Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.
Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.
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DOI：
10.1016/j.stemcr.2016.10.003
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发表时间：
2016-11-08
影响因子：
5.9
通讯作者：
Barbaric, Ivana
中科院分区：
文献类型：
--
作者：
Baker, Duncan;Hirst, Adam J.;Gokhale, Paul J.;Juarez, Miguel A.;Williams, Steve;Wheeler, Mark;Bean, Kerry;Allison, Thomas F.;Moore, Harry D.;Andrews, Peter W.;Barbaric, Ivana
培养过程中获得的人类多能干细胞 (hPSC) 的基因变化可能会混淆实验结果，并可能危及临床治疗的结果。 hPSC 中特别常见的变化是 1、12、17 和 20 号染色体三体性。因此，应定期筛查 hPSC 是否存在此类畸变。尽管有多种方法用于评估 hPSC 基因型，但尚未对检测 hPSC 培养物中低水平嵌合体的常用技术的敏感性进行系统评估。我们进行了混合实验来模拟自然发生的嵌合体，并评估了染色体显带、qPCR、荧光原位杂交和数字液滴 PCR 在检测变异方面的敏感性。我们的分析强调了常用方法检测嵌合体的局限性，这是解释 hPSC 遗传状态和制定 hPSC 在再生医学中安全应用标准的关键要求。 hPSC 符合用于核型分析的随机采样规则 排除 <1% 水平的嵌合体需要采样 >500 个中期 qPCR 是一种快速检测方法，用于检测 hPSC 中常见扩增区域 通过常用方法评分为正常的培养物可能含有高达 10% 的变异 Barbaric 和同事测试了需要对多少个中期进行评分才能检测不同水平的嵌合体 hPSC 培养物。他们还设计了 qPCR 检测作为检测常见染色体异常的快速方法。 qPCR、数字液滴 PCR 和 FISH 的灵敏度测试表明，这些方法可以漏掉群体中多达 10% 的异常细胞。
Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine. hPSCs conform to random sampling rules used for karyotyping Excluding mosaicism at <1% level requires sampling >500 metaphases qPCR is a rapid assay for detection of commonly amplified regions in hPSCs Cultures scored as normal by commonly used methods could harbor up to 10% variants Barbaric and colleagues tested how many metaphases need to be scored to detect different levels of mosaicism in hPSC cultures. They also devised qPCR assays as a rapid means of detecting common chromosomal abnormalities. Testing of the sensitivity of qPCR, digital droplet PCR, and FISH revealed that these methods can miss as many as 10% abnormal cells in the population.
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