Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

Abstract

Abstract. We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.

Intravital microscopy of biosensor activities and intrinsic metabolic states

“…This substantially limits the ability of intensity measurements to distinguish the individual intrinsic fluorescent species with certainty. In striking contrast the fluorescence lifetimes of the intrinsic auto-fluorescence can be distinguished by phasor analysis [20,21,29,30]. The phasor approach enables intravital FLIM to be used to map cell-specific metabolic signatures in the tissues of live animals without the need to determine the lifetimes for individual species.…”

“…Solutions of individual cellular fluorophores were used to determine the phasor lifetime positions for each intrinsic fluorescent species [20,21,29,30]. The intrinsic fluorophore standards were purchased from Sigma Aldrich (St. Louis, MO), and their concentrations in solutions were determined by UV–VIS spectrophotometry using the extinction coefficients supplied by the manufacturer or from the literature [31,32].…”

A Global Assessment of Stem Cell Engineering

“…66 For this reason, an increasing interest and effort has shifted to robust, label-free detection methods that can effectively delineate different populations of cells from varying degrees of heterogeneous starting populations. 67,68 Finally, stem cell manufacturing infrastructure and facilities that accommodate the bioprocessing systems could also benefit from systems engineering analysis. More ''closed'' culture systems are needed in to ensure the sterility and safety of stem cell-derived products, particularly for cell therapies.…”

Spectral phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR

“…The work of Stringari (Stringari and others, 2011; Stringari and others, 2012) further exploits this approach by fingerprinting a large number of optical biomarkers with a unique lifetime phasor locations. The wide range of applications here listed underlines the usefulness of the method.…”