相衬荧光寿命显微镜研究游离态和蛋白结合态 NADH 揭示神经干细胞分化潜能
Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential.
机构信息
Laboratory of Fluorescence Dynamics, Biomedical Engineering Department, University of California Irvine, Irvine, California, United States of America.
出版信息
PLoS One. 2012;7(11):e48014. doi: 10.1371/journal.pone.0048014. Epub 2012 Nov 5.
In the stem cell field there is a lack of non invasive and fast methods to identify stem cell's metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors.
在干细胞领域,缺乏非侵入性和快速的方法来识别干细胞的代谢状态、分化状态和细胞谱系决定。在这里,我们描述了一种无标记的方法,该方法使用 NADH 作为内源性生物标志物和荧光寿命显微镜的 Phasor 方法来测量细胞的代谢指纹。我们表明,不同的代谢状态与不同的细胞分化阶段以及干细胞向神经元和神经胶质命运的偏向有关,而谱系标记的表达之前。我们的数据表明,NADH FLIM 特征可在两个不同的发育阶段(E12 和 E16)非侵入性地区分神经元与未分化的神经前体细胞和干细胞(NPSCs)。NPSCs 通过不同的分化阶段从糖酵解表型转变为氧化磷酸化表型。NPSCs 的特征是游离/结合 NADH 比高,而分化的神经元的特征是游离/结合 NADH 比低。我们证明了 NPSCs 的代谢特征与其分化潜能相关,表明神经元前体细胞和神经胶质前体细胞具有不同的游离/结合 NADH 比。NPSCs 中的还原条件与它们的神经发生潜能相关,而氧化条件与神经胶质潜能相关。我们首次表明,FLIM NADH 代谢指纹为干细胞潜能提供了一种新颖的、定量的测量方法,并且是一种无标记的、非侵入性的方法,可以识别偏向神经元或神经胶质的祖细胞。