电离辐射暴露后诱导多能干细胞来源的皮肤角质细胞中的 DNA 损伤反应。

DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells.

机构信息

Laboratory for Future Interdisciplinary Research of Science and Technology, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan; Cosmetic Research and Development Department, TAKARA BELMONT Corporation, Tokyo, Japan; Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan.

Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan.

出版信息

Int J Radiat Oncol Biol Phys. 2019 Sep 1;105(1):193-205. doi: 10.1016/j.ijrobp.2019.05.006. Epub 2019 May 11.

DOI:10.1016/j.ijrobp.2019.05.006

PMID:31085283

Abstract

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 γ-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 α6 and CD71 (real-time (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 P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 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 P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). 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 decreased in derived keratinocytes compared with iPSCs.

CONCLUSIONS

We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.

摘要

目的

表皮细胞位于体表，因此容易受到遗传毒性应激的影响，包括电离辐射（IR）、紫外线和化学化合物。IR 对皮肤组织的生物学效应是放射治疗等医学应用的一个重大问题。在癌症治疗的放射治疗中，角质形成细胞干细胞和祖细胞有发生 IR 依赖性肿瘤形成的风险。为了阐明表皮细胞基因组稳定性的分子机制，我们从人诱导多能干细胞（iPSCs）中衍生出皮肤角质形成细胞，并分析了它们的 DNA 损伤反应（DDR）。

方法与材料

从 iPSCs 中衍生出皮肤角质形成细胞，并指定为第一代（P1）、第二代（P2）和第三代（P3）传代细胞，以比较 DDR 的分化状态。在 2 Gy 伽马射线照射后，用 DNA 双链断裂标记物 γ-H2AX/53BP1 和细胞衰老标记物 p16/p21 对细胞进行免疫染色，以分析 DDR。通过 Western blot、WST-8 测定和 TUNEL 测定分别分析 DDR 蛋白表达水平、细胞存活和细胞凋亡。还分析了构建的 3D 类器官建模的 DDR。

结果

P1、P2 和 P3 角质形成细胞用免疫荧光法用角蛋白 14 和 p63 标志物进行了特征鉴定，所有细胞均对这两种标志物呈阳性。衍生的角质形成细胞表现出高整合素 α6 和 CD71 的表达（实时（qRT）-PCR 比值：iPSCs：整合素 α6：1.12，CD71：1.25，P1：整合素 α6：7.80，CD71：0.43，P2：整合素 α6：5.53，CD71：0.48），表明 P1 和 P2 角质形成细胞具有作为角质形成细胞祖细胞的潜力。同时，P3 角质形成细胞表现出低整合素 α6 和 CD71 的表达（qRT-PCR 比值：P3：整合素 α6：0.55，CD71：0.10），表明分化的角质形成细胞。在 IR 暴露后，P1 和 P2 角质形成细胞通过 γ-H2AX/53BP1 焦点测定显示 DNA 修复活性增加（P1：γ-H2AX：28.0%，53BP1：17.0%，P2：γ-H2AX：37.7%，53BP1：28.3%），但 P3 角质形成细胞（P3：γ-H2AX：74.7%，53BP1：63.7%）则没有与 iPSCs（γ-H2AX：57.0%，53BP1：55.0%）相比。此外，与 iPSCs 相比，衍生的角质形成细胞中细胞衰老标志物 p16 和 p21 的表达增加（P16：非辐照，iPSCs：0%，P1：12.5%，P2：14.5%，P3：29.7%，IR，iPSCs：0%，P1：19.5%，P2：34.8%，P3：64.5%）。与 iPSCs 相比，DDR 蛋白表达、细胞敏感性和细胞凋亡活性在衍生的角质形成细胞中降低。