1. Cell death, non-invasively assessed by intrinsic fluorescence intensity of NADH, is a predictive indicator of functional differentiation of embryonic stem cells.
- Author
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Buschke DG, Squirrell JM, Fong JJ, Eliceiri KW, and Ogle BM
- Subjects
- Animals, Cell Differentiation, Cell Line, Embryoid Bodies drug effects, Embryoid Bodies metabolism, Fluorescence, Mice, Myocytes, Cardiac cytology, NAD metabolism, Protein Kinase Inhibitors pharmacology, Staurosporine pharmacology, Cell Death drug effects, Embryoid Bodies cytology, NAD analysis, Spectrometry, Fluorescence methods
- Abstract
Background Information: 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., Results: 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., Conclusions: 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., (Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.)
- Published
- 2012
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