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Title: Modulation of canonical Wnt signalling in mesenchymal stem cells using a GSK3beta inhibitor
Author: Cook, David
ISNI:       0000 0004 2744 9342
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2013
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Multipotent stromal cells/mesenchymal stem cells (MSCs) can differentiate into multiple lineages including osteogenic and adipogenic cells. Wnt signalling has been implicated in controlling MSC fate, but the mechanism is unclear and apparently conflicting data exists. Here I show that a glycogen synthase kinase 3β inhibitor, AR28, is a potent activator of canonical Wnt signalling using β-catenin translocation studies and TCF-reporter assays. AR28 induced axis duplication and secondary regions of chordin expression in Xenopus laevis embryos, when injected into the ventral marginal zone, indicative of canonical Wnt signalling. When human MSCs were grown under adipogenic conditions, AR28 caused a significant dose-dependent reduction in FABP5/BODIPY double-positive cells with a corresponding rescue of proliferation. In assays to determine the effects of AR28 on MSC osteogenesis using standard differentiation inducers (β-glycerophosphate, L-ascorbic acid and dexamethasone), AR28 caused a significant decrease in alkaline phosphatase (ALP) activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when using mild osteogenic stimulation, excluding dexamethasone, increases in both ALP and Alizarin Red mineral staining were identified following AR28 treatment, with corresponding increases in proliferation and cell number. This AR28-induced osteogenic response was blocked by mitomycin C, identifying cell proliferation as an important step in Wnt-induced osteogenesis under these conditions. Pre-treatment of MSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. BMP2 treatment of MSCs was capable of inducing both osteogenic and chondrogenic differentiation, to which AR28 caused a switch towards the osteogenic lineage, with synergistic increases in ALP. AR28 simultaneously caused a decrease in the chondrogenic differentiation of MSCs treated with BMP2 through the down regulation of Sox9 transcription. Together these results highlight the potential of GSK3β inhibitors as therapeutic modulators of canonical Wnt signalling, and there use to treat a multitude of bone related disorders.
Supervisor: Genever, Paul ; O'Shea, Patrick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available