Gene reactivation upon erosion of X-chromosome inactivation in female hiPSCs is predictable yet variable and persists through differentiation

Female human induced pluripotent stem cells frequently undergo X-chromosome inactivation (XCI) erosion, marked by XIST RNA loss and partial reactivation of the inactive X (Xi). This overlooked phenomenon limits our understanding of its impact on stem cell applications. Here, we show that XCI erosion is frequent and heterogeneous, leading to the reactivation of several X-linked genes. These are primarily located on the short arm of the X chromosome, particularly near escape genes and within H3K27me3-enriched domains, with reactivation linked to reduced promoter DNA methylation. Interestingly, escape genes further increase their expression from Xi upon XCI erosion, highlighting the critical role of XIST in their dosage regulation. Importantly, global (hydroxy)methylation levels and imprinted regions remain unaffected, and analysis of trilineage commitment and cardiomyocyte formation reveals that XCI erosion persists across differentiation. These findings underscore the need for greater awareness of the implications of XCI erosion for stem cell research and clinical applications. 1) RNA-seq analysis was conducted on biological triplicates of a diverse range of hiPSC lines representing various erosion states of the X-chromosome inactivation. Cell lines exhibiting XIST expression (XIST+) consist of ASD and F7cl15, and cell lines without XIST expression (XIST-) comprise F002, F7cl4, CD, and CE. RNA-seq was also performed before (day0- D0) and after ectodermal differentiation (day7- D7) for one replicate of the isogenic pair: F7cl4 (XIST-) and F7cl15 (XIST+). 2) IMPLICON was conducted for a diverse range of human pluripotent stem cell lines (hiPSCs) representing different erosion states of the X-chromosome inactivation. Specifically, two cell lines exhibiting XIST expression (XIST+): F7cl15, 2042cl9; and five cell lines without XIST expression (XIST-) and showing varying levels of erosion: F7cl4, CD, CE, 2040cl6, 2042cl1. As controls, two lines of human fibroblasts from which some hiPSCs lines were originated, were included: 2040 and 2042 fibroblasts. Of note, the following samples are isogenic: 2040 fibroblasts and 2040cl6 hiPSCs; F7cl15 and F7cl14; 2042 fibroblasts, 2042cl9 hiPSCs and 2042cl1 hiPSCs. 3) RNA Amplicon Sequencing was conducted for four (4) human pluripotent stem cell lines (hiPSCs) representing different erosion states of the X-chromosome inactivaton and for its respective derived cardiomyocytes (hiPSC-CMs). The cell line ASD presents XIST expression (XIST+) and the cell lines F002, CD and CE are XIST- and display varied levels of erosion.