Epidermal cells are positioned on the outermost layer of skin and thus risk exposure to environmental factors. The biological effect of ionizing radiation on the skin tissue is a significant problem in medical applications such as radiation therapy for cancer treatment. In this study, we established humaninduced pluripotent stem cells and skin keratinocytes and analyzed DNA damage response and cell senescence after exposure to ionizing radiation.Purpose: Epidermal cells are positioned on the body surface and thus risk being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR). Methods and Materials: Skin keratinocytes were derived from iPSCs and designated as first-(P1), second-(P2), and third-(P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers g-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed. Results: P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin a6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin a6: 1.