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Evaluation of Genotoxicity and Mutagenic Effects of Vector/DNA Nanocomplexes in Transfected Mesenchymal Stem Cells by Flow Cytometry
Acta Biomaterialia ( IF 9.7 ) Pub Date : 2018-05-19 , DOI: 10.1016/j.actbio.2018.05.029
Alireza Nomani 1 , Xuguang Chen 1 , Arash Hatefi 2
Affiliation  

In recent years, there has been a great deal of interest in ex-vivo genetic modification of mesenchymal stem cells (MSCs) to meet various biomedical needs. Considering the self-renewal potential of MSCs, it is critically important to ensure that transfection vectors (gene carriers) do not induce genotoxicity because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable, unbiased, and quantitative method to measure genotoxicity (micronuclei formation) of gene carriers directly in transfected MSCs. Consequently, it has not been possible to study the correlation of vectors’ physicochemical characteristics with their impact on stem cell genome stability. To address this deficiency, a flow cytometry-based method with a specialized gating protocol was developed that not only measures micronuclei formation, but also determines the mechanism of mutagenesis (i.e., clastogenic vs. aneugenic) of each vector in transfected MSCs. This gating protocol effectively eliminates all interfering signals associated with aggregated nanoparticles (viral and non-viral), exogenous DNA, and apoptotic/necrotic bodies from the micronuclei measurement process. The presented gating protocol for flow cytometry, which is provided as a template, enables investigators in academia, industry and regulatory bodies to rapidly and reliably evaluate the genosafety profiles of gene carriers. The findings of this study also indicate that highly positively charged lipid- and polymeric-based vectors can induce genotoxicity even without manifesting substantial somatic toxicity. Thus, extreme care must be taken before implanting ex-vivo-modified MSCs back into a patient’s body.

Statement of Significance

There is a great interest in genetic modification of stem cells (SCs) by using vectors for various biomedical needs. Considering the self-renewal potential of SCs, it is essential to ensure that such vectors do not induce genetic aberrations (genotoxicity) because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable method to measure genotoxicity of vectors directly in transfected SCs. To address this deficiency, a specialized flow cytometry-based method was developed that quantitatively analyzed genotoxicity and determined the mechanism of mutagenesis that occurred in transfected SCs during the transfection process. The developed technique will enable scientists to design safer vectors for genetic modification of stem cells.



中文翻译:

流式细胞术评估载体/DNA纳米复合物在转染间充质干细胞中的遗传毒性和致突变作用

近年来,人们对间充质干细胞 (MSCs) 的离体基因改造以满足各种生物医学需求产生了极大的兴趣。考虑到 MSC 的自我更新潜力,确保转染载体(基因载体)不会诱导基因毒性至关重要,因为它们理论上可以将单个干细胞转变为癌症起始细胞。不幸的是,目前没有可靠、公正和定量的方法来直接测量转染的 MSC 中基因载体的遗传毒性(微核形成)。因此,不可能研究载体的理化特性与其对干细胞基因组稳定性的影响之间的相关性。为了弥补这一不足,开发了一种基于流式细胞术的具有专门门控协议的方法,该方法不仅可以测量微核的形成,还可以确定转染的 MSC 中每个载体的诱变机制(即,致裂性与非内生性)。该门控协议有效地消除了微核测量过程中与聚集的纳米粒子(病毒和非病毒)、外源 DNA 和凋亡/坏死体相关的所有干扰信号。所提供的流式细胞术门控协议作为模板提供,使学术界、工业界和监管机构的研究人员能够快速可靠地评估基因携带者的基因安全概况。这项研究的结果还表明,高正电荷的基于脂质和聚合物的载体即使不表现出实质性的体细胞毒性,也可以诱导基因毒性。因此,在将体外修饰的 MSCs 植入患者体内之前,必须格外小心。

重要性声明

通过使用载体满足各种生物医学需求,人们对干细胞 (SC) 的遗传修饰产生了浓厚的兴趣。考虑到 SCs 的自我更新潜力,必须确保此类载体不会诱发遗传畸变(基因毒性),因为它们理论上可以将单个干细胞转变为癌症起始细胞。不幸的是,目前没有可靠的方法来直接在转染的 SCs 中测量载体的遗传毒性。为了解决这一缺陷,开发了一种基于流式细胞术的专门方法,可定量分析基因毒性并确定转染过程中转染 SCs 中发生的诱变机制。开发的技术将使科学家能够设计更安全的载体,用于干细胞的基因改造。

更新日期:2018-05-19
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