Live tracking of human senescent cells using gene editing
2Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel
A key hallmark of organismal aging is cellular senescence – the stable arrest of the cell cycle (proliferation) which occurs due to stress factors that inflict DNA damage. Recent research has shown that senescence is a pleiotropic phenomenon: it is crucial to proper wound healing, embryonic development, and tissue regeneration, but it has also been strongly linked to various age-related diseases, such as cardiovascular diseases. The detection of senescent cells can be difficult, and it usually requires fixation of the cells, thus preventing them from being live-tracked over time. Here, we devised a new method to detect senescent cells “online” without any need for fixation or single-use labeling methods. After showing that CDKN2A gene expression is elevated in senescent cells, we genetically tagged its expression with a fluorescent protein. Using the TALEN genome editing tool, we inserted a DNA cassette containing a CDKN2A promoter followed by a tagRFP gene into a safe harbor site of human iPSCs. After differentiation of the TALEN-iPSCs to cardiac fibroblasts and subsequent induction to senescence with Doxorubicin, the cells expressed the tagRFP in a way that allowed us to distinguish them from their non-senescent counterparts. By that, we have created a unique iPSCs line that enables tracking of live senescent cells over time in all batches of its differentiated lineages without any additional staining or labeling.