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

During culture, female human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs), exhibit a propensity for erosion of X-chromosome inactivation (XCI). This phenomenon is characterized by the loss of XIST RNA expression and reactivation of a subset of X-linked genes from the inactive X chromosome (Xi). Despite its common occurrence, XCI erosion is often overlooked by the stem cell community, hindering a complete understanding of its impact on both fundamental and translational applications of hiPSCs. Our study investigates erosion dynamics in female hiPSCs and reveals that XCI erosion is a frequent yet heterogeneous phenomenon, resulting in the 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 the loss of XIST RNA and XCI erosion. This implies that XIST RNA normally restrains 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. 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+).