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. 2021 Dec 5;13(2):102–119. doi: 10.1007/s13238-021-00890-3

Figure 6.

Figure 6

Enhanced proliferation of cells with hypermethylated H19-DMR accounts for increased developmental potential of late-passage haploid cells. (A) DNA methylation levels of H19-DMR and IG-DMR in the two Dusp9-HA-tagged AG-haESC lines (5’HA-120 and 5’HA-138) with distinct developmental potential. (B) The body weights of E18.5 SC pups produced by ICAHCI (from TSa2i-14, TSa2i-C57, and TSa2i-F1 AG-haESCs). Pups derived from DKO-AG-haESCs (2i-O48) and sperm are as controls. (C) DNA methylation levels of H19 and IG DMRs in subclones from TSa2i-C57 (sc1 to sc21) were obtained through single-cell expansion. (D) DNA methylation levels of H19-DMR in the 2nd subclones expanded from single cells of the 1st subclones with high (sc6), middle (sc13), and low (sc16) levels of H19-DMR methylation. (E) Increased DNA methylation levels of H19-DMR in sc13 during culture. (F) Growth curve of the cells from subclones with high (sc6 and sc8), middle (sc13), and low (sc16) levels of H19-DMR methylation. ***, P < 0.0001. (G) Expression of Igf2 and H19 in sc6, sc8, sc13, and sc16 were analyzed by qPCR. ****, P < 0.00001; ns, not siginificant. (H) Growth curve of sc16-originated cells with hypomethylated H19-DMR and the same cells with H19 KO (sc16-H19-KO-40, 72, or 84 representing three different KO lines). ****, P < 0.00001. I Birth rate of SC mice generated from TSa2i AG-haESCs (TSa2i-14, p60 and TSa2i-C57, p58), and TSa2i AG-haESCs with H19 and IG DMR deletions (DKO-20, p68 and DKO-187, p68) or H19-DMR deletion (H19KO-40, p68-69). ns, not significant