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Primary Cilium-Mediated Mesenchymal Stem Cell Mechanobiology in Bone

Final Report Summary - CILIAMECHANOBIO (Primary Cilium-Mediated Mesenchymal Stem Cell Mechanobiology in Bone)

Every 30 seconds a person suffers an osteoporosis-related bone fracture in the EU. Osteoporosis arises when adult marrow stem cells (MSC) fail to produce sufficient numbers of bone forming osteoblasts. A key regulator of MSC behaviour is physical exercise, yet the mechanisms by which MSCs sense and respond to changes in their physical environment are virtually unknown. Primary cilia sensory cellular extensions important in adult bone, but to date, their role in MSC behaviour is poorly understood. Therefore, the objective of this project is to determine the role of the stem cell primary cilium in exercise-induced bone formation. The identification of such will lead to the direct manipulation of MSCs via novel cilia-targeted therapeutics that mimic the regenerative influence of exercise. The achievements and outcomes of this project to date are summarised below.

Theme 1: In this theme we have developed novel bioreactors and manufacturing technologies to produce fibrous materials that replicate the complex physical environment in the laboratory that the stem cell would experience within the body. Utilising these technologies, we have demonstrated that the application of physical stimuli such as cyclic fluid shear and pressure to MSCs can induce bone formation in a laboratory setting and have demonstrated that the stem cell utilised the primary cilium to sense these physical cues.

Theme 2: The objective of Theme 2 is to determine how the cilium is required for exercise-induced bone formation. We have shown that mechanically sensitive calcium channels, TRPV4 and PC2, along with a novel mechanosensitive G-protein couple receptor (GPR161) localise to the primary cilium of MSCs. Furthermore, we have shown that these mechanosensors are required for MSCs to respond to mechanical stimuli,and have delineated a complex mechanotransduction pathway encompassing GPR161-AC6-cAMP-HH which leads to MSC osteogenesis. The identification of these critical molecules involved in cilia-mediated mechanosensing, has lead us to the development of new cilia-targeted therapeutics that activate these components biomechanically, mimicking the beneficial effect of mechanical stimuli, promoting bone formation.

Theme 3: In this theme, we investigated the contribution of osteocytes in regulating MSC recruitment and differentiation and the role of the cilium in this response. Initially we demonstrated that osteocytes secrete factors which regulate MSC recruitment, proliferation, and differentiation, in addition to angiogenesis and that the release of these pro-regenerative factors was dependent on the mechanical stimulation. We identified extracellular vesicles (EVs) as the mechanism by which these factors are delivered, and importantly demonstrated that these mechanically activated-EVs (MA-EVs) can be isolated and utilised as a mechanotherapeutic to enhance bone formation. In addition to MSC mechanotransduction, we also investigated the role of the cilium in MSC recruitment. We have demonstrated that low levels of Transforming Growth Factor β1 (TGFβ1) induce the recruitment of human MSCs and that this recruitment relies on proper formation of the primary cilium. Furthermore, we demonstrated that TGFβ signalling occurs at the ciliary base and subsequent translocation to the nucleus also relies on the proper formation of the primary cilium. These data highlights the primary cilium as a potential therapeutic target to enhance MSC recruitment and bone formation.
Theme 4: Lastly in Theme 4 we developed in-vivo models to specifically track and target MSCs. In particular, by specifically deleting the primary cilium and AC6 within the stem cell population we were able to demonstrate the critical role of the stem cell and these cellular components in exercise-induced bone formation. Through the validation of our findings in these advanced models, we further validate the development of novel cilia-targeted therapeutics to enhance bone formation and treat osteoporosis.