Fourth, nuclear transfer-derived ESC are more faithfully reprogrammed than most iPSC generated from adult somatic tissues. Like the immediate and rapid demethylation of the sperm pronucleus following fertilization, somatic nuclei are rapidly demethylated by nuclear transfer into ooplasm, prompting speculation that the egg harbors an active demethylase14. In contrast, demethylation is a late phenomenon in factor-based reprogramming, and likely occurs passively13. Studying how ooplasm erases methylation might identify biochemical functions that would enhance factor-based reprogramming. Failure to demethylate pluripotency genes is associated with intermediate or partial states of reprogramming122940, and knock-down of the maintenance methyltranferase DNMT1 or treatment with the demethylating agent 5-AZA can convert intermediate states to full pluripotency. Demethylation appears passage dependent13, and reprogramming efficiency correlates with the rate of cell division and the passage number41. In our experiments, we compared pluripotent stem cells of comparable low passage number (Supplemental Table 5), but continued serial passage may homogenize the differentiation potential of pluripotent cell types.
