The significance of the physical environment in regulating stem cell behavior has only recently come to be appreciated. This is particularly evident in bone, where physical loading is a key regulator of bone adaptation through the activation of osteoprogenitors during development [52
], in adult tissue in vivo [3
] and regenerative medicine strategies in vitro [10
]. In order to fully capitalize on the positive effect of physical loading, a greater understanding of how this signal is transduced into an osteogenic response is required. In this study, we demonstrate that physical loading in the form of OFF-induced shear stress is a pro-osteogenic stimulus for hMSCs. In addition, we demonstrate for the first time that osteoprogenitors use the primary cilium in the mechanotransduction of fluid flow by mediating loading-induced changes in osteogenic gene expression and proliferation. Our findings therefore highlight OFF as a potential component of bioreactor-based strategies to form bone-like tissues suitable for regenerative medicine and furthermore highlight the primary cilium as a potential therapeutic target for efforts to mimic loading in vivo with the aim of preventing bone-loss during diseases such as osteoporosis.
Short periods of mechanical stimulation significantly enhanced osteogenic gene expression and the proliferation rate of hMSCs. Although the vast majority of studies to date have investigated the effect of long-term exposure of mechanical stimulation in MSCs [19
], this study demonstrates that as little as 2 hours of OFF was sufficient to significantly upregulate COX2 and BMP2 mRNA over static and osteogenic differentiation media controls and remained upregulated for 2 hours following the cessation of flow. COX2 is an enzyme that catalyzes the synthesis of prostaglandin-E2 (PGE2). COX2, through the production of PGE2, mediates loading-induced bone formation and is critically involved in bone fracture repair in vivo [23
]. COX2 knockout mice display a marked reduction in osteoblastogenesis which correlates with significantly reduced levels of OSTERIX and RUNX2 gene expression, two pivotal early transcription factors in the osteogenic lineage. Interestingly, PGE2 and BMP2 treatment rescue this defect and enhance the expression of both transcription factors, indicating a role for BMP2 in osteoblastogenesis downstream of COX2 yet upstream of OSTERIX and RUNX2 [54
]. Therefore, this study captured very early-stage markers of osteogenic differentiation. It must be noted that despite increases in osteogenic gene expression in response to flow, due to the transient nature of their expression, the duration and magnitude of flow used in this study may not be sufficient to fully drive the osteogenic lineage commitment of hMSC. Therefore, longer periods of mechanical stimulation and/or greater postincubation periods may be required to observe changes in the other markers assayed and verify osteogenic differentiation. In addition, only high magnitude mechanical stimulation generated a significant increase in the proliferation rate of hMSCs. This data is consistent with a series of studies by Riddle et al. where high flow rate OFF resulted in the release of ATP which acted in an autocrine/paracrine manner to enhance proliferation [25
]. ATP-induced proliferation activates phosphoinositide 3-kinase (PI3K)/Akt-, mTOR (mammalian target of rapamycin)/p70/S6K-, and ERK1/2-dependent signaling pathways in fibroblasts [56
], all of which have been implicated in mechanically-mediated changes in hMSC and osteoblast proliferation [26
]. Therefore, numerous pathways may regulate mechanically-mediated changes in MSC proliferation via ATP-mediated purinergic signaling. As loading-induced bone formation is magnitude dependent [58
], it is interesting to speculate that higher magnitude loading such as 2Pa shear represents a mechanosensing threshold whereby, in order to meet the bodies demands of greater bone formation, osteoprogenitor proliferation is initiated to provide sufficient numbers of bone forming cells. Although determining this is beyond the scope of this study, it is evident that OFF is a potent stimulus regulating hMSC behavior. In summary, short periods of OFF significantly enhanced osteogenic gene expression and proliferation of hMSCs and therefore supports the use of OFF as an effective component of bioreactor based tissue engineering strategies.
