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Int J Radiat Oncol Biol Phys. 2019 May 11. pii: S0360-3016(19)30706-0. doi: 10.1016/j.ijrobp.2019.05.006. [Epub ahead of print]

DNA damage response after ionizing radiation exposure in skin keratinocytes derived from human induced pluripotent stem cells.

Author information

1
Laboratory for Future Interdisciplinary Research of Science and Technology, Institute of Innovative Research, Tokyo Institute of Technology; Cosmetic R&D Department, Takara Belmont Corp; Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology.
2
Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology. Electronic address: mshimada@lane.iir.titech.ac.jp.
3
Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology.
4
Laboratory for Future Interdisciplinary Research of Science and Technology, Institute of Innovative Research, Tokyo Institute of Technology.

Abstract

PURPOSE:

Epidermal cells are positioned on the body surface and thus have the potential risk of being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet (UV) rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications, such as radiotherapy. Keratinocyte stem cells and progenitors are at a risk of 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).

MATERIALS AND METHODS:

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 2Gy gamma-ray exposure, cells were immune-stained with DNA double strand breaks markers γ-H2AX/53BP1 and cell senescence marker p16/p21 for DDR analysis. DDR protein expression level, cell survival and, apoptosis were analyzed by western blotting, WST-8 assay, and TUNEL assay, 3D organoid model respectively.

RESULTS:

First, second, and third passage keratinocytes were characterized with keratinocyte markers K14 and p63 using immunofluorescence and all cells were positive to the both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (qRT-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that first and second passage keratinocytes have potential as keratinocyte progenitors. Meanwhile, third passage keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the first and second passage keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28%, 53BP1: 17%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%), but not in third passage keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%), compared with iPSCs (γ-H2AX: 57%, 53BP1: 55%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity and apoptosis activity was decreased in derived keratinocytes compared with iPSCs.

CONCLUSIONS:

We here demonstrated that derivation of keratinocytes from iPSCs and its characterization of differentiated state and DDR response. Derived keratinocytes showed progenitors like character to the DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effect of IR such as DDR on the skin tissue from radiotherapy for cancer.

KEYWORDS:

3D organoid; DNA damage response; cellular senescence; induced pluripotent stem cells; ionizing radiation; skin keratinocytes

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