Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation

Metabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM.

In this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.

Differentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation.

Based on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates. Conclusions: In this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.