Epigenetic biomarkers to track differentiation of pluripotent stem cells [methylation array]

Quality control of induced pluripotent stem cells remains a challenge. For validation of the pluripotent state, it is crucial to determine trilineage differentiation potential toward endoderm, mesoderm, and ectoderm. Here, we report GermLayerTracker, a combination of site-specific DNA methylation (DNAm) assays that serve as biomarker for early germ layer specification. CG dinucleotides (CpGs) were identified with characteristic DNAm at pluripotent state and after differentiation into endoderm, mesoderm, and ectoderm. Based on this, a pluripotency score was derived that may be indicative for differentiation capacity, as well as lineage-specific differentiation scores to monitor either directed differentiation, or self-organized multilineage differentiation in embryoid bodies. Furthermore, we established pyrosequencing assays for fast and cost-effective analysis. In the future, the GermLayerTracker could be used for quality control of pluripotent cells and to estimate lineage-specific commitment during initial differentiation events. 3 iPSC lines were differentiated into the three germ layer with directed differentiation. Additionally, cells (4 cell lines) were also used to generate embryoid bodies (EBs) for undirected differentiation (4 and 7 days). DNA and RNA was isolated for Illumina Methylation BeadChips and RNA-seq.