Mesenchymal stromal cells (MSC) are studied as possible immunotherapy after solid organ transplantation. However, to obtain sufficient numbers, cells need to be expanded in culture. Furthermore, MSC may be pre-treated in culture with inflammatory cytokines to optimise their immunomodulatory properties. It is unknown whether culture expansion and inflammatory challenge induce epigenetic changes in MSC, which could affect their function by alterations in gene expression. In this study, changes in DNA methylation patterns in MSC after cytokine stimulation and culture expansion were investigated.

Human umbilical cord derived MSC were cultured for 3 days with IFNγ, TGFβ or a combination of cytokines (IFNγ, TGFβ and retinoic acid). Next, cytokines were removed and MSC were cultured for a further 14 days. Genome-wide DNA methylation was measured (single CpG site, gene ontology and regions) directly after stimulation (day 3) and after long term removal of cytokines (day 17) with Infinium MethylationEpic Beadchip array and analysed with the use of R.

At day 3, CpG site located on chromosome 2 (Cg00221794), within active enhancer regions according to roadmap epigenomics consortium, was significantly hypermethylated in IFNγ treated MSC and the cytokine combination treated MSC compared to unstimulated MSC. At day 17, this site remained hypermethylated when comparing unstimulated with IFNy and the cytokine combination, with an additional 5 and 16 differently methylated CpG sites, respectively. TGFβ stimulation did not lead to changes in DNA methylation. Comparison of unstimulated MSC between day 3 and day 17 resulted in >4000 significantly differentially methylated CpG sites and >100 significantly differentially methylated regions, corresponding to membrane, cytoskeleton, mitochondria, apoptosis and immune related genes.

DNA methylation patterns of MSC are altered by various cytokines, some of which persist after removal of cytokines, indicating imprinting. Interestingly, we observed a large impact of in vitro culturing on the DNA methylation patterns of MSC. These findings impact the way we currently look at and implement MSC stimulation, as well as the manner of culturing of MSC, which greatly influence them.

CITATION INFORMATION: de Witte S, Peters F, Merino A, Korevaar S, Elliman S, Newsome P, Meurs J, Boer K, Baan C, Hoogduijn M. Impact of Culture Expansion and Inflammatory Cytokine Challenge on DNA Methylation Profiles in MSC. Am J Transplant. 2017;17 (suppl 3).

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