The totipotent 2C-like state safeguards genomic stability of mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) sporadically transition to a transient totipotent state that resembles blastomeres of the 2-cell embryo stage, which has been proposed to contribute to exceptional genomic stability, one of the key features of mouse ESCs. However, the biological significance of the rare population of 2C-like cells (2CLCs) in ESC cultures remains to be tested. Here, we generated an inducible reporter cell system for specific elimination of 2CLCs from the ESC cultures to disrupt the equilibrium between ESCs and 2CLCs. We show that removing 2CLCs from the ESC cultures leads to dramatic accumulation of DNA damage, genomic mutations and rearrangements, indicating impaired genomic instability. Furthermore, 2CLCs removal results in increased apoptosis and reduced proliferation of mouse ESCs. Together, our data reveal that transition to the privileged 2C-like state is a major component of the intrinsic mechanisms that maintain exceptional genomic stability of mouse ESCs for long-term self-renewal. Overall design: To investigate the effects of removal of 2C-like cells (2CLCs) on the long-term self renewal of mouse embryonic stem cells (ESCs), we generated an inducible reporter cell system (2C::HSVTK reporter cell line) to eliminate 2CLCs specifically by adding Ganciclovir to ESC culture medium. After removal of 2CLCs, we collected Ganciclovir and DMSO treated 2C::HSVTK reporter cells (each two repeats) for whole genome sequencing (WGS) to dissect the genomic mutations and rearrangements after removel of 2CLCs.