Multispectral Imaging of Collagen, NAD(P)H and Flavin Autofluorescence in Mesenchymal Stem Cells Undergoing Trilineage Differentiation

Jared M. Campbell; Saabah B. Mahbub; Ayad G. Anwer; Abbas Habibalahi; Stan Gronthos; Sharon Paton; Shane T. Grey; Lindsay E. Wu; Robert B. Gilchrist; Ewa M. Goldys

doi:10.3390/cells13201731

Cells (Oct 2024)

Multispectral Imaging of Collagen, NAD(P)H and Flavin Autofluorescence in Mesenchymal Stem Cells Undergoing Trilineage Differentiation

Jared M. Campbell,
Saabah B. Mahbub,
Ayad G. Anwer,
Abbas Habibalahi,
Stan Gronthos,
Sharon Paton,
Shane T. Grey,
Lindsay E. Wu,
Robert B. Gilchrist,
Ewa M. Goldys

Affiliations

Jared M. Campbell: Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia
Saabah B. Mahbub: Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia
Ayad G. Anwer: Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia
Abbas Habibalahi: Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia
Stan Gronthos: Mesenchymal Stem Cell Laboratory, School of Biomedicine, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA 5000, Australia
Sharon Paton: Mesenchymal Stem Cell Laboratory, School of Biomedicine, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA 5000, Australia
Shane T. Grey: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia
Lindsay E. Wu: School of Biomedical Sciences, University of New South Wales, Sydney, NSW 2052, Australia
Robert B. Gilchrist: School of Clinical Medicine, University of New South Wales, Sydney, NSW 2052, Australia
Ewa M. Goldys: Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia

DOI: https://doi.org/10.3390/cells13201731
Journal volume & issue: Vol. 13, no. 20
p. 1731

Abstract

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Understanding the molecular mechanisms of differentiation is important for regenerative medicine and developmental biology. This study aims to characterise the role of the glycolysis/oxidative phosphorylation balance as a driver of mesenchymal stem cell (MSC) differentiation. Cells were maintained in normal conditions or stimulated towards the MSC trilineage cell types over 21 days. Multispectral imaging of cell autofluorescence was applied as a non-invasive methodology to continuously image cultures in situ. Spectral signals for collagen, NAD(P)H, and flavins were unmixed. MSCs cultured under chondrogenic conditions exhibited increased collagen levels relative to controls. Following osteogenic induction, MSCs showed increased collagen levels relative to controls during the earlier stages of culture; however, control cells increased their collagen levels as they became confluent. MSCs cultured under adipogenic conditions exhibited lower levels of collagen than controls. The redox ratio (RR; NAD(P)H/flavins) immediately decreased during chondrogenesis, with this early effect persisting throughout the culture compared to control cells, which appeared to increase their RR, similar to osteogenesis. Adipogenesis resulted in a small increase in RR on day 2 relative to control cells, followed by a persistent decrease. Chondrogenic and adipogenic differentiation favoured oxidative phosphorylation, whereas osteogenesis and MSC overgrowth resulted in a glycolytic metabolism. Following consideration of these findings, as well as the diverse reports in the literature, it is concluded that neither enhanced oxidative phosphorylation nor glycolysis are fundamental to the canonical modes of differentiation, and researchers should avoid interpreting shifts as indicating differentiation.

Published in Cells

ISSN: 2073-4409 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Biology (General): Cytology
Website: https://www.mdpi.com/journal/cells

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