Primary cilia were shown to protrude 4-6 μm in length from the apical surface of hMSCs at high incidence. This high incidence is consistent with previous findings in hMSCs and human embryonic stem cells [44
] and is considerably higher than what has been detected in more differentiated cells of the osteogenic lineage [41
] suggesting a greater role for the primary cilium in the progenitor cell. For example, during development the HH signaling pathway, which is regulated by the primary cilium, is known to play pivotal roles in the commitment of different lineages and it has been shown that stem cells which do not possess a primary cilium have a reduced capacity for neurogenesis [61
] and cardiogenesis [62
] due to impaired HH signaling. In addition to cilium incidence, primary cilia were shown to extend from the mother centriole, which is intimately connected with the cells cytoplasmic microtubule network, into the extracellular space. These observations suggest that stem cell primary cilia possess physical characteristics consistent with extracellular-sensing in more differentiated cells such as the osteoblast and osteocyte [41
hMSCs which do not possess a primary cilium display an inhibited osteogenic response to mechanical stimulation demonstrating an important role for the cilium in regulating stem cell mechanotransduction. Flow-mediated increases in COX2 and BMP2 mRNA expression is lost in hMSCs with removal of the primary cilium yet the basal expression of these genes remained unaltered. This inhibition of osteogenic gene expression would not only suppress osteogenic differentiaiton in cells exposed to OFF but also through the potential loss of PGE2 (through COX2) and BMP2 secretion may directly affect the differentiation of adjacent cells throughout the stem cell niche. Therefore, these data point to a pro-osteogenic mechanosensory role for the primary cilium in hMSCs. Several mechanosensitive pathways regulated by the primary cilium have been shown to be important in the osteogenic differentiation of stem cells. Interestingly, a recent study demonstrated that activation of the HH signaling pathway is inversely correlated with osteogenic differentiation in hMSCs [59
] despite a strong positive correlation in murine cells [27
]. In this study, the application of mechanical stimulation, which was sufficient to enhance osteogenic gene expression, resulted in an inhibition of HH signaling as measured by PTCH1 and GLI1 mRNA expression. These data therefore suggest that fluid flow counters HH suppression of osteogenic differentiation in hMSCs. Surprisingly, the flow-mediated inhibition of HH was not lost with removal of the primary cilium, indicating that HH signaling is not playing a direct role in our studies findings. Further work is required to fully elucidate the role of HH signaling in mechanically mediated osteogenic differentiation of hMSCs. Despite many cell types using the cilium in a mechanosensory role, it is becoming apparent that the underlying molecular mechanism differs depending on the cell type. For example, cilia-mediated mechanosensing in kidney epithelial cells is dependent upon intracellular calcium while in osteocytes cAMP is the second messenger molecule. It is distinctly possible that the molecular mechanism of cilia-based mechanosensing is preserved throughout the osteogenic lineage (MSC–osteoblast–osteocyte). Therefore, that would indicate AC6/cAMP signaling as the molecular mechanism [43
]. In fact, the application of 30 minutes of OFF was sufficient to significantly enhance cAMP production in hMSCs (data not shown). Future work aims to delineate the exact molecular mechanosensing components involved in cilia-mediated hMSC mechanotransduction, the identification of which could allow direct pharmaceutical manipulation, resulting in therapeutic treatments for bone loss in diseases such as osteoporosis.
Unexpectedly, inhibiting primary cilia formation and function significantly enhanced flow-mediated hMSC proliferation, yet once again did not affect basal proliferation. Therefore, it seems that the cell uses the primary cilium to control mechanically mediated changes in proliferation. A similar phenomenon has been reported in epithelial cells of the kidney where defects in the primary cilium results in uncontrolled proliferation characterized by high mTOR activity, cyst formation, and ultimately in polycystic kidney disease (PKD). mTOR is a kinase belonging to the PI3K-related kinase family of proteins and has essential roles in protein translation, cell growth, and proliferation [65
] and is activated in several types of tumors [66
]. Boehlke et al. [67
] recently demonstrated that bending of the primary cilium under flow results in the downregulation of mTOR activity. Shillingford et al. [68
] showed that the ciliary protein, polycystin-1 (PC1), forms a complex with mTOR also inhibiting its activity and subsequently cell proliferation. This has led to speculation that defects in the primary cilium and/or PC1 leaves mTOR in an uncontrolled state, where it is susceptible to activation from other kinases such as ERK1/2 [69
] which is known to be phosphorylated in hMSCs in response to OFF [26
]. In effect, this would hypersensitize the cell to a pro-proliferative stimulus such as fluid flow. Our data supports this model indicating that this phenomenon is not tissue specific and therefore has far reaching significance as many ciliopathies such as PKD are characterized by uncontrolled proliferation and cyst formation.
Some limitations of this study should be mentioned. The primary cilium was required for OFF-mediated increases in early osteogenic gene expression, but the role of cilium in regulating OFF-mediated osteogenic lineage commitment at later time points was not verified. Although the data presented strongly indicate a role for the cilium in this response, further work is necessary to fully characterize the role of the cilium in mechanically-mediated stem cell differentiation. Inhibition of primary cilia formation was achieved by siRNA knockdown of Polaris. Although siRNA treatment significantly reduced primary cilium formation, 30% of hMSCs imaged post-transfection still possessed a primary cilium demonstrating that this technique is not completely effective in removing the primary cilium. However, a 65% reduction in cilium incidence was sufficient to significantly blunt hMSC responsiveness to fluid flow and therefore demonstrates this organelles role in hMSC mechanotransduction. Previous studies have used chloral hydrate treatment to remove cilia in parallel with siRNA. Chloral hydrate acts to disrupt the cilia/basal body connection but has also demonstrated nonspecific effects on cell behavior. Given that previous studies have not demonstrated different outcomes between the two methods and the potential nonspecific effects of a chemical treatment, chloral hydrate was not used in this study. Although this study demonstrates a role for the primary cilium in hMSC mechanotransduction, it is important to acknowledge that there are numerous other mechanosensitive organelles/molecules, which the stem cell could use for mechanotransduction. For example, adult bone cells which do not possess β1 integrin and/or focal adhesion kinase do not respond to fluid shear with an increase in osteogenic gene expression [70
]. Depending on the form, frequency or magnitude of stimulation the cell may use different mechanotransduction mechanisms, or, intriguingly, in some cases there may be dependent mechanisms. This may indeed be the case in tissues where β1 integrins have been shown to localize to the primary cilium, including chondrocytes [72
] and kidney epithelial cells [73
]. Although the investigation of this possible crosstalk is beyond the scope of this study, future work aims to explore other potential mechanisms of mechanotransduction in hMSCs and their potential involvement in cilia mediated mechanotransduction.