Date Published: September 25, 2011
Publisher: Impact Journals LLC
Author(s): Anne Schellenberg, Qiong Lin, Herdit Schüler, Carmen M. Koch, Sylvia Joussen, Bernd Denecke, Gudrun Walenda, Norbert Pallua, Christoph V. Suschek, Martin Zenke, Wolfgang Wagner.
Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications – these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled.
Fifty years ago, Leonhard Hayflick discovered that the number of cell divisions is limited for cells in culture  – after about 40 to 60 divisions the proliferation rate decays until the cells ultimately enter a senescent state. This phenomenon applies to all somatic cells in culture which are not immortalized.
Human MSC are relatively stable towards chromosomal aberrations whereas long-term culture induces highly reproducible epigenetic modifications at specific sites in the genome. These epigenetic modifications may govern senescence-associated functional changes such as loss of differentiation potential upon several passages. Therefore, culture expansion and passaging clearly have to be taken into account for cellular therapy.