Epigenetic drift during long-term culture of cells in vitro [4C]

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. These changes might be directly regulated or caused by gradual deregulation of the epigenetic state, which is often referred to as “epigenetic drift”. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated in the course of culture expansion of mesenchymal stem cells (MSCs) and other cell types. During reprogramming into induced pluripotent stem cells (iPSCs) particularly the culture-associated hypomethylation is reversed simultaneously with age-associated and pluripotency-associated DNAm changes. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that upon passaging the DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpolulations of MSCs at later passages. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no preferential interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results indicate that DNAm changes during culture-expansion resembles epigenetic drift. Overall design: Examination of chromatin interactions in two donors of mesenchymal stromal cells in early (P2 and P3) and late (P7 and P9) passages at four senescence-associated regions (associated with the genes ALOX12, CASR, LTC4S and KRTAP13.3)