DNA Methylation Analysis as a Tool for Cell Typing

Abstract

Cell therapeutic approaches currently lack definitive quality control measures which guarantee safety in clinical applications and create consistent standards for regulatory approval. These approaches rely on isolation, purification and possibly ex vivo manipulation of donor cells. Since such cells are exposed to artificial environments, there is potential for deviations from natural growth processes. The resulting heterogeneity of cell cultures is an inherent problem. Therefore, verification of cell identity and quantification of subpopulations is mandatory. Focusing on cultured human primary cells, we tested whether DNA methylation patterns serve as distinctive cell type markers. We identified panels of cell type specific differentially methylated gene regions (CDMs) which produce unambiguous profiles for these cell types. Applying methylation sensitive single nucleotide primer extension generated binary cell type descriptors ("barcodes") which allow quantification of cell mixtures. Thus, methylation based analytics suggest themselves as promising tools for the characterization and quality control of ex vivo manipulated cells.

Epigenetic quantification of tumor-infiltrating T-lymphocytes

“…30 During bisulfiteconversion of genomic DNA, unmethylated CpGs are converted to TpG ("TpG-variant"), whereas methylated CpGs remain as CpG residues ("CpG-variant") 31 allowing the discrimination between both variants. Exploitation of epigenetic mechanisms as tool for cell quantification was suggested 32,33 and "epigenetic immunophenotyping" based on measurement of bisulfite-converted FOXP3 TSDR to identify and distinguish Treg (as TpG variant) from all other cell types, including activated, FOXP3-expressing effector T cells (as CpG variant) in mice and humans was established. 34 FOXP3 TSDR was confirmed as Treg marker of unequalled specificity.…”

DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3+ conventional T cells

“…Epigenetic modification, notably DNA demethylation, and the resulting chromatin remodeling appear to be mechanisms crucial for fixation of gene expression patterns [34][35][36][37]. Chromatin remodeling upon epigenetic modification is thought to determine the accessibility of genes by transcriptional activators or repressors.…”

“…DNA methylation constitutes a biologically and chemically stable epigenetic modification, resulting in long-term gene expression changes [34][35][36][37]. Thus, we here asked whether differential methylation of the FOXP3 locus might be less susceptible to short-term fluctuation of gene expression, and, therefore, be superior to determination of mRNA or protein expression for the unambiguous identification of FOXP3 + Treg.…”

Quantitative DNA Methylation Analysis of FOXP3 as a New Method for Counting Regulatory T Cells in Peripheral Blood and Solid Tissue

“…DNA methylation is a biologically and chemically stable epigenetic modification, locking long-term gene expression patterns (30)(31)(32). Demethylation at a highly conserved region within the human FOXP3 gene (Treg-specific demethylated region, TSDR; ref.…”