Real-Time Analysis of Endogenous Wnt Signalling in 3D Mesenchymal Stromal Cells

Figure 3

Wnt pathway activation is observed reproducibly in 3D MSC culture and during osteogenic differentiation but not during adipogenic differentiation. (a) Lightsheet microscopy of cell tracker red stained human Y201 Wnt EGFP MSCs showing sporadic activation of Wnt signalling increasing with time in culture, with Wnt3a-treated controls (right panels). Scale bar = 100 μm. (b) Confocal microscopy of murine C3H10T1/2 MSC 3D cultures showing Wnt signalling is active in basal conditions and during osteogenic differentiation but not adipogenic differentiation. Scale bar = 100 μm. (c) Immunofluorescence of sectioned spheroids stained for EGFP demonstrates location of EGFP fluorescence in 3D culture. Scale bar = 100 μm. Adipogenic differentiation causes spheroids to enlarge in size; a lower magnification image of the whole spheroid is included (top right, merge panel). (d) Adipogenic C3H10T1/2 MSC spheroids show inverse relationship between adipogenesis and Wnt signalling by Oil Red O staining.

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