Establishing extracellular milieus to stimulate neuronal regeneration is a critical need in neuronal tissue engineering. Many studies have used a soluble factor (such as nerve growth factor or retinoic acid [RA]), micropatterned substrate, and electrical stimulation to induce enhanced neurogenesis in neuronal precursor cells. However, little attention has been paid to mechanical stimulation because neuronal cells are not generally recognized as being mechanically functional, a characteristic of mechanoresponsive cells such as osteoblasts, chondrocytes, and muscle cells. In this study, we performed proof-of-concept experiments to demonstrate the potential anabolic effects of mechanical stretch to enhance cellular neurogenesis. We cultured human neuroblastoma (SH-SY5Y) cells on collagen-coated membrane and applied 10% equibiaxial dynamic stretch (0.25 Hz, 120 min/d for 7 days) using a Flexcell device. Interestingly, cell stretch alone, even without a soluble neurogenic stimulatory factor (RA), produced significantly more and longer neurites than the non–RA-treated, static control. Specific neuronal differentiation and cytoskeletal markers (e.g., microtubule-associated protein 2 and neurofilament light chain) displayed compatible variations with respect to stretch stimulation.

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The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flowinduced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

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Despite the important role of mechanical signals in bone remodeling, relatively little is known about how fluid shear affects osteoblastic cell migration behavior. Here we demonstrated that MC3T3-E1 osteoblast migration could be activated by physiologically-relevant levels of fluid shear in a shear stress-dependent manner. Interestingly, shear-sensitive osteoblast migration behavior was prominent only during the initial period after the onset of the steady flow (for about 30 min), exhibiting shear stress-dependent migration speed, displacement, arrest coefficient, and motility coefficient. For example, cell speed at 1 min was 0.28, 0.47, 0.51, and 0.84 μm min-1 for static, 2, 15, and 25 dyne cm-2 shear stress, respectively. Arrest coefficient (measuring how often cells are paused during migration) assessed for the first 30 min was 0.40, 0.26, 0.24, and 0.12 respectively for static, 2, 15, and 25 dyne cm-2. After this initial period, osteoblasts under steady flow showed decreased migration capacity and diminished shear stress dependency. Molecular interference of RhoA kinase (ROCK), a regulator of cytoskeletal tension signaling, was found to increase the shear-sensitive window beyond the initial period. Cells with ROCK-shRNA had increased migration in the flow direction and continued shear sensitivity, resulting in greater root mean square displacement at the end of 120 min of measurement. It is notable that the transient osteoblast migration behavior was in sharp contrast to mesenchymal stem cells that exhibited sustained shear sensitivity (as we recently reported, J. R. Soc. Interface. 2015; 12:20141351). The study of fluid shear as a driving force for cell migration, i.e., “flowtaxis”, and investigation of molecular mechanosensors governing such behavior (e.g., ROCK as tested in this study) may provide new and improved insights into the fundamental understanding of cell migration-based homeostasis.

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BackgroundNephron progenitor cells (NPCs) give rise to all segments of functional nephrons and are of great interest due to their potential as a source for novel treatment strategies for kidney disease. Fibroblast growth factor (FGF) signaling plays pivotal roles in generating and maintaining NPCs during kidney development, but little is known about the molecule(s) regulating FGF signaling during nephron development. Sprouty 1 (SPRY1) is an antagonist of receptor tyrosine kinases. Although SPRY1 antagonizes Ret-GDNF signaling, which modulates renal branching, its role in NPCs is not known.MethodsSpry1, Fgf9, and Fgf20 compound mutant animals were used to evaluate kidney phenotypes in mice to understand whether SPRY1 modulates FGF signaling in NPCs and whether FGF8 functions with FGF9 and FGF20 in maintaining NPCs.ResultsLoss of one copy of Spry1 counters effects of the loss of Fgf9 and Fgf20, rescuing bilateral renal agenesis premature NPC differentiation, NPC proliferation, and cell death defects. In the absence of SPRY1, FGF9, and FGF20, another FGF ligand, FGF8, promotes nephrogenesis. Deleting both Fgf8 and Fgf20 results in kidney agenesis, defects in NPC proliferation, and cell death. Deleting one copy of Fgf8 reversed the effect of deleting one copy of Spry1, which rescued the renal agenesis due to loss of Fgf9 and Fgf20.ConclusionsSPRY1 expressed in NPCs modulates the activity of FGF signaling and regulates NPC stemness. These findings indicate the importance of the balance between positive and negative signals during NPC maintenance.

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