Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brainderived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca 2+ , a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells. (5) and that the mechanical properties of the culturing environment before transplantation can influence the outcome of in vivo stem cell transplants (6). Hence, a molecular and mechanistic understanding of how stem cells process mechanical cues and how this processing results in downstream signaling events and ultimately in fate decisions is needed for greater control over the fate of transplanted cells.Studies in mesenchymal and neural stem cells have revealed the involvement of focal adhesion zones and cytoskeletal proteins, such as integrins, nonmuscle myosin II (7), Rho GTPases (8-10), and vinculin (11), that participate in the generation of cellular traction forces. Recent work also has identified the nucleoskeletal protein lamin-A (12) and the transcriptional coactivators Yap (Yesassociated protein) and Taz (transcriptional coactivator with PDZbinding motif) (13) in mechanotransduction in mesenchymal stem cells. However, the mechanisms by which mechanical cues detected by cellular traction forces are transduced to downstream intracellular pathways of differentiation remain unclear.Ion channels are involved, directly or indirectly, in the transduction of all forms of physical stimuli-including sound, light, temperature, mechanical force, and even gravity-into intracellular signaling pathways. Hence, we wondered whether ion channels could be involved in transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification. In particular, we focused here on cationic stretch-activated channels (SACs) because they are known to detect mechanical forces with high sensitivity and broad dynamic range and because they are permeable to Ca 2+ , an important second messenger implicated in cell fate (14,15). We examined the role of SACs in neural stem cells, for which mechanical cues influence specification alo...

Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of “outside-in” mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca 2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca 2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues.

Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs.

Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been reported. We delivered a 10% static equibiaxial stretch to NSPCs and examined effects on differentiation. We found static stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes, and this effect is dependent on particular extracellular matrix (ECM)-integrin linkages. Generation of oligodendrocytes from NSPCs was reduced on laminin, an outcome likely mediated by the α6 laminin-binding integrin, whereas similar effects were not observed for NSPCs on fibronectin. Our data demonstrate a direct role for tensile strain in dictating the lineage choice of NSPCs and indicate the dependence of this phenomenon on specific substrate materials, which should be taken into account for the design of biomaterials for NSPC transplantation.