Live-cell imaging of DNA damage and cell cycle progression uncovers distinct responses during neural differentiation of hiPSCs.

Ionizing radiation induces DNA double-strand breaks, which compromise genomic stability and trigger programmed cell death. The cell's differentiation state modulates DNA damage response (DDR) mechanisms, including DNA repair pathways and cell cycle regulation. The accumulation of p53-binding protein 1 (53BP1) at DSB sites serves as a reliable biomarker for such damage. Previously, we developed a fluorescent live-cell imaging system, termed "Focicle," which monitors 53BP1 foci dynamics and cell cycle phases, utilizing fluorescent ubiquitination-based cell cycle indicators (hCdt1 and hGmnn) in mouse cells. In the current study, to investigate the relationship between differentiation state and DDR activity, we generated Focicle-integrated human induced pluripotent stem cells and further differentiated them into neural progenitors and mature neurons using an optimized Focicle cassette adapted for human cell lines. Using laser microirradiation, we observed differentiation-dependent alterations in 53BP1 foci accumulation dynamics and cell cycle progression. The newly established Focicle system represents a valuable tool for elucidating DDR activity during organ development.