Primed-to-naive conversion of pluripotent stem cells can be tracked by specific DNA methylation changes for optimized culture conditions

Abstract During early embryonic development, cells transition from naive to primed pluripotent state. Various culture conditions have been established to revert primed cells back to naive state, to increase differentiation potential and to reset epigenetic abnormalities. In this study, we modified culture conditions to allow primed-to-naive conversion under feeder-independent and normoxic conditions (FINO medium), which exemplified the need for a quantitative measure of pluripotent states. DNA methylation (DNAm) profiling revealed extensive hypomethylation at naive state, but also significant gains of methylation at specific sites in the genome. We demonstrate that DNAm patterns can be used to benchmark culture protocols. Furthermore, we developed a naive-score based on DNAm at two genomic sites, which can be analyzed by digital PCR to monitor transition between pluripotent states. Our study describes a simplified culture protocol for primed-to-naive conversion, offers insights into the specific DNAm changes, and introduces a robust DNAm-based biomarker to track this process effectively.