Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1-4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ß1-chain-derived fragment interacts via α3ß1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3ß1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.

Model based variable selection as a tool to highlight biological differences in Raman spectra of cells.

In vitro Raman spectroscopy used for non-invasive, non-destructive characterization of single cells and tissues has proven to be a powerful tool for understanding the complex biochemical processes within these biological systems. Additionally it enables the comparison of a wide range of in vitro model systems by discriminating them based on their biomolecular differences. However, one persistent challenge in Raman spectroscopy has been the highly complex structure of cell and tissue spectra, which comprise signals from lipids, proteins, carbohydrates and nucleic acids, which may overlap significantly. This leads to difficulty in discerning which molecular components are responsible for the changes seen between experimental groups. To address this problem, we introduce a technique to highlight the significant biochemical changes between sample groups by applying a novel approach using Partial Least Squares - Discriminant Analysis (PLS-DA) Variable Importance Projection (VIP) scores normally used for variable selection as heat maps combined with group difference spectra to highlight significant differences in Raman band shapes and position. To illustrate this method we analyzed single HeLa cells in their live, fixed, fixed and ethanol dehydrated, to the fixed, dehydrated and then rehydrated states respectively. Fixation, ethanol dehydration and rehydration are known to induce molecular changes in the lipids and proteins within each cell.

Scarring vs. functional healing: Matrix-based strategies to regulate tissue repair.

All vertebrates possess mechanisms to restore damaged tissues with outcomes ranging from regeneration to scarring. Unfortunately, the mammalian response to tissue injury most often culminates in scar formation. Accounting for nearly 45% of deaths in the developed world, fibrosis is a process that stands diametrically opposed to functional tissue regeneration. Strategies to improve wound healing outcomes therefore require methods to limit fibrosis. Wound healing is guided by precise spatiotemporal deposition and remodelling of the extracellular matrix (ECM). The ECM, comprising the non-cellular component of tissues, is a signalling depot that is differentially regulated in scarring and regenerative healing. This Review focuses on the importance of the native matrix components during mammalian wound healing alongside a comparison to scar-free healing and then presents an overview of matrix-based strategies that attempt to exploit the role of the ECM to improve wound healing outcomes.

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information.

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin ß1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the ß1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor ß1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Basement membrane fragments in the context of the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) enables cells of epithelial phenotype to become motile and change to a migratory mesenchymal phenotype. EMT is known to be a fundamental requisite for tissue morphogenesis, and EMT-related pathways have been described in cancer metastasis and tissue fibrosis. Epithelial structures are marked by the presence of a sheet-like extracellular matrix, the basement membrane, which is assembled from two major proteins, laminin and collagen type IV. This specialized matrix is essential for tissue function and integrity, and provides an important barrier to the potential pathogenic migration of cells. The profound phenotypic transition in EMT involves the epithelial cells disrupting the basement membrane. Matrix metalloproteinases (MMPs) are known to cleave components of basement membranes, but MMP-basement membrane crosstalk during EMT in vivo is poorly understood. However, MMPs have been reported to play a role in EMT-related processes and a variety of basement membrane fragments have been shown to be released by specific MMPs in vitro and in vivo exhibiting distinct biological activities. This review discusses general considerations regarding the basement membrane in the context of EMT, a possible role for specific MMPs in EMT and highlights biologically active basement membrane fragments liberated by MMPs.

Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells.

Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

A designer peptide as a template for growing Au nanoclusters.

A peptide was designed to generate a sub-nanometric template that guides the growth of fluorescent gold nanoclusters. The peptide was endorsed with nucleating moieties and a three-dimensional structure that arrests the growth of ultrasmall nanoparticles. The nanoclusters are not cytotoxic and can be found in the cytosol of cells.