Mouse embryonic stem cells maintain lineage immortality by sporadically entering a two-cell-like state that allows for asymmetric distribution of DNA damage among daughter cells. One is destined to be removed and the other is functionally rejuvenated.

A fundamental paradox in biology is how cell lineages achieve immortality while individual cells inevitably accumulate damage. Embryonic stem cells (ESCs) are one of nature’s solutions to this problem. ESCs can be expanded indefinitely in culture while maintaining genomic stability and developmental potential. The mechanisms underlying this remarkable property remain enigmatic, especially given that ESCs are continuously exposed to replication stress and naturally occurring DNA damage.

In this issue, cell researchMin, Zhang, and colleagues report an elegant solution to this contradiction.¹ Using advanced long-term live-cell imaging combined with a fluorescent reporter, they demonstrated that mouse ESCs periodically enter a transient two-cell-like (2C-like) state that serves as a critical quality control checkpoint for stem cell lineages.

Conditions like 2C have long puzzled stem cell biologists. It is characterized by the expression of genes such as MERVL and MERVL. Zscan4,² This condition occurs naturally in approximately 1% of cultured ESCs at any given time. Previous research has shown that blocking entry into this state causes a cultural crisis.³ On the other hand, cells in a 2C-like state paradoxically exhibit increased DNA damage and increased apoptosis.⁴ These seemingly contradictory observations raised a fundamental question: How do conditions characterized by high damage and death contribute to long-term stem cell regeneration?

By meticulously tracing single cell lineages over a four-day movie, the authors discovered that 2C-like cells undergo functionally asymmetric divisions, giving rise to two daughter lineages with dramatically different fates (Figure 1). 1). Approximately 60% of cells that enter a 2C-like state divide asymmetrically, generating one daughter lineage (termed “2C death”) that accumulates high levels of MERVL expression, exhibits extensive DNA damage, and ultimately undergoes cell death. The sister line (termed “2C-survived”) exhibits reduced MERVL expression, reduced DNA damage, and returns to a pluripotent state with enhanced functional properties.

Important mechanistic insights were gained from live-cell imaging of DNA damage using the 53BP1-mVenus reporter. The authors found that damaged DNA, visualized as 53BP1 foci, segregated asymmetrically during 2C-like cell division and that the majority of damage-containing foci were preferentially inherited by the 2C-dead lineage (Figure 1). 1). Remarkably, this asymmetric separation requires an intact DNA damage response pathway. Inhibiting ATM, ATR, CHEK, or PARP significantly reduced both the frequency of asymmetric divisions and the asymmetry of damage between sisters.

The functional implications of this asymmetric partitioning are significant. Using an ingenious fluorescent protein timer system that distinguishes cells based on how recently they have exited the 2C-like state, the authors demonstrated that cells that survive 2C exhibit rejuvenating characteristics: reduced DNA damage by comet assay, enhanced alkaline phosphatase activity, increased expression of pluripotency markers such as Nanog and Oct4, and Improved clonogenicity in vitro and, most impressively, chimera efficiency of 73% when injected into cells. In blastocysts, only 13% of cells had not recently transitioned to a 2C-like state.

This study elegantly links cellular aging mechanisms across phylogeny. Just as Saccharomyces cerevisiae asymmetrically sequesters damaged proteins to produce rejuvenated daughters at the expense of aging mothers.⁵ ESCs utilize asymmetric division to maintain lineage youth as individual cells age. This similarity extends to the molecular mechanism, where the authors show that old centrosomes preferentially segregate into the 2C-death lineage, which is reminiscent of spindle polar body aging in yeast.

Several important questions emerge from this study. First, what molecular mechanisms drive asymmetric partitioning of damaged DNA? The requirement for DNA damage response proteins suggests active sensing and sorting, but the downstream effectors remain unknown. Second, does asymmetric division in a 2C-like state represent a conserved rejuvenation mechanism in other stem cell types or even in vivo during early development? Third, can this process be manipulated to enhance asymmetric division to counter stem cell depletion?⁶ Or could blocking cancer stem cells be used for treatment?

The quantitative framework provided by the authors is particularly convincing. They calculate that rejuvenation lasts for 8 to 10 generations. This means that only 0.1% to 0.4% of cells need to undergo rejuvenation each generation to maintain population health, well within the observed 1% 2C-like fraction. This indicates that sporadic transitions to a 2C-like state are quantitatively sufficient to promote long-term ESC self-renewal.

This research fundamentally reshapes the way we understand cellular immortality. Rather than relying solely on efficient damage repair, ESCs employ division-based strategies that concentrate damage to disposable lineages while regenerating the original lineage. Importantly, these findings suggest that “Zscan4⁺ The state is a process that restores force, but the process itself (i.e., Zscan4⁺ condition) is ineffective or has low efficacy.”⁷: Cells in a 2C-like state are indeed functionally impaired, but asymmetric division allows one daughter (2C survivor) to downregulate 2C marker expression, increase potency and escape, while the damaged sister (2C dead) retains high levels of 2C markers and is eliminated. This study, which first demonstrates functionally asymmetric division that promotes rejuvenation in mammalian cells, establishes ESCs as a powerful model to study cellular aging and regeneration, with potential implications for regenerative medicine and cancer biology.