DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift

Julia Franzen,Miloš Nikolić,Alina Ostrowska,Matthias Begemann,Axel Schambach,Chao-Chung Kuo,Anton Selich,Eduardo Fernandez-Rebollo,Wolfgang Wagner,Theodoros Georgomanolis,Clara Grezella,Reinhard Stöger,Lilija Brant,Anthony D Ho,Björn Rath,Michael Rothe,Melita Sara Mulabdić,Argyris Papantonis

doi:10.1038/s42003-021-02116-y

Abstract

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.

Highlights

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes
We have previously identified DNAm changes during culture expansion of mesenchymal stromal cells (MSCs) based on 27k Illumina BeadChip datasets, and thereby established a 6 CpG predictor to estimate passage numbers for MSCs7
In continuation of this work, we utilized the available 450k Illumina BeadChip datasets, which interrogate ~16 times more CpGs than the 27k version of the chip, and we considered datasets of additional types of primary cells

Summary

Introduction

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. Long-term culture is reflected by highly reproducible DNA methylation (DNAm) changes at specific sites in the genome[5,6]. We referred to these epigenetic modifications as senescenceassociated DNAm changes, albeit it is unclear if the cultureassociated DNAm changes are linked to the state of cellular senescence—we changed the terminology into cultureassociated DNAm changes It is so far unclear how DNAm patterns evolve during culture expansion and why they occur at specific genomic regions. We have demonstrated that senescence entry upon extensive culture expansion is associated with a reorganization of CTCF into large senescence-induced CTCF clusters (SICCs)[20] It remains unclear if and how the binding of CTCF changes during long-term culture

Methods

Results

Conclusion