Stable maintenance of MERVL-positive embryonic stem cells reveals sustained transcriptional programs and enhancer remodeling

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 demonstrate the transiently stable maintenance of MERVL-positive ESCs that exhibit persistent MERVL expression and activation of 2C-associated genes. Live-cell imaging revealed uniform and sustained MERVL activity in these MERVL-positive ESCs, 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 altered developmental trajectories. Single-cell RNA sequencing revealed clear separation of control ESCs from MERVL-positive populations and redistribution across distinct transcriptional states, with Red and Mosaic lines showing graded shifts within a shared transcriptional manifold. Epigenomic profiling further revealed distinct chromatin states, specialized super-enhancer landscapes, and active enhancer marking at MERVL loci. Together, these findings demonstrate that stable maintenance of MERVL-positive ESCs is achievable in vitro, providing a powerful model to dissect ERV-driven transcriptional regulation, epigenomic remodeling, and 2C-like transcriptional and epigenetic programs.