Figure 2

Analyses of the origin of ovarian colony-forming cells (OCCs). (A) Single nucleotide polymorphism (SNP) genotyping of OCCs and control cells. The heterozygosity or homozygosity of SNP loci of OCC-1 and OCC-2 of B6D2F1 strain was compared with that of B6D2F1 embryonic stem cells (ESCs), somatic fibroblasts of DBA2 and C57BL6 mice, and parthenogenetic ESCs (pESCs). Both homozygosity and heterozygosity were concomitantly detected in the OCC line. ESCs of F1 strain showed heterozygosity alone, and only homozygotic SNP loci were detected in the fibroblasts of the inbred strain. The pESC line possessed both homozygotic and heterozygotic chromosomes. (B). Methylation status of OCCs, ESCs, and pESCs. Genomic DNA isolated from these cells was subjected to bisulfite genomic sequencing analysis. The methylation levels of the promoter regions of stemness-related genes (Oct-4 and Nanog) and imprinted genes expressed differentially after parthenogenetic activation (H19, Peg3, Snrpn, and Gtl2) were compared. The PCR products were cloned, and 10 plasmid clones were sequenced for each sample. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Stemness-related genes were demethylated in all cell lines, whereas the expression of other genes differed markedly among the cell lines. The methylation in OCCs was significantly different from that in ESCs or pESCs; OCCs had more methylated H19 and Gtl2 compared with pESCs and less methylated Peg3 and Snrpn compared with ESCs. (Reprinted with permission from Gong et al., 2010; 93:2564-601).