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Autofluorphores and telomerase, a hyperspectral signature of in vitro mesenchymal stem cell ageing

Jared Campbell (1, 2), Abbas Habibalahi (1, 2), Saabah Mahbub (1, 2), Sharon Paton (3, 4), Stan Gronthos (3, 4) and Ewa Goldys (1, 2)

  1. ARC Centre of Excellence in Nanoscale Biophotonics, The University of New South Wales, Sydney, New South Wales, Australia

  2. Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, New South Wales, Australia

  3. Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia

  4. South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia

Increasing age is associated with the depletion of the mesenchymal stem cell (MSC) niche, compromising the body’s capacity for self-repair. Autologous transplantation of hMSCs, following in vitro expansion, is a strong candidate regenerative therapy for several diseases of ageing, but this depletion and the concomitant loss of differentiation capacity compromises their utility. We undertook to monitor the changes undergone by five in vitro cultured bone marrow derived hMSC lines as they were expanded through sequential passages. Hyperspectral microscopy measures endogenous cell autofluoresence, giving a direct window into the biological processes of hMSC ageing. We used this data to develop a model which distinguished young (passages 2-4) middle (passages 5-7) and old (passages 8-10) hMSCs. The model was trained on four lines, then validated on the fifth to test generalisability. With it we were able to distinguish between the three groups with high overall accuracy (ROC AUC= 0.96). It was particularly accurate for distinguishing young from old cells (ROC AUC= 0.99) as well as young from middle (ROC AUC= 0.97). However, the middle and old hMSCs were less distinct (ROC AUC= 0.87), indicating that the age-related changes with the biggest impact occur early in hMSCs’ replicative lifespans. Additionally, as the metabolic co-enzymes NA(P)DH and FAD are autofluorescent we were able to unmix their signals from the hyperspectral data to estimate concentrations and calculate optical Redox Ratio. Hyperspectral microscopy is non-destructive and does not rely on the introduction of exogenous fluorophores, as such we were then able to undertake immunohistochemical staining of telomerase reverse transcriptase (TERT) protein expression on the same samples. TERT has been shown to be inactive – with respect to telomere extension – in cultured hMSCs. Nonetheless, its expression increased significantly with age, suggesting that it does play a role in hMSC ageing, but through its telomere independent, non-canonical functions.