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

Chiara Stringari; Robert Sierra; Peter J. Donovan; Enrico Gratton

doi:10.1117/1.JBO.17.4.046012

20 April 2012 Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

Chiara Stringari, Robert Sierra, Peter J. Donovan, Enrico Gratton

Author Affiliations +

Funded by: National Institutes of Health, NIH-P41, Laser Microbeam Medical Program, LAMMP, NIH RO1, NIH PO1

Journal of Biomedical Optics, Vol. 17, Issue 4, 046012 (April 2012). https://doi.org/10.1117/1.JBO.17.4.046012

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.

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Chiara Stringari, Robert Sierra, Peter J. Donovan, and Enrico Gratton "Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy," Journal of Biomedical Optics 17(4), 046012 (20 April 2012). https://doi.org/10.1117/1.JBO.17.4.046012

Published: 20 April 2012

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