CpGs that become hypermethylated in normal aging. Furthermore, the apparent epigenetic age-predictions correlate with clinical parameters and overall survival in several types of cancer, indicating that regulation of DNAm patterns in age-associated CpGs is relevant for cancer development.

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.

SummaryReplicative senescence has fundamental implications on cell morphology, proliferation, and differentiation potential. Here, we describe a simple method to track long-term culture based on continuous DNA-methylation changes at six specific CpG sites. This epigenetic senescence signature can be used as biomarker for various cell types to predict the state of cellular senescence with regard to the number of passages, population doublings, or days of in vitro culture.Key words: cellular senescence; epigenetic; DNA-methylation; mesenchymal stem cells; fibroblast.Cellular senescence in the course of culture expansion has functional relevance: The proliferation rate and in vitro differentiation potential of various cell types such as mesenchymal stem cells (MSC) declines with higher passages. Furthermore, cells may acquire genetic aberrations (MezaZepeda et al., 2008), and there is evidence for epigenetic modifications upon long-term culture . Therefore, replicative senescence needs to be considered for quality control of cell preparationsespecially in cellular therapy (Tarte et al., 2010).Commonly used parameters for cellular senescence are (i) passage number, (ii) cumulative population doublings (cPD), and (iii) the time of in vitro culture. These parameters need to be carefully documented throughout culture expansion-otherwise, it was so far impossible to retrospectively determine the state of senescence. Expression of senescence-associated beta galactosidase (SA-b-gal) can discern cells at the senescent stage but hardly provides a quantitative measure throughout culture expansion (Dimri et al., 1995). Cellular senescence is associated with a loss of telomere integrity. However, telomere length and telomerase expression vary in different cell types and have not proven as reliable quantitative measure for replicative senescence (Prowse & Greider, 1995). Long-term culture was also associated with highly consistent gene expression changes but this method requires comparative samples of early passage and differential gene expression did not facilitate absolute quantification of cellular senescence (Schallmoser et al., 2010). Recently, it has been demonstrated that long-term culture is associated with specific epigenetic modifications in DNA-methylation profiles Noer et al., 2007; Schellenberg et al., 2011;Koch et al., 2011).Therefore, we hypothesized that methylation at specific cytosine residues might provide an epigenetic signature to track the state of cellular senescence.We have used various human cell preparations to identify a generally applicable senescence signature: Fibroblasts from different dermal regions, MSC from bone marrow (BM; iliac crest or caput femoris) or from adipose tissue (AT); culture medium was supplemented with either fetal calf serum (FCS; 2% or 10%) or human platelet lysate. Long-term growth curves demonstrated that the maximal number of cPD depends on cell type, tissue of origin, and culture medium (Fig. 1A). Senescent cells acquired typical morphological changes, stained positive fo...