Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage
Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage
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DOI：
10.1089/bio.2015.0072
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发表时间：
2016-04-01
影响因子：
1.6
通讯作者：
Nakata, Koh
中科院分区：
文献类型：
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作者：
Kawase, Tomoyuki;Hayama, Kazuhide;Nakata, Koh
在制备用于再生治疗的细胞产品时，应严格控制细胞质量。评估细胞活性、身份和纯度的方法是建立并常规使用的，而目前评估细胞安全性的方法，特别是遗传完整性或致瘤性，是耗时和相对不敏感的。作为开发更实用的筛查系统的一部分，作者先前证明了伽马-H_2AX和p53是评估DNA损伤历史的有用标记物。为了进一步验证这些标记并开发一种更定量的方法，现在已经使用流式细胞术(FCM)检测了这些标记和另外两个候选标记在单细胞中的表达。流式细胞仪分析和免疫荧光染色显示，伽玛射线照射抑制细胞增殖、细胞增大和细胞核，并立即上调大量细胞中的伽马-H_2AX和p21(Waf1)，持续12天。在许多受累细胞的细胞核中形成了伽马-H_2AX焦点。最初急剧增加的P53表达在12天后缓慢下降，而Rb的表达呈线性增加。目前的发现表明，这种高通量、基于细胞的蛋白质标志物和细胞大小的组合评估能够从非常大的细胞群体中快速和常规地检测到少量有DNA损伤历史的细胞。使用这种筛选方法将提高验证再生治疗中使用的基于细胞的产品质量的能力。
In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that gamma-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that gamma-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated gamma-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.