In humans, XY males and XX females show marked differences in incidence and manifestations of many neurodevelopmental disorders (NDDs). These sex differences may arise from hormonal influences but also from differential regulation of sex chromosomes. The X chromosome in particular is enriched in genes involved in brain functions and associated with NDD compared to other chromosomes. In addition, one X chromosome undergoes epigenetic silencing during early female development thereby re-equilibrating the dosage of X-linked products with males. However, some genes escape from this X-chromosome inactivation (XCI) which may contribute to sex-biased differences in neural development and functions. This project aims to characterize XCI during neural differentiation and to perturb this process and analyse the consequences on cerebral development.

Using a clonal female human embryonic stem cell (hESC) line (H9) with a known inactive X (Xi), we established 2D neural progenitor cell (hNPC) and 3D cerebral organoid (hCO) differentiation models. To disrupt XCI, we used H9 cells with a deletion of XIST, the non-coding RNA that mediates XCI. Allele-specific RNA sequencing and RNA-FISH identified a subset of X-linked genes that escape from XCI in hNPCs, showing differential regulation compared to hESCs and suggesting cell-type-specific escape mechanisms.

In XIST KO cells, hNPCs formed with normal efficiency, but showed reactivation of specific Xi genes and altered expression of autosomal genes, indicating that XIST loss does not impair initial neural specification but may affect downstream differentiation. In hCOs, XIST deletion caused early appearance of pigmented structures—potentially retinal progenitors—and loss of specific neural populations, as revealed by single-cell RNA sequencing.

These findings provide the first in-depth allelic characterization of XCI dynamics during human neural differentiation and suggest that perturbing XCI alters cell composition and may impair neurodevelopment. This work highlights the importance of dosage regulation of X-linked genes during brain development and its potential contribution to sex-biased NDDs.