When human embryonic stem cells are plated on specific nanopatterns, they can effectively and rapidly differentiate into a neuronal lineage without the use of differentiation-inducing agents. Thus, ECM nanoscale topography not only regulates cell morphology but also cell fate. While the combination of such nanotopographic cues with biochemical cues such as retinoic acid further enhances neuronal differentiation, nanotopography showed a stronger effect compared to retinoic acid alone on an unpatterned surface. The mechanisms by which nanotopographic ECM cues influence differentiation appear to involve changes in cytoskeletal organization and structure, potentially in response to the geometry and size of the underlying features of the ECM. This might influence the clustering of integrins in focal adhesions and the formation of actin stress fibers, and thus the adhesion and spreading of cells. Secondary effects, such as alterations in the effective stiffness perceived by the cell or differences in protein adsorption due to the Niraparib structural features of the substrate are also possible. However, the cellular mechanisms of cell fate control by ECM nanotopography remain largely unexplored. One of the best characterized example of control of cell behavior by ECM topology has been observed during fibroblast cell migration. It is well described that fibroblasts migrate about 1.5 times faster on ECM fibrils in 3D cell-derived matrices compared to the same ECM presented in a classic 2D environment. In this study, 1D micro-patterned ECM lines with precise size features have been shown to recapitulate the cell migration behavior observed in cell-derived 3D ECM WZ4002 clinical trial environments. This most likely occurs because these ECM lines are able to mimic the fibrillar nