Cell death, non-invasively assessed by intrinsic fluorescence intensity of NADH, is a predictive indicator of functional differentiation of embryonic stem cells

• Published in: Biology of the cell 2012-06, Vol.104 (6), p.352-364
• Main Authors: Buschke, David G., Squirrell, Jayne M., Fong, Jimmy J., Eliceiri, Kevin W., Ogle, Brenda M.
• Format: Article
• Language: English
• Subjects: Animals; Autofluorescence; Cell aggregate; Cell Death - drug effects; Cell Differentiation; Cell Line; Embryoid Bodies - cytology; Embryoid Bodies - drug effects; Embryoid Bodies - metabolism; Fluorescence; Mice; Myocytes, Cardiac - cytology; NAD - analysis; NAD - metabolism; NADH; Protein Kinase Inhibitors - pharmacology; Spectrometry, Fluorescence - methods; Staurosporine - pharmacology; Stem cells; Viability
• ISSN: 0248-4900; 1768-322X
• DOI: 10.1111/boc.201100091

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. The use of NADH as an intrinsic biomarker of cell death in stem cell aggregates is validated by comparing NADH fluorescence intensity to that of a known extrinsic live/dead probe using multi‐photon fluorescence microscopy. This type of non‐invasive analysis of stem cell behaviour could prove very useful because stem cell populations with a high degree of cell death could be excluded or removed to improve both basic and clinica