(A) Fluorescence-activated cell sorting (FACS) analysis of the DNA content of phESCs and ahESCs. Diploid ESCs were stained as control.

(B) Distribution of DNA methylation levels in oocytes, sperm, TTFs, E10.5fPGCs (female PGCs at E10.5), E10.5mPGCs (male PGCs at E10.5), E13.5fPGCs (female PGCs at E13.5), E13.5mPGCs (male PGCs at E13.5), E16.5fPGCs (female PGCs at E16.5), E16.5mPGCs (male PGCs at E16.5), early-passage ESCs (passage 20, n = 2), early-passage ahESCs (passage 20, n = 2), early-passage phESCs (passages 19 and 20), late-passage ESCs (passage 42, n = 2), late-passage ahESCs (passages 40 and 42), and late-passage phESCs (passage 40, n = 2).

(C) DNA methylation levels at promoters (top) and imprinted regions (bottom) in all related samples.

(D) Heatmap of imprinted region methylation in all related samples. Gray squares indicate a lack of valid reads.

(E) Bisulfite sequencing analysis of imprinted regions at the H19, IG, Igf2r, Impact, Peg1, and Snrpn loci in ESCs (passage 20), ahESCs (passage 24), and phESCs (passage 20). Filled circles represent methylated cytosines, and open circles represent unmethylated cytosines.

(F) Principal-component analyses of the CpG methylation levels in all related samples covered with no less than 10 reads. Each sample has two independent repeats. PC1, PC2, and PC3 represent the top three principal components. Three groups were determined by unsupervised k-means clustering.