(iPS3 and iPS7) that had intermediate levels of XIST expression at late passage (†, Figure S5B), but showed focal loss of XCI, consistent with the XIST threshold effect suggested above (Figure 5C–D). Using allele-specific RT-PCR, we confirmed that loss of DNA methylation was associated with biallelic expression of genes located in regions of XCI (Figure 6A–E). We observed loss of XIST expression (and loss of DNA methylation on the X chromosome) in most of the hiPSC clones, and retention of XIST expression (with preservation of X chromosome DNA methylation) in a minority of the clones, even though all the clones were generated and passaged in the same manner and at the same time. In contrast to the examined imprinted genes, no significant associations for the loss of XCI with specific cell culture or derivation conditions were detected in the multiple linear regression models.
