Human naïve pluripotent stem cells exhibit X chromosome dampening and X-inactivation (RNA-Seq)

Naïve human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X-chromosome state has remained unresolved. We found that the inactive X-chromosome (Xi) of primed hESCs was reactivated in naïve culture conditions. Similar to cells of the blastocyst, resulting naive cells exhibited two active X-chromosomes with XIST expression and chromosome-wide transcriptional dampening, and initiated XIST-mediated X-inactivation upon differentiation. Both establishment and exit from the naïve state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X-chromosome dosage compensation processes in early human development: X-dampening and X-inactivation. Furthermore, the naïve state reset Xi abnormalities of primed hESCs, providing cells better suited for downstream applications. However, naïve hESCs displayed differences to the embryo because XIST expression was predominantly mono-allelic instead of bi-allelic, and X-inactivation was non-random, indicating the need for further culture improvement. Overall design: Fourteen total independent samples were analyzed. Ten are from UCLA1 hESC line and four are from UCLA4 hESC line. Independently processed samples from same culture condition are called replicates and labedled as rep1/rep2, where available. All UCLA1 clones are from 5iLAF culture condition, and all UCLA4 samples are from 5iLAF culture condition.