Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
Biol Cell. 2012 Jun;104(6):352-64. doi: 10.1111/boc.201100091. Epub 2012 Mar 23.
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.
干细胞生物学和干细胞移植的持续进步依赖于非侵入性生物标志物来描述细胞和干细胞聚集体。此类生物标志物的非侵入性质量至关重要，因为外源性标签、探针或报告器可能会无意中极大地改变干细胞状态，细胞-细胞和细胞-基质相互作用的中断也是如此。在这里，我们研究了利用多光子光学方法检测的自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、内在生物标志物的效用。为了检验这种可能性，我们使用 ATP 竞争型电子传递链激酶抑制剂(staurosporine)诱导早期分化的鼠胚体 (EB) 中的细胞死亡 (第 3 天)。在 staurosporine 处理数小时后，用单一种类的死活探针对 EB 进行染色。对每个 EB 的单个横截面平面进行成像，以检测死活探针的荧光强度（外源性荧光）以及 NADH 的荧光强度（内源性荧光）。在随后的时间点（第 6-12 天）评估 EB 形成搏动区域的情况，作为功能性分化的指标。
统计比较表明，死活染色的外源性荧光强度与 NADH 的内源性荧光强度之间存在很强的正相关，这表明 NADH 荧光强度可以可靠且非侵入性地评估 EB 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 EB 在后期时间点产生功能性心肌细胞的能力有限。
我们证明了使用多光子激发测量时，NADH 荧光强度可作为干细胞聚集体中细胞死亡的非侵入性指标。此外，我们表明分化早期干细胞死亡的程度可预测功能性心肌细胞的形成。
