De-erosion of X chromosome dosage compensation by the editing of regulatory regions restores the differentiation potential in hPSCs.

Human pluripotent stem cells (hPSCs) regularly and irreversibly show the erosion of X chromosome inactivation (XCI) by long non-coding RNA (lncRNA) silencing, causing challenges in various applications of female hPSCs. Here, we report reliable methods to reactivate with monoallelic expression in female hPSCs. Surprisingly, we find that the editing of regulatory regions by Cas9-mediated non-homologous end joining is sufficient for the reactivation of by endogenous systems. Proliferated hPSCs with reactivation show XCI from an eroded X chromosome, suggesting that hPSCs with normal dosage compensation might lead to a growth advantage. Furthermore, the use of targeting vectors, including the regulatory region sequences and selection cassette, enables reactivation in hPSCs with high efficiency. -reactivated hPSCs can show the restoration of differentiation potential. Thus, our findings demonstrate that re-expression is a beneficial method to maximize the use of female hPSCs in various applications, such as proper disease modeling.