Cell death, non-invasively assessed by intrinsic fluorescence intensity of NADH, is a predictive indicator of functional differentiation of embryonic stem cells.
Cell death, non-invasively assessed by intrinsic fluorescence intensity of NADH, is a predictive indicator of functional differentiation of embryonic stem cells.
复制标题
DOI：
10.1111/boc.201100091
复制
发表时间：
2012-06
影响因子：
2.7
通讯作者：
Ogle BM
中科院分区：
文献类型：
--
作者：
Buschke DG;Squirrell JM;Fong JJ;Eliceiri KW;Ogle BM
干细胞生物学和干细胞移植方面的持续进展依赖于非侵入性生物标记物来表征细胞和干细胞聚集体。这种生物标记物的非侵入性是必不可少的，因为外源性的标记、探针或报告可能会无意中戏剧性地改变干细胞的状态，也可能会破坏细胞与细胞和细胞与基质的相互作用。在这里，我们研究了自身荧光代谢物烟酰胺腺嘌呤二核苷酸(NADH)作为细胞死亡的非侵入性、内在生物标记物的实用性，当用多光子光学方法检测时。为了测试这种可能性，在分化的早期阶段(第3天)，用星形孢子素诱导小鼠类胚体(EBS)细胞死亡，星形孢子素是一种ATP竞争性激酶电子传递抑制剂。在星状孢子素处理数小时后，用单色活/死探针对EBS进行染色。每个EB的单个横切面成像以检测活的/死的探针的荧光强度(非本征荧光)以及NADH的荧光强度(本征荧光)。在随后的时间点(第6-12天)对EBS进行评估，以形成搏动区域作为功能分化的指标。统计学比较表明，活/死染色的外在荧光强度与NADH的内源荧光强度呈显著正相关，提示NADH的荧光强度可用于可靠、无创性地评估EBS细胞的死亡。此外，在聚集体形成后不久细胞死亡水平较高的EBS在以后的时间点上产生功能心肌细胞的能力有限。我们证明了当使用多光子激发测量时，NADH荧光强度作为干细胞聚集体中细胞死亡的非侵入性指示器的实用性。此外，我们还表明，分化早期干细胞死亡的程度对功能性心肌细胞的形成具有预测作用。
Continued advances in stem cell biology and stem cell transplantation rely on non-invasive biomarkers to characterise cells and stem cell aggregates. The non-invasive quality of such biomarkers is essential because exogenous labels, probes or reporters can unintentionally and dramatically alter stem cell state as can disruption of cell-cell and cell-matrix interactions. Here, we investigate the utility of the autofluorescent metabolite, nicotinamide adenine dinucleotide (NADH), as a non-invasive, intrinsic biomarker of cell death when detected with multi-photon optical-based approaches. To test this possibility, cell death was induced in murine embryoid bodies (EBs) at an early stage (day 3) of differentiation using staurosporine, an ATP-competitive kinase inhibitor of electron transport. Several hours after staurosporine treatment, EBs were stained with a single-colour, live/dead probe. A single-cross-sectional plane of each EB was imaged to detect the fluorescence intensity of the live/dead probe (extrinsic fluorescence) as well as the fluorescence intensity of NADH (intrinsic fluorescence). EBs were assessed at subsequent time points (days 6–12) for the formation of beating areas as an indicator of functional differentiation. Statistical comparison indicated a strong positive correlation between extrinsic fluorescence intensity of the live/dead stain and intrinsic fluorescence of NADH, suggesting that the intensity of NADH fluorescence could be used to reliably and non-invasively assess death of cells of EBs. Furthermore, EBs that had high levels of cell death soon after aggregate formation had limited ability to give rise to functional cardiomyocytes at later time points. We demonstrate the utility of NADH fluorescence intensity as a non-invasive indicator of cell death in stem cell aggregates when measured using multi-photon excitation. In addition, we show that the degree of stem cell death at early stages of differentiation is predictive for the formation of functional cardiomyocytes.
登录
查看更多内容
影响因子：
2.2
作者：
Heikal AA
通讯作者：
Heikal AA
影响因子：
2.7
作者：
Collins, Tony J.
通讯作者：
Collins, Tony J.
影响因子：
9
作者：
通讯作者：
--
影响因子：
3.5
作者：
Levitt, Jonathan M.;Baldwin, Amy;Georgakoudi, Irene
通讯作者：
Georgakoudi, Irene
影响因子：
6.6
作者：
Kondoh, Hiroshi;Lleonart, Matilde E.;Beach, David
通讯作者：
Beach, David