Stable Maintenance of Two-Cell-Like Cells from Embryonic Stem Cells Reveals Chromatin and Super Enhancer Regulation of MERVL Elements

Mouse embryonic stem cells (ESCs) occasionally transit into a rare two-cell-like (2C) state characterized by transient activation of endogenous retroviruses such as MERVL and expression of 2C-specific genes including the Zscan4 cluster. These 2C-like cells (2CLCs) resemble early blastomeres and display expanded developmental potential, but their unstable and sporadic nature has hindered mechanistic studies. Here, we establish stable 2CLCs (s2CLCs) that maintain persistent MERVL expression and homogeneous 2C gene activation. Live-cell imaging revealed uniform and sustained MERVL activity in s2CLCs, contrasting with the heterogeneous and transient expression observed in conventional ESCs. Transcriptome profiling demonstrated robust induction of 2C-specific regulatory networks, and embryoid body differentiation combined with machine learning uncovered increased lineage variability and expanded developmental trajectories. Epigenomic profiling further revealed unique chromatin states, distinctive super enhancer landscapes, and active enhancer marking at MERVL loci. Together, these findings demonstrate that stable maintenance of the 2C-like state is achievable in vitro, providing a powerful model to dissect ERV-driven transcriptional regulation, epigenomic remodeling, and totipotent-like developmental potential.