Achieving Stable X-chromosome Inactivation in Human Embryonic Stem Cells

X-inactivation is a paradigm of epigenetic transcriptional regulation. Human embryonic stem cells (hESCs) that harbor an inactivated X-chromosome often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that the composition of culture media dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined medium stably maintained XIST RNA coating, whereas hESCs cultured in the widely-used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of LiCl or inhibitors of glycogen synthase kinase 3 (GSK-3) proteins, which are inhibited by lithium, to the defined hESC culture medium impeded XIST expression. Together, these data may reconcile the observed variations in X-inactivation in hESCs and inform the culture of pluripotent stem cells for disease modeling and regenerative medicine. Overall design: Eight samples weree analyzed by RNA-Seq. Six are female hESC line UM90-14. Two samples are human foreskin fibroblast feeders. Two samples were collected at passage 6 and six samples were collected at passage 28.