Establishment of a doxycycline-inducible p16INK4a knock-in human embryonic stem cell line, WAe009-A-3Y

Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a) is a senescence-associated cell-cycle regulator that inhibits CDK4/6-dependent RB phosphorylation and cell-cycle progression. Here, we established a doxycycline-inducible p16INK4a knock-in human embryonic stem cell line by targeting a Tet-On expression cassette into the AAVS1 safe-harbor locus. The generated line retained pluripotency marker expression, tri-lineage differentiation potential, and a normal karyotype. Doxycycline treatment induced dose-dependent increases in p16INK4a expression and ZsGreen reporter expression. This cell line provides a foundational resource for developing p16INK4a-associated cellular senescence models across multiple stem cell-derived lineages.