Smith Rosanna C G, Stumpf Patrick S, Ridden Sonya J, Sim Aaron, Filippi Sarah, Harrington Heather A, MacArthur Ben D
Centre for Human Development, Stem Cells, and Regeneration, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.
Mathematical Sciences, University of Southampton, Southampton, United Kingdom.
Biophys J. 2017 Jun 20;112(12):2641-2652. doi: 10.1016/j.bpj.2017.05.005.
A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, because temporal changes in intracellular protein abundances cannot be measured directly in live cells, fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systematically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog, we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight the problem of measurement in live cells.
人们观察到，包括转录因子Nanog在内的许多重要多能性调节因子在单个胚胎干细胞中会随机波动。通过短暂地使细胞为分化成不同谱系做好准备，这些波动被认为对多能性的维持和退出很重要。然而，由于无法在活细胞中直接测量细胞内蛋白质丰度的时间变化，波动通常使用基因工程报告细胞系来评估，这些细胞系产生荧光信号作为蛋白质表达的替代指标。在这里，通过结合数学建模和实验，我们表明，广泛使用的报告策略可能会以不可预见的方式系统性地干扰它们旨在监测的动态，有时会给出极具误导性的结果。以Nanog为例，我们展示了基因报告基因如何损害重要的多能性维持正反馈回路的行为，并在潜在动态中引发分岔，从而在报告细胞系中产生异质的Nanog表达模式，而这些模式并不代表野生型。这些发现有助于解释已发表的关于Nanog变异性的一系列观察结果，并突出了活细胞测量的问题。
