Abstract
Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells. Here we report that a reduction in non-CG methylation is associated with impaired differentiation capacity into endodermal lineages. Genome-wide analysis of 2,670 non-CG sites in a discovery cohort of 25 phenotyped human induced pluripotent stem cell (hiPSC) lines revealed unidirectional loss (Δβ=13%, P<7.4 × 10−4) of non-CG methylation that correctly identifies endodermal differentiation capacity in 23 out of 25 (92%) hiPSC lines. Translation into a simplified assay of only nine non-CG sites maintains predictive power in the discovery cohort (Δβ=23%, P<9.1 × 10−6) and correctly identifies endodermal differentiation capacity in nine out of ten pluripotent stem cell lines in an independent replication cohort consisting of hiPSCs reprogrammed from different cell types and different delivery systems, as well as human embryonic stem cell (hESC) lines. This finding infers non-CG methylation at these sites as a biomarker when assessing endodermal differentiation capacity as a readout.
Highlights
Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells
We used a two-stage design consisting of a discovery cohort of 25 human induced pluripotent stem cell (hiPSC) lines, and a replication cohort of 7 hiPSC lines from the HipSci study and 3 human embryonic stem cell lines
We extended the source of pluripotent stem cells to include human embryonic stem cell (hESC) as well as diversifying the range of donor cell types and OSKM delivery methods from which hiPSC lines were derived; donor cells included erythroblasts (N 1⁄4 4) and dermal fibroblasts (N 1⁄4 3) obtained from five unrelated individuals, and OSKM delivery systems included a DNA-based vector (episomal (N 1⁄4 3)) in addition to a virus-based vector (Sendai virus (N 1⁄4 4)); confirmation of pluripotency, differentiation protocol and differentiation capacity scoring are detailed in Methods
Summary
Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells. Here we report that a reduction in non-CG methylation is associated with impaired differentiation capacity into endodermal lineages. Translation into a simplified assay of only nine non-CG sites maintains predictive power in the discovery cohort (Db 1⁄4 23%, Po9.1 Â 10 À 6) and correctly identifies endodermal differentiation capacity in nine out of ten pluripotent stem cell lines in an independent replication cohort consisting of hiPSCs reprogrammed from different cell types and different delivery systems, as well as human embryonic stem cell (hESC) lines. This finding infers non-CG methylation at these sites as a biomarker when assessing endodermal differentiation capacity as a readout. This finding infers nonCG methylation can be used as a biomarker for assessing endodermal differentiation capacity
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