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

摘要

The changes in cell metabolism can affect the epigenome-modifying enzymes activity during iPSCs differentiation andthus control the functional potential of the final cell. Therefore, for therapeutic applications, the restoration of a fullyfunctional mitochondrial network specific for the cell types derived from iPSCs will be required to support the energy andother mitochondrial factors. Recently, FLIM method allows to study the metabolic changes that accompanying celldifferentiation noninvasively and without additional labels. In this study, we investigated the metabolic changes in iPSCsduring neural differentiation using two-photon fluorescence microscopy and FLIM. Cellular metabolism was examined bymonitoring the optical redox ratio (FAD/NAD(P)H), the fluorescence lifetime contributions of the free and bound formsof 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 NADHand NADPH in neuron associated with metabolic switch to oxidative phosphorylation and the biosynthetic processes oroxidative stress, respectively. We also found that the optical redox ratio FAD/NAD(P)H decreased during neuraldifferentiation, and this was likely to be explained by the intensive lipid membrane synthesis or ROS generating and theenhanced NADPH production associated with them. The biochemical analysis was carried out to verify the metabolicstatus of iPSCs and their neural derivatives. Based on the data on glucose consumption, lactate and ATP amount weregistered the trend to the metabolic pathways redistribution towards the oxidative phosphorylation in neuron.