DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells
DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells
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DOI：
10.1016/j.ijrobp.2019.05.006
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发表时间：
2019-09-01
影响因子：
7
通讯作者：
Okino, Akitoshi
中科院分区：
文献类型：
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作者：
Miyake, Tomoko;Shimada, Mikio;Okino, Akitoshi
目的：表皮细胞位于人体表面，因此有暴露于遗传毒性应激的危险，包括电离辐射、紫外线和化合物。红外对皮肤组织的生物效应是放射治疗等医学应用中的一个重要问题。在癌症放射治疗期间，角质形成细胞干细胞和前体细胞面临IR依赖性肿瘤发生的风险。为了阐明表皮细胞基因组稳定性的分子机制，我们从人诱导的多能干细胞(IPSCs)中分离出皮肤角质形成细胞，并分析了其DNA损伤反应(DDR)。方法与材料：从IPSCs中分离出皮肤角质形成细胞，命名为第一代(P1)、第二代(P2)和第三代(P3)细胞，以比较DDR的分化状态。2Gy射线照射后，用DNA双链断裂标记物Gamma-H2AX/53BP1和细胞衰老标记物p16/p21免疫组织化学染色进行DDR分析。分别用Western blotting、WST-8和TUNEL法检测DDR蛋白表达水平、细胞存活率和细胞凋亡率。结果：免疫荧光显示P1、P2、P3角质形成细胞均表达角质形成细胞标志物角蛋白14和P63，且全部细胞均为阳性。培养的角质形成细胞高表达整合素α6和CD71(实时定量聚合酶链式反应比率：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)，提示P1和P2角质形成细胞具有作为角质形成细胞前体的潜能。同时，P3角质形成细胞低表达整合素α6和CD71(qRT-PCR比值：P3：整合素α6：0.55，CD71：0.10)，提示角质形成细胞分化。与IPSCs(γ-H2AX：57.0%，53BP1：55.0%)相比，IR暴露后，P1和P2角质形成细胞的DNA修复活性(P1：Gamma-H2AX：28.0%，53BP1：17.0%，P2：Gamma-H2AX：37.7%，53BP1：28.3%)明显增强，而P3角质形成细胞(P3：Gamma-H2AX：74.7%，53BP1：63.7%)的DNA修复活性无明显变化。在来源角质形成细胞中，细胞衰老标记物p16和p21的表达高于IPSCs(P16：未照射，IPSCs：0%，P2：12.5%，P2：14.5%，P3：29.7%，IR，IPSCs：0%，P1：19.5%，P2：34.8%，P3：64.5%)。与IPSC相比，分化角质形成细胞的DDR蛋白表达、细胞敏感性和细胞凋亡活性降低。结论：本研究证实了IPSCs来源的角质形成细胞及其分化状态和DDR的特征。分化后的角质形成细胞表现出类祖细胞的特征。这些结果表明，来源的角质形成细胞是分析IR的有用工具，例如DDR对癌症放射治疗后皮肤组织的影响。(C)2019 Elsevier Inc.保留所有权利。
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 gamma-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 alpha 6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin alpha 6: 1.12, CD71: 1.25, P1: integrin alpha 6: 7.80, CD71: 0.43, P2: integrin alpha 6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin alpha 6 and CD71 (qRT-PCR ratio: P3: integrin alpha 6: 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 gamma-H2AX/53BP1 focus assay (P1: gamma-H2AX: 28.0%, 53BP1: 17.0%, P2: gamma-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: gamma-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (gamma-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. (C) 2019 Elsevier Inc. All rights reserved.