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Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched 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 support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.Julia Franzen et al. investigate if changes in DNA methylation at specific genetic loci during cell culture expansion are due to a specific mechanism or gradual deregulation of an epigenetic state. Their results suggest that changes in CpG methylation are due to indirect epigenetic drift, rather than a consequence of targeting by DNA methyltransferases.

Abstract Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes at specific sites in the genome. These changes might be due to an directly regulated epigenetic process, or to 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, which seems to occur in relation to chromatin conformation. Competing Interest Statement WW is cofounder of Cygenia GmbH (www.cygenia.com), which can provide service for epigenetic analysis to other scientists. JF contributes to this company, too. All other authors do not have a conflict of interest to declare. Footnotes * We noticed a mistake in the script for the hemimethylation analysis. In fact, hemimethylation at the culture-associated CpGs was much less pronounced. We have therefore tempered this aspect in a revised manuscript. The title and discussion was changed accordingly. We dearly apologize for this confusion.