3D-models based on patient-specific induced pluripotent stem cells are widely used for biomedical research. Thus, non-invasive and continuous comprehensive description of these of 3D-models using FLIM and multiphoton microscopy requires.
3D models based on cells differentiated from patient-specific induced pluripotent stem cells (iPSCs) are widely used to identify disease phenotypes, to accurately analyze dysfunctions at the level of human tissues and organs, to screen new drugs, which makes them more promising tool for biomedical research tasks than monolayer cultures, which is associated with their proximity to in vivo. The metabolic activity with oxygenation level of cells, assessed by optical imaging methods, can be used as markers of cell viability, proliferative activity and the degree of differentiation in 3D culture conditions. In this paper we used fluorescence and phosphorescence lifetime imaging microscopies (FLIM and PLIM) to study the metabolic status and the oxygenation level of derived from iPSCs neural stem cells (NSC) cultured in 3D condition. An analysis of the fluorescence intensities and FLIM data showed that NSCs in monolayer and at the periphery of large spheroids have more glycolytic phenotypes, NSCs in the center of large spheroids and NSCs grouped into small spheroids have more oxidative state. For determination of the relative oxygen level in spheroids PLIM of BPTDM stained neurospheres was carried out. As it was supposed, oxygen transport in the spheroid depended on it size. In neurospheres with an average size 600 μm O2 distribution is radial, with the lowest concentration in the center. Thus, the metabolic status and oxygenation level of the NSC in the spheroid composition was assessed in a life-time and noninvasive manner.
Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.
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