Abstract
The ability of stem cells to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Because traditional assessments of stem cells are destructive, time consuming, and logistically intensive, the use of a non-invasive, label-free approach to study of cell differentiation provides a powerful tool for rapid, high-content characterization of cell and tissue cultures. Here, we elucidate the metabolic changes in MSCs during adipogenic differentiation, based on the fluorescence of the metabolic co-factors NADH, NADPH, and FAD using the methods of two-photon fluorescence microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH is required. In our study we demonstrated, for the first time, an increased contribution of protein-bound NADPH in adipocytes that is associated with lipogenesis. The optical redox ratio FAD/NAD(P)H decreased during adipogenic differentiation, and that this was likely to be explained by the intensive biosynthesis of lipids and the enhanced NADPH production associated with this. Based on the data on the fluorescence lifetime contribution of protein-bound NAD(P)H, we registered a metabolic switch from glycolysis to oxidative phosphorylation in adipocytes.
Highlights
The ability of stem cells both to self-renew and to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology
As, during the differentiation of stem cells, the energy metabolism switches from glycolysis to oxidative phosphorylation, we suggested that the fluorescence contribution from the free form of NADH should decrease, while that from the protein-bound form of NADH should increase
In this study we investigated the metabolic changes in living mesenchymal stem cells (MSCs) during adipogenic differentiation, using two-photon fluorescence microscopy and fluorescence lifetime imaging (FLIM)
Summary
The ability of stem cells both to self-renew and to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Proliferating stem cells have increased needs for energy (ATP) and reducing cofactors as well as for carbon, nitrogen and hydrogen in order to support intensive biosynthesis[3,4]. Partial breakdown of glucose through glycolysis and the pentose phosphate pathway provides a compromise between the catabolic generation of ATP and reducing cofactors and the production of biosynthetic substrates to meet the cells’ anabolic requirements[5]. Differentiated cells no longer need to sustain a high rate of replication and have lower anabolic demands. They require large amounts of energy to support cellular homeostasis and their increasingly specialized functions and typically produce ATP through oxidative phosphorylation[6]. Metabolic imaging is generally based on analysis of the reducing cofactors nicotinamide adenine dinucleotide NAD(P)H and flavin adenine dinucleotide FAD14–16
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