Ageing human bone marrow mesenchymal stem cells have depleted NAD(P)H and distinct multispectral autofluorescence

Stem cell exhaustion plays a major role in the ageing of different tissues. Similarly, in vitro cell ageing during expansion prior to their use in regenerative medicine can severely compromise stem cell quality through progressive declines in differentiation and growth capacity. We utilized non-destructive multispectral assessment of native cell autofluorescence to investigate the metabolic mechanisms of in vitro mesenchymal stem cell (MSC) ageing in human bone marrow MSCs over serial passages (P2-P10). The spectral signals for NAD(P)H, flavins and protein-bound NAD(P)H were successfully isolated using Robust Dependent Component Analysis (RoDECA). NAD(P)H decreased over the course of hMSC ageing in absolute terms as well as relative to flavins (optical redox ratio). Relative changes in other fluorophore levels (flavins, protein-bound NAD(P)H) suggested that this reduction was due to nicotinamide adenine dinucleotide depletion rather than a metabolic shift from glycolysis to oxidative phosphorylation. Using multispectral features, which are determined without cell fixation or fluorescent labelling, we developed and externally validated a reliable, linear model which could accurately categorize the age of culture-expanded hMSCs. The largest shift in spectral characteristics occurs early in hMSC ageing. These findings demonstrate the feasibility of applying multispectral technology for the non-invasive monitoring of MSC health in vitro.