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The trans‐spliced noncoding RNA  RMST  (ts RMST ) is an emerging regulatory lncRNA in the human pluripotency circuit. Previously, we found that ts RMST  represses lineage‐specific transcription factors through the PRC2 complex and NANOG in human pluripotent stem cells (hESCs). Here, we demonstrate that ts RMST  also modulates noncanonical Wnt signaling to suppress the epithelial‐to‐mesenchymal transition (EMT) and in vitro differentiation of embryonic stem cells (ESCs). Our results demonstrate that disruption of ts RMST  expression in hESCs results in the upregulation of WNT5A, EMT, and lineage‐specific genes/markers. Furthermore, we found that the PKC inhibitors Go6983 and Go6976 inhibited the effects of WNT5A, indicating that WNT5A promotes the EMT and in vitro differentiation although conventional and novel PKC activation in hESCs. Finally, we showed that either antiserum neutralization of WNT5A or Go6983 treatment in ts RMST  knockdown cells decreased the expression of mesenchymal and lineage‐specific markers. Together, these findings indicate that ts RMST  regulates Wnt and EMT signaling pathways in hESCs by repressing WNT5A, which is a potential EMT inducer for promoting in vitro differentiation of hESCs through PKC activation. Our findings provide further insights into the role of trans‐spliced RNA and WNT5A in hESC differentiation, in which EMT plays an important role. S tem  C ells   2016;34:2052–2062

stem cells

The Trans‐Spliced Long Noncoding RNA ts RMST  Impedes Human Embryonic Stem Cell Differentiation Through WNT5A‐Mediated Inhibition of the Epithelial‐to‐Mesenchymal Transition