AbstractHuman pluripotent stem cells (hPSCs) exist in multiple, transcriptionally distinct states and serve as powerful models for studying human development. Despite their significance, the molecular determinants and pathways governing these pluripotent states remain incompletely understood. Here, we demonstrate that transposable elements act as sensitive indicators of distinct pluripotent cell states. We engineered hPSCs with fluorescent reporters to capture the temporal expression dynamics of two state-specific transposable elements, LTR5_Hs, and MER51B. This dual reporter system enables real-time monitoring and isolation of stem cells transitioning from naïve to primed pluripotency and further towards differentiation, serving as a more accurate readout of pluripotency states compared to conventional systems. Unexpectedly, we identified a rare, metastable cell population within primed hPSCs, marked by transcripts related to preimplantation embryo development and which is associated with a DNA damage response. Moreover, our system establishes the chromatin factor NSD1 and the RNA-binding protein FUS as potent molecular safeguards of primed pluripotency. Our study introduces a novel system for investigating cellular potency and provides key insights into the regulation of embryonic development.
