Primary cilia-mediated mechanotransduction in human mesenchymal stem cells

Abstract

Physical loading is a potent stimulus required to maintain bone homeostasis, partly through the renewal and osteogenic differentiation of mesenchymal stem cells (MSCs). However, the mechanism by which MSCs sense a biophysical force and translate that into a biochemical bone forming response (mechanotransduction) remains poorly understood. The primary cilium is a single sensory cellular extension, which has recently been shown to demonstrate a role in cellular mechanotransduction and MSC lineage commitment. In this study, we present evidence that short periods of mechanical stimulation in the form of oscillatory fluid flow (OFF) is sufficient to enhance osteogenic gene expression and proliferation of human MSCs (hMSCs). Furthermore, we demonstrate that the cilium mediates fluid flow mechanotransduction in hMSCs by maintaining OFF-induced increases in osteogenic gene expression and, surprisingly, to limit OFF-induced increases in proliferation. These data therefore demonstrate a pro-osteogenic mechanosensory role for the primary cilium, establishing a novel mechanotransduction mechanism in hMSCs. Based on these findings, the application of OFF may be a beneficial component of bioreactor-based strategies to form bone-like tissues suitable for regenerative medicine and also highlights the cilium as a potential therapeutic target for efforts to mimic loading with the aim of preventing bone loss during diseases such as osteoporosis. Furthermore, this study demonstrates a role for the cilium in controlling mechanically mediated increases in the proliferation of hMSCs, which parallels proposed models of polycystic kidney disease. Unraveling the mechanisms leading to rapid proliferation of mechanically stimulated MSCs with defective cilia could provide significant insights regarding ciliopathies and cystic diseases.

Figure 1

Effect of 2 hours of oscillatory fluid flow-induced 1 Pa of shear stress on the mRNA expression of (A) COX2, (B) BMP2, (C) RUNX2, and (D) OPN at 30 minutes, 2 hours, 24 hours, and 48 hours post-cessation of flow. Cells were cultured statically and in osteogenic differentiation media as controls. Furthermore, the effect of 2 hours of oscillatory flow-induced (E) 1 Pa and (F) 2 Pa of shear stress on the proliferation rate of human mesenchymal stem cells as assayed by 1 hour of EdU incorporation 20 hours post-cessation of flow. Mean ± SEM, * indicates significantly different than controls (*, p < .05; **, p < .01; ***, p < .001). Abbreviations: COX2, cyclo-oxygenase-2; EdU, 5-ethynyl-2′-deoxyuridine.

Figure 2

Confirmation of the existence of primary cilia on human mesenchymal stem cells (hMSCs). (A): Primary cilia stained with antiacetylated α-tubulin were present on 86% of hMSCs in the perinuclear region of the cell. (B, C): Live microtubule imaging demonstrated that hMSC primary cilia are intricately connected to the microtubule cytoskeleton and extend from the mother centriole 4–6 μm into the extracellular space. Open arrowhead: primary cilia; close arrowhead: centrioles. (D): Polaris mRNA expression of hMSCs and (C) number of ciliated cells 72 hours following transfection with scrambled small-interfering RNA (siRNA) and siRNA targeting Polaris. Mean ± SEM, * indicates significantly different than scrambled control (*, p < .05; ***, p < .001). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Effect of 2 hours of oscillatory fluid flow-induced 1 Pa of shear stress on the mRNA expression of (A) COX2 and (B) BMP2 2 hours post-cessation of flow in human mesenchymal stem cells transfected with scrambled small-interfering RNA (siRNA) and siRNA targeting POLARIS. Mean ± SEM, * indicates significantly different than no flow control (*, p < .05; ***, p < .001). Abbreviation: COX2, cyclo-oxygenase-2.

Figure 4

Effect of oscillatory fluid flow-induced 1 Pa of shear stress on the mRNA expression of (A) PTCH1 and (B) GLI1 30 minutes and 2 hours post-cessation of flow. Effect of oscillatory fluid flow-induced 1 Pa of shear stress on the mRNA expression of (C) PTCH1 and (D) GLI1 2 hours post-flow in human mesenchymal stem cells transfected with scrambled small-interfering RNA (siRNA) and siRNA targeting POLARIS. Mean ± SEM, * indicates significantly different than no flow control (*, p < .05; **, p < .01). Abbreviation: PTCH1, Patched1.

Figure 5

Effect of 2 hours of oscillatory fluid flow-induced 2 Pa of shear stress on the proliferation rate of human mesenchymal stem cells transfected with scrambled small-interfering RNA (siRNA) and siRNA targeting POLARIS. Mean ± SEM, * indicates significantly different than no flow control (*, p < .05; **, p < .01). Abbreviation: EdU, 5-ethynyl-2′-deoxyuridine.