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

Applications of embryonic stem cells (ESCs) require faithful chromatin changes during differentiation, but the fate of the 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 (Xi XIST+ 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 induce 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 Xi XIST+ Xa ESCs are most suited for downstream applications and show that all other X states are abnormal byproducts of our ESC derivation and propagation method.