Microenvironments to study migration and somal translocation in cortical neurons.

Migrating post-mitotic neurons of the developing cerebral cortex undergo terminal somal translocation (ST) when they reach their final destination in the cortical plate. This process is crucial for proper cortical layering and its perturbation can lead to brain dysfunction. Here we present a reductionist biomaterials platform that faithfully supports and controls the distinct phases of terminal ST in vitro. We developed microenvironments with different adhesive molecules to support neuronal attachment, neurite extension, and migration in distinct manners. Efficient ST occurred when the leading process of migratory neurons crossed from low-to high-adhesive areas on a substrate, promoting spreading of the leading growth cone. Our results indicate that elementary adhesive cell-substrate interactions strongly influence migratory behavior and the final positioning of neurons during their developmental journey. This in vitro model allows advanced experimentation to reveal the microenvironmental requirements underlying cortical layer development and disorders.

Bifunctional Hydrogels Containing the Laminin Motif IKVAV Promote Neurogenesis.

Engineering of biomaterials with specific biological properties has gained momentum as a means to control stem cell behavior. Here, we address the effect of bifunctionalized hydrogels comprising polylysine (PL) and a 19-mer peptide containing the laminin motif IKVAV (IKVAV) on embryonic and adult neuronal progenitor cells under different stiffness regimes. Neuronal differentiation of embryonic and adult neural progenitors was accelerated by adjusting the gel stiffness to 2 kPa and 20 kPa, respectively. While gels containing IKVAV or PL alone failed to support long-term cell adhesion, in bifunctional gels, IKVAV synergized with PL to promote differentiation and formation of focal adhesions containing ß1-integrin in embryonic cortical neurons. Furthermore, in adult neural stem cell culture, bifunctionalized gels promoted neurogenesis via the expansion of neurogenic clones. These data highlight the potential of synthetic matrices to steer stem and progenitor cell behavior via defined mechano-adhesive properties.

Phototriggered fibril-like environments arbitrate cell escapes and migration from endothelial monolayers.

Cell detachment and migration from the endothelium occurs during vasculogenesis and also in pathological states. Here, we use a novel approach to trigger single cell release from an endothelial monolayer by in-situ opening of adhesive, fibril-like environment using light-responsive ligands and scanning lasers. Cell escapes from the monolayer were observed on the fibril-like adhesive tracks with 3-15 µm width. The frequency of endothelial cell escapes increased monotonically with the fibril width and with the density of the light-activated adhesive ligand. Interestingly, treatment with VEGF induced cohesiveness within the cell layer, preventing cell leaks. When migrating through the tracks, cells presented body lateral reduction and nuclear deformation imposed by the line width and dependent on myosin contractility. Cell migration mode changed from mesenchymal to amoeboid-like when the adhesive tracks narrowed (≤5 µm). Moreover, cell nucleus was shrunk showing packed DNA on lines narrower than the nuclear dimensions in a mechanisms intimately associated with the stress fibers. This platform allows the detailed study of escapes and migratory transitions of cohesive cells, which are relevant processes in development and during diseases such as organ fibrosis and carcinomas.

Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding.

The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvß3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.