Label-free sorting of iPS cells during neuronal differentiation using FLIM and multiphoton fluorescence microscopy

A. Meleshina; S. Rodimova; E. Dashinimaev; A. Artyuhov; N. Mescheryakova; A. Kashina; E. Kryukov; V. Elagin; D. Reunov; E. Vorotelyak; E. Zagaynova

doi:10.1117/12.2544797

21 February 2020 Label-free sorting of iPS cells during neuronal differentiation using FLIM and multiphoton fluorescence microscopy

A. Meleshina, S. Rodimova, E. Dashinimaev, A. Artyuhov, N. Mescheryakova, A. Kashina, E. Kryukov, V. Elagin, D. Reunov, E. Vorotelyak, E. Zagaynova

Author Affiliations +

Proceedings Volume 11226, Neural Imaging and Sensing 2020; 112261F (2020) https://doi.org/10.1117/12.2544797
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

The changes in cell metabolism can affect the epigenome-modifying enzymes activity during iPSCs differentiation and thus control the functional potential of the final cell. Therefore, for therapeutic applications, the restoration of a fully functional mitochondrial network specific for the cell types derived from iPSCs will be required to support the energy and other mitochondrial factors. Recently, FLIM method allows to study the metabolic changes that accompanying cell differentiation noninvasively and without additional labels. In this study, we investigated the metabolic changes in iPSCs during neural differentiation using two-photon fluorescence microscopy and FLIM. Cellular metabolism was examined by monitoring the optical redox ratio (FAD/NAD(P)H), the fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. Given that neural differentiation is also accompanied by synthetic processes and oxidative stress, this process was included in the scope of this work. We demonstrated an increased contribution of protein-bound NADH and NADPH in neuron associated with metabolic switch to oxidative phosphorylation and the biosynthetic processes or oxidative stress, respectively. We also found that the optical redox ratio FAD/NAD(P)H decreased during neural differentiation, and this was likely to be explained by the intensive lipid membrane synthesis or ROS generating and the enhanced NADPH production associated with them. The biochemical analysis was carried out to verify the metabolic status of iPSCs and their neural derivatives. Based on the data on glucose consumption, lactate and ATP amount we registered the trend to the metabolic pathways redistribution towards the oxidative phosphorylation in neuron.

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A. Meleshina, S. Rodimova, E. Dashinimaev, A. Artyuhov, N. Mescheryakova, A. Kashina, E. Kryukov, V. Elagin, D. Reunov, E. Vorotelyak, and E. Zagaynova "Label-free sorting of iPS cells during neuronal differentiation using FLIM and multiphoton fluorescence microscopy", Proc. SPIE 11226, Neural Imaging and Sensing 2020, 112261F (21 February 2020); https://doi.org/10.1117/12.2544797

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