Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation (Bisulfite-seq)

During culture, female human pluripotent stem cells (hPSCs), including human induced PSCs (hiPSCs) exhibit a propensity for erosion of X-chromosome inactivation (XCI). This phenomenon is characterized by loss of XIST RNA expression and reactivation of a subset of X-linked genes from the inactive X chromosome (Xi). XCI erosion, despite its common occurrence, is often overlooked by the stem cell community, hindering a complete understanding of its impact on both fundamental and translational applications of hiPSCs. Investigating erosion dynamics in female hiPSCs, our study reveals that XCI erosion is a frequent yet heterogeneous phenomenon, resulting in reactivation of several X-linked genes. The likelihood of a gene to erode increases for those located on the short arm of the X chromosome and within H3K27me3-enriched domains. Paradoxically, genes that typically escape XCI are hypersensitive to loss of XIST RNA and XCI erosion. This implies that XIST RNA normally restrain expression levels of these genes on the Xi. Importantly, increased X-linked gene expression upon erosion does not globally impact (hydroxy)methylation levels in hiPSCs or at imprinted regions. By exploring diverse differentiation paradigms, such as trilineage commitment and cardiac differentiation, our study reveals the persistence of abnormal XCI patterns throughout differentiation. This finding has significant implications for fundamental research, translational applications, and clinical use of stem cells. We underscore the importance of raising awareness within the stem cell community regarding XCI erosion and advocate for its inclusion in comprehensive hiPSC quality control. 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.