Targeted chromosome shredding to explore X chromosome aneuploidies in aging
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Abstract
Aging is characterized by a gradual decline in cellular functions, and growing evidence shows that it does not affect men and women equally. Although women live, on average, five years longer than men, they often experience poorer health overall. Many age-associated diseases display sex biases. One contributing factor is the sex chromosome complement. Male mammals carry one X and one Y chromosome, whereas females possess two X chromosomes, one of which is largely transcriptionally inactivated, though some genes escape silencing. These escaper genes contribute to female resilience, yet their misregulation can also increase disease susceptibility. Notably, female cells with different X chromosome aneuploidies accumulate with age, with the preferential loss of the inactive X the most common. The causal relationship between X chromosome loss and aging remains unknown, and the immediate cellular consequences upon X chromosome loss are largely unexplored.
This work reports the first successful derivation and characterization of C3H/Bl6 hybrid mouse embryonic stem cells. Both male and female C3H/Bl6 cells express pluripotency markers, with only a minor differentiation bias toward the germ layers. The differentiation potential of these cells was evaluated through random differentiation into embryoid bodies, which expressed markers of all lineages; however, fewer beating cardiomyocyte-like structures were observed in embryoid bodies derived from female cells. Immunostainings confirmed the presence of cell types from all three germ layers. Analysis of X chromosome dynamics during the differentiation of female cells into epiblast-like stem cells showed that X chromosome silencing occurred progressively over seven days, following a heterogenous yet overall consistent kinetics.
Genome editing in the C3H/Bl6 background proved challenging and yielded low efficiencies. Nevertheless, we established a cellular system enabling allele-specific chromosome targeting and shredding in female hybrid cells. The system was activated through Cre recombinase delivery via baculovirus. We found that segmental aneuploidies of the targeted X chromosome constituted the predominant outcome, followed by complete X chromosome loss. Evidence of genomic instability was also observed, with a small proportion of cells exhibiting micronuclei containing X chromosome fragments.
Overall, we established a chromosome targeting and shredding system in bona fide C3H/Bl6 hybrids mouse embryonic stem cells. We demonstrated that generating X chromosome aneuploidies is feasible, with chromosome segmentation representing the primary event. This tool provides a foundation to dissect the causal relationship of sex chromosome loss and aging. In addition, these hybrid cells are a valuable model for studying stem cell biology, X chromosome dynamics and sex differences.