Epigenetic modifications play a central role in differentiation and development. This study indicates that modification of DNA methylation is also involved in replicative senescence upon long-term culture of MSC. Furthermore, a similar pattern of differential methylation was observed between MSC from young and elderly donors. These results support the perception that replicative senescence and aging are not only due to stochastic accumulation of cellular defects but rather represent a developmental program. On the other hand, the process is overlaid by stress-induced senescence changes e.g. due to high oxygen content or the artificial environment. Furthermore, the cellular aging of MSC might be accompanied by a different composition of heterogeneous subpopulations and loss of self-renewal potential under in vitro conditions. Hence, senescence of MSC has many facets that need to be further elucidated (Wagner et al., review submitted).
Methylation was analyzed by using the novel HumanMethylation27 BeadChip representing 27 578 CpGs that are associated with promoter regions of more than 13 500 annotated genes. This array was designed to cover CpG dinucleotides in the promoter region of genes and therefore is clearly biased toward the assessment of gene-specific methylation. Differential CpG methylation was highly reproducible in different donor samples and was verified by pyrosequencing for all sequences tested. Thus, this technique facilitates reliable profiling of global methylation patterns.
Other authors have reported that global DNA methylation levels decrease upon long-term culture (Wilson & Jones, 1983
; Young & Smith, 2001
). Therefore, it has been speculated that maintenance of DNA methylation by DNA methyltransferase 1 (DNMT1) is incomplete resulting in a gradual decrease of DNA methylation with repeated cell cycles (Nilsson et al., 2005
). However, our results did not reveal an overall decrease in DNA methylation levels. In fact, methylation remained constant over the whole culture period at most CpG sites represented on the BeadChip. To our knowledge this study demonstrates for the first time that only specific CpG islands become differentially methylated upon long-term culture. DNA methylation changes upon aging may also depend on individual differences although differential methylation was verified by pyrosequencing in independent donor samples. It is also likely, that age-related changes are influenced by the different cellular composition upon aging as well as by the different tissue and isolation technique.
Methylation changes in our study were often associated with homeobox genes. Expression of these genes is tightly regulated under temporal control during vertebrate development. It has been shown that chromatin modifications play an important role for regulation of the ‘Hox clock’ that coordinates body patterning (Illingworth et al., 2008
; Soshnikova & Duboule, 2009
). Furthermore, age-associated gene expression changes have been described in homeobox genes (Stelnicki et al., 1998
; Wagner et al., 2009
) and it is conceivable that they are involved in the timing of aging. Exemplarily, we have verified differential methylation for HOXA5
upon long-term culture and aging by pyrosequencing. The homeobox gene DLX5
is also involved in differentiation toward the osteoblastic lineage (Lee et al., 2005
). Therefore, hypermethylation and decreased gene expression of DLX5
might be relevant for the impaired differentiation capacity of MSC upon long-term culture. It has been shown that methylation changes at specific CpG sites can affect differentiation of MSC (Noer et al., 2007
; Zimmermann et al., 2008
). In addition, age-related methylation changes have been described. Several tumor suppressor genes are hypermethylated upon aging with potential implications for cancer susceptibility in the elderly (So et al., 2006
). Among these was RUNX3
that revealed age-associated changes in our analysis. Another differentially methylated gene was the cycline-dependent kinase inbibitor 2B (CDKN2B
) that has been implicated in senescence in our previous work (Schallmoser et al., 2009
Several genes such as RUNX3
were more than 20% hypermethylated at specific CpG sites and more than 20% hypomethylated in others. These antagonistic modifications within the same promoter region indicate that DNA modifications are restricted to relatively short and specific sequences. On the other hand, pyrosequencing revealed that methylation changes are not restricted to unique CpG sites but also include neighboring cytidines of the same CpG island. It has been suggested that senescence can also be induced in immortal cells by treatment with 5aza-2deoxycytidine (5AZA) (Vogt et al., 1998
). However, it is difficult to discern toxic effects of 5AZA. The complex pattern of hypermethylation and hypomethylation observed in this study might explain why replicative senescence cannot be controlled by demethylating agents.
methylation is facilitated by DNA methyltransferases DNMT3A and DNMT3B but it is mostly unknown how the extent of de novo
methylation is regulated and where it takes place. Active DNA hypomethylation is also considered to occur but a specific enzyme that removes the methyl group from 5-methylcytosine has not yet been described. It has been suggested that DNA methyltransferases exhibit dual actions in CpG methylation and demethylation of 5mCpGs through deamination (Kangaspeska et al., 2008
; Metivier et al., 2008
). DNA methylation patterns may vary drastically within CpG islands and only methylation of a defined core-region silences gene expression (Gonzalgo et al., 1998
; Deng et al., 1999
; Ushijima & Okochi-Takada, 2005
). We have shown that age-associated hypermethylated and hypomethylated genes are enriched in SP1 transcription factor binding sites and it has been proposed that binding of the SP1 transcription factor protects CpG islands from methylation (Brandeis et al., 1994
; Macleod et al., 1994
). ‘Seeds of methylation’ is another interesting mechanism that leads to increasing methylation of CpG islands. This hypothesis is based on the observation that little methylation is induced in an initially nonmethylated region whereas a high degree of methylation is induced in a sparsely methylated region (Song et al., 2002
; Stirzaker et al., 2004
). This mechanism might serve as a cell-cycle counter.
It is commonly accepted that DNA methylation silences gene expression. This is also reflected by the correlation of the two different levels investigated in our study: nonmethylated genes revealed higher signal intensity in mRNA expression profiling. However, differential DNA methylation at specific CpG sites does not necessarily correlate with observed gene expression changes. Relevant fold-changes in gene expression can only be expected if the CpG sites are predominantly methylated. It is also conceivable that selective modification of specific promoter regions either activates or inactivates binding of transcription regulators.
We have previously described age-related gene expression changes in human stem and progenitor cells and observed a moderate but significant concordance in the expression profiles upon aging in vivo
and replicative senescence in vitro
(Wagner et al., 2009
). However, the proliferation rate was not higher in MSC from younger donors and they did not reach a higher number of cumulative population doublings. This might be attributed to the relatively low number of samples per group as other authors reported an inverse relationship between donor age and the replicative life span in vitro
for fibroblasts or MSC (Schneider & Mitsui, 1976
; Stenderup et al., 2003
; Mareschi et al., 2006
; Shibata et al., 2007
). Also the doubling time of either freshly isolated marrow cells or MSC of early passage has been described to be significantly lower in samples from elderly people (Zhou et al., 2008
) whereas this was not observed in our study. Analysis of age-specific changes might be affected by the different sources for MSC that were available for the two age groups. MSC were either isolated from human bone marrow from the iliac crest or from the femoral head. There were no differences in growth, cell morphology, immunophenotype or differentiation potential (Wagner et al., 2009
). Thus, MSC from BM and HIP fulfilled the criteria that are commonly used for definition of MSC although these cell preparations are heterogeneous and may comprise different subpopulations (Dominici et al., 2006
; Wagner & Ho, 2007
). However, we can not exclude that some of the methylation changes are due to the different bone marrow regions. Notably, however, there was a relevant overlap of methylation changes upon long-term culture and aging of MSC. Furthermore, there is a significant concordance of our age-related changes in MSC and age-associated methylation changes in ovarian cancer samples (Teschendorff et al.
, manuscript in preparation). Further integrative analysis of methylation profiles of different cell types and tissues will help to discern the existence of a distinct methylation signature for aging.
Bone marrow is a complex tissue with various different cell types. Hence, it is apparent that aging and replicative senescence are not identical despite molecular similarities of the two processes. Long-term culture has been shown to impair differentiation potential of MSC, especially of adipogenic differentiation (Baxter et al., 2004
; Bonab et al., 2006
; Wagner et al., 2008
). By contrast, other authors demonstrated that aging activates adipogenic differentiation and suppresses osteogenic differentiation (Moerman et al., 2004
; Zhou et al., 2008
). These authors also demonstrated that expression of the master transcription factor for osteoblast differentiation RUNX2 and DLX5 decreased upon aging and this is in line with higher methylation as observed in our data. It is conceivable, that the higher propensity for adipogenesis in BM of elderly people results in a lower number of osteogenic progenitor cells.
Aging is not an inevitable fate of all cells. Our germ line cells and their progeny are sustained through the generations and embryonic stem cells as well as induced pluripotent stem cells do not show signs of replicative senescence upon long-term culture (Zeng, 2007
). It is therefore conceivable, that the loss of pluripotency is directly accompanied with limitation of the replicative live span (Marciniak-Czochra et al., 2009
). Both mechanisms might be regulated by similar epigenetic modifications that govern differentiation and development. Our results demonstrate that specific CpG sites become either methylated or demethylated upon long-term culture. Analysis of these specific CpG sites might facilitate monitoring of the replicative lifespan of cells. It will be of central interest to further increase our understanding of the mechanisms regulating senescence-associated and age-associated epigenetic modifications.