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Live Simultaneous Monitoring of Mineral Deposition and Lipid Accumulation in Differentiating Stem Cells

De Melo, Nigel; McGinlay, Sarah; Markus, Robert; Macri-Pellizzeri, Laura; Symonds, Michael E.; Ahmed, Ifty; Sottile, Virginie

Authors

Nigel De Melo

Sarah McGinlay

Robert Markus

Laura Macri-Pellizzeri

Michael E. Symonds

Ifty Ahmed

Virginie Sottile



Abstract

Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells.

Journal Article Type Article
Publication Date Jul 10, 2019
Journal Biomimetics
Print ISSN 2313-7673
Publisher MDPI
Peer Reviewed Peer Reviewed
Volume 4
Issue 3
Article Number 48
Pages 1-10
Institution Citation De Melo, N., McGinlay, S., Markus, R., Macri-Pellizzeri, L., Symonds, M. E., Ahmed, I., & Sottile, V. (2019). Live Simultaneous Monitoring of Mineral Deposition and Lipid Accumulation in Differentiating Stem Cells. Biomimetics, 4(3), 1-10. doi:10.3390/biomimetics4030048
DOI https://doi.org/10.3390/biomimetics4030048
Keywords live monitoring; stem cell; mineralisation; lipid detection
Publisher URL https://www.mdpi.com/2313-7673/4/3/48

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