We observed a large degree of variability in X chromosome CpG methylation in female hPSCs, which appeared to be dependent on the level of XIST expression. Our results were consistent with a loss of XIST expression with time in culture, followed by erosion of DNA methylation, originating in several sub-segments of the X chromosome and spreading to involve larger regions. Our prediction that loss of XCI may affect the fidelity of hPSC-based X-linked disease models is consistent with the findings reported in an accompanying manuscript from Mekhoubad et al. In their studies, a hiPSC-based disease model of the X-linked disease, Lesch-Nyhan Syndrome, lost the ability to recapitulate hallmark biochemical characteristics of the disease with time in culture. These researchers showed that this phenomenon was due to loss of XCI and reactivation of the wild-type HPRT gene in the late passage female hiPSCs, consistent with our observations that loss of XCI at the HPRT locus was a common feature that occurred in more than half of the female hESC and hiPSC samples we analyzed.
