To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices (ECM), we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibers, and bulk contraction of the material. While increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation, increasing fiber stiffness instead suppressed spreading and proliferation depending on network architecture. Lower fiber stiffness permitted active cellular forces to recruit nearby fibers, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signaling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fiber recruitment as a novel mechanism by which cells probe and respond to mechanics in fibrillar matrices.

Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM). A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled. Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20 microm diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 microm diameter) islands. Differentiation did not depend on ECM composition or density. Rather, the actin cytoskeleton mediated shape-induced differentiation by regulating serum response factor (SRF) transcriptional activity. Knockdown of SRF or its co-factor MAL inhibited differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues are transduced into transcriptional responses that determine epidermal cell fate.

Contractile forces exerted on the surrounding extracellular matrix (ECM) lead to the alignment and stretching of constituent fibers within the vicinity of cells. As a consequence, the matrix reorganizes to form thick bundles of aligned fibers that enable force transmission over distances larger than the size of the cells. Contractile force-mediated remodeling of ECM fibers has bearing on a number of physiologic and pathophysiologic phenomena. In this work, we present a computational model to capture cell-mediated remodeling within fibrous matrices using finite element-based discrete fiber network simulations. The model is shown to accurately capture collagen alignment, heterogeneous deformations, and long-range force transmission observed experimentally. The zone of mechanical influence surrounding a single contractile cell and the interaction between two cells are predicted from the strain-induced alignment of fibers. Through parametric studies, the effect of cell contractility and cell shape anisotropy on matrix remodeling and force transmission are quantified and summarized in a phase diagram. For highly contractile and elongated cells, we find a sensing distance that is ten times the cell size, in agreement with experimental observations.

A major challenge in tissue engineering is the development of materials that can support angiogenesis, wherein endothelial cells from existing vasculature invade the surrounding matrix to form new vascular structures. To identify material properties that impact angiogenesis, here we have developed an in vitro model whereby molded tubular channels inside a synthetic hydrogel are seeded with endothelial cells and subjected to chemokine gradients within a microfluidic device. To accomplish precision molding of hydrogels and successful integration with microfluidics, we developed a class of hydrogels that could be macromolded and micromolded with high shape and size fidelity by eliminating swelling after polymerization. Using this material, we demonstrate that matrix degradability switches three-dimensional endothelial cell invasion between two distinct modes: single-cell migration and the multicellular, strand-like invasion required for angiogenesis. The ability to incorporate these tunable hydrogels into geometrically constrained settings will enable a wide range of previously inaccessible biomedical applications.

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

The control of the cell microenvironment on model patterned substrates allows the systematic study of cell biology in well defined conditions, potentially using automated systems. The extreme protein resistance of poly(oligo(ethylene glycol methacrylate)) (POEGMA) brushes is exploited to achieve high fidelity patterning of single cells. These coatings can be patterned by soft lithography on large areas (a microscope slide) and scale (substrates were typically prepared in batches of 200). The present protocol relies on the adsorption of extra-cellular matrix (ECM) proteins on unprotected areas using simple incubation and washing steps. The stability of POEGMA brushes, as examined via ellipsometry and SPR, is found to be excellent, both during storage and cell culture. The impact of substrate treatment, brush thickness and incubation protocol on ECM deposition, both for ultra-thin gold and glass substrates, is investigated via fluorescence microscopy and AFM. Optimised conditions result in high quality ECM patterns at the micron scale, even on glass substrates, that are suitable for controlling cell spreading and polarisation. These patterns are compatible with state-of-the-art technologies (fluorescence microscopy, FRET) used for live cell imaging. This technology, combined with single cell analysis methods, provides a platform for exploring the mechanisms that regulate cell behaviour.

In this paper we report on the synthesis of a new family of nonionic dendritic amphiphiles that self-assemble into defined supramolecular aggregates. Our approach is based on a modular architecture consisting of different generations of hydrophilic polyglycerol dendrons [G1-G3] connected to hydrophobic C(11) or C(16) alkyl chains via mono- or biaromatic spacers, respectively. All amphiphiles complex hydrophobic compounds as demonstrated by solubilization of Nile Red or pyrene. The structure of the supramolecular assemblies as well as the aggregation numbers are strongly influenced by the type of the dendritic headgroup. While the [G1] amphiphiles form different structures such as ringlike and fiberlike micelles, the [G2] and [G3] derivatives aggregate toward spherical micelles of low polydispersity clearly proven by transmission electron microscopy (TEM) measurements. In the case of the biaromatic [G2] derivative, the structural persistence of the micelles allowed a three-dimensional structure determination from the TEM data and confirmed the aggregation number obtained by static light scattering (SLS) measurements. On the basis of these data, molecular packing geometries indicate a drastic mass deficit of alkyl chains in the hydrophobic core volume of spherical micelles. It is noteworthy that these highly defined micelles contain as little as 15 molecules and possess up to 74% empty space. This behavior is unexpected as it is very different from classical detergent micelles such as sodium dodecyl sulfate (SDS), where the hydrophobic core volume is completely filled by alkyl chains.