A diverse array of environmental factors contribute to the overall control of stem cell activity. In particular, new data continues to mount on the influence of the extracellular matrix (ECM) on stem cell fate through physical interactions with cells, such as the control of cell geometry, ECM geometry/topography at the nanoscale, ECM mechanical properties, and the transmission of mechanical or other biophysical factors to the cell. Here we review some of the physical processes by which cues from the ECM can influence stem cell fate, with particular relevance to the use of stem cells in tissue engineering and regenerative medicine.

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core [1]. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture [2–4]. Here, we 3D printed rigid filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks which could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization, and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices (ECMs), and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional (3D) encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular-traction, independent of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that either permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). In addition, switching the permissive hydrogel to a restrictive state via delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Also, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell-matrix and cell-cell contact. While increased mechanical loading at cell-matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell-cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell-cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell-cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.adherens junction | mechanotransduction | myosin | PDMS | traction force

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure threedimensional traction forces exerted by cells fully encapsulated within well-defined elastic hydrogel matrices. We apply this approach to measure tractions from a variety of cell types and contexts, and reveal patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.Cells are constantly probing, pushing and pulling on the surrounding extracellular matrix (ECM). These cell-generated forces drive cell migration and tissue morphogenesis and maintain the intrinsic mechanical tone of tissues 1, 2. Such forces not only guide mechanical and structural events, but also trigger signaling pathways that promote functions ranging from proliferation to stem cell differentiation3 , 4. Therefore, precise measurements of the spatial and temporal nature of these forces are essential to understanding when and where mechanical events come to play in both physiological and pathological settings.Methods employing planar elastic surfaces or arrays of flexible cantilevers have mapped, with subcellular resolution, the forces that cells generate against their substrates1 , 5 -7 . However, many processes are altered when cells are removed from native three-dimensional (3D) environments and cultured on two-dimensional (2D) substrates. At a structural level, cells encapsulated within a 3D matrix exhibit dramatically different morphology, cytoskeletal organization, and focal adhesion structure from those on 2D substrates 8 . Even the initial means by which cells attach and spread against a 2D substrate are quite different from the invasive process required for cells to extend inside a 3D matrix. These differences suggest that dimensionality alone may significantly impact how cellular forces are generated and transduced into biochemical or structural changes. Yet, although the mechanical properties of 3D ECMs and the cellular forces generated therein have been shown to * Correspondence should be addressed to C.S. Chen, [C.S. Chen (chrischen@seas.upenn.edu, Tel: 01-215-746-1750, Fax: 01-215-746-1752 COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests. Here, we quantitatively measure the traction stresses (force per area), hereafter tractions, exerted by cells embedded within a hydrogel matrix. GFP-expressing fibroblasts were encapsulated within mechanically well-defined polyethylene glycol (PEG) hydrogels that incorporate proteolytically degradable domains in the polymer backbone and pendant adhesive ligands 10 . The incorporation of adhesive and degradable domains permits the cells to invade, spread, and adopt physiologically relevant morphologies ( Fig. 1a and Supplementary Movie 1). The hydrogels used in this study had a Young's modulus of 600 to 1,000 Pa ( Supplementary Fig. 1), a range similar to commonly used ECMs such as reconstituted collagen ...

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.

The density and architecture of capillary beds that form within a tissue depend on many factors, including local metabolic demand and blood flow. Here, using microfluidic control of local fluid mechanics, we show the existence of a previously unappreciated flowinduced shear stress threshold that triggers angiogenic sprouting. Both intraluminal shear stress over the endothelium and transmural flow through the endothelium above 10 dyn/cm 2 triggered endothelial cells to sprout and invade into the underlying matrix, and this threshold is not impacted by the maturation of cell-cell junctions or pressure gradient across the monolayer. Antagonizing VEcadherin widened cell-cell junctions and reduced the applied shear stress for a given transmural flow rate, but did not affect the shear threshold for sprouting. Furthermore, both transmural and luminal flow induced expression of matrix metalloproteinase 1, and this up-regulation was required for the flow-induced sprouting. Once sprouting was initiated, continuous flow was needed to both sustain sprouting and prevent retraction. To explore the potential ramifications of a shear threshold on the spatial patterning of new sprouts, we used finite-element modeling to predict fluid shear in a variety of geometric settings and then experimentally demonstrated that transmural flow guided preferential sprouting toward paths of draining interstitial fluid flow as might occur to connect capillary beds to venules or lymphatics. In addition, we show that luminal shear increases in local narrowings of vessels to trigger sprouting, perhaps ultimately to normalize shear stress across the vasculature. Together, these studies highlight the role of shear stress in controlling angiogenic sprouting and offer a potential homeostatic mechanism for regulating vascular density.angiogenesis | force | mechanotransduction | migration | morphogenesis T he density of capillary blood vessels varies widely across different organs and tissues and is determined by the ability of unmet local metabolic needs to trigger angiogenesis. Perhaps most well characterized is the induction of VEGF expression by parenchymal hypoxia, leading to angiogenesis that persists until the hypoxia is relieved by subsequent enhanced tissue perfusion (1). Indeed, significant advances have been made in understanding the mechanisms by which numerous biochemical stimuli induce endothelial sprouting (2). Importantly, excess metabolic demand also triggers enhanced local circulation by relaxation of upstream arterioles (3) and results in increased blood flow to these regions. In addition to enhanced delivery of nutrients, the increased blood flow also increases shear stress on the luminal surface of the endothelium, which in some studies has been shown to induce capillary growth in skeletal muscle (4, 5), whereas others showed shear stress enhances endothelial barrier function and inhibits sprouting (6-9).Unlike luminal shear, transmural flow, or fluid flow exiting the wall of the vessel, is universally accepted to induce sprouting o...

Stem cells that adopt distinct lineages cannot be distinguished based on traditional cell shape. This study reports that higher-order variations in cell shape and cytoskeletal organization that occur within hours of stimulation forecast the lineage commitment fates of human mesenchymal stem cells (hMSCs). The unique approach captures numerous early (24 h), quantitative features of actin fluororeporter shapes, intensities, textures, and spatial distributions (collectively termed morphometric descriptors). The large number of descriptors are reduced into "combinations" through which distinct subpopulations of cells featuring unique combinations are identified. We demonstrate that hMSCs cultured on fibronectin-treated glass substrates under environments permissive to bone lineage induction could be readily discerned within the first 24 h from those cultured in basal-or fat-inductive conditions by such cytoskeletal feature groupings. We extend the utility of this approach to forecast osteogenic stem cell lineage fates across a series of synthetic polymeric materials of diverse physicochemical properties. Within the first 24 h following stem cell seeding, we could successfully "profile" the substrate responsiveness prospectively in terms of the degree of bone versus nonbone predisposition. The morphometric methodology also provided insights into how substrates may modulate the pace of osteogenic lineage specification. Cells on glass substrates deficient in fibronectin showed a similar divergence of lineage fates, but delayed beyond 48 h. In summary, this high-content imaging and single cell modeling approach offers a framework to elucidate and manipulate determinants of stem cell behaviors, as well as to screen stem cell lineage modulating materials and environments.biomaterials | differentiation | imaging and modeling | stem cells | actin organization