Dynamic heterogeneity of DNA methylation and hydroxymethylation in embryonic stem cell populations captured by single-cell 3D high-content analysis

Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a 10-day differentiation course in vitro: by means of confocal and super-resolution imaging together with 3D high-content analysis, an essential tool in single-cell screening. In summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC. 2) While average Oct-4 levels stagnated on a cell-population base (0.015 lnIU/day), Sox17 and FoxA2 increased 22-fold and 3-fold faster, respectively (Sox17: 0.343 lnIU/day; FoxA2: 0.046 lnIU/day). In comparison, global DNA methylation levels increased 4-fold faster (0.068 lnIU/day), and global hydroxymethylation declined at 0.046 lnIU/day, both with a better explanation of the temporal profile. 3) This progression was concomitant with the occurrence of distinct nuclear codistribution patterns that represented a heterogeneous spectrum of states in differentiation; converging to three major coexisting 5mC/5hmC phenotypes by day 10: 5hmC⁺/5mC^-, 5hmC⁺/5mC⁺, and 5hmC^-/5mC⁺ cells. 4) Using optical nanoscopy we could delineate the respective topologies of 5mC/5hmC colocalization in subregions of nuclear DNA: in the majority of 5hmC⁺/5mC⁺ cells 5hmC and 5mC predominantly occupied mutually exclusive territories resembling euchromatic and heterochromatic regions, respectively. Simultaneously, in a smaller subset of cells we observed a tighter colocalization of the two cytosine variants, presumably delineating chromatin domains in remodeling. We conclude that 1) 5mC emerges as the most differential marker in our model system. 2) However, the combined enrollment of the two DNA modifications provided higher-definition screening and lead to the identification of cell subpopulations based on differential 5hmC/5mC phenotypes corresponding to different 5hmC/5mC ratios. The results encourage: a) assessing the regenerative potential of early-endodermal cells enriched for the three DNA methylation/hydroxymethylation categories, and b) exploring the universality of this type of epigenetic phenotyping across other lineage-specific differentiations.