Human Embryonic Stem Cells Do Not Change Their X Inactivation Status during Differentiation

Abstract

SUMMARY Applications of ESCs require faithful chromatin changes during differentiation but the fate of each X-chromosome-state in differentiating ESCs is unclear. Female human ESC-lines either carry two active X-chromosomes (XaXa), an Xa and inactive-X-chromosome with or without XIST-RNA-coating (XiXIST+Xa;XiXa), or an Xa and an eroded-Xi (XeXa) where the Xi no longer expresses XIST-RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC-lines and followed their X-chromosome-state during differentiation. Surprisingly, we found that the X-state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not initiate X-inactivation, and displayed higher X-linked gene-expression than XiXa cells. These results demonstrate that X-chromosome-dosage-compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X-states are abnormal byproducts of our ESC-derivation and propagation methods.

X-chromosome dosage as a modulator of pluripotency, signalling and differentiation?

“…The use of human ESCs for studying X-dosage-dependent effects has so far been hampered by the fact that most cell lines adopt a state where one X-chromosome is at least partially silenced [54]. A recent study has characterized the X-inactivation status and the differentiation potential of a series of female human ESC lines in detail [55]. The finding that lines with two active X-chromosomes maintain this state during differentiation, shows that double X-dosage in human ESCs does not completely block differentiation.…”

New Advances in Human X Chromosome Status from a Developmental and Stem Cell Biology

“…The inactive X chromosome (Xi) that has fully undergone XCI, is highly enriched for H3K27me3 and H3K9me3 histone modifications (39). However, these XCI-linked epigenetic features are not stable over extended passages (40, 41), and over time, primed hESCs will lose XIST coating and H3K27me3 marking of their inactive X chromosomes, resulting in an X chromosome in epigenetically eroded state (Xe) (40). The Xe may maintain the Xi’s pattern of H3K9me3 enrichment, but has reduced peaks called for this mark.…”

“…Importantly, it lacks almost all H3K27me3 marks entirely (40). In severe cases of erosion, there is a severe depletion of CpG island methylation across the entirety of the Xe (41), which may result in higher gene reactivation than the remaining H3K9me3 marking may suggest. As a result, genes that were previously repressed XCI-derived effects can be spuriously reactivated, causing aberrations in gene load (40, 41).…”

Memories of an X-chromosome

“…Nevertheless, the partial XCI reprogramming accomplished under 5iLAF conditions was able to erase Xi abnormalities of primed hPSCs. In particular, the authors were able to induce XCI following differentiation from the naïve state in some hPSCs lines that failed to achieve XCI when the differentiation started from primed cells (14). Besides this achievement, the article reveals other interesting and unexpected observations.…”

“…However, the role of XIST in X-chromosome dampening is also controversial, as most naïve cells showed monoallelic XIST expression, so dampening should only affect one X-chromosome, a question that remains unresolved in the manuscript. The XIST negative XaXa transitional state is not observed when naïve cells are derived straight from human blastocysts, but it can be observed in the derivation of primed cells from human blastocysts (14), so a similar transition may occur in the developing human embryo undergoing XCI.…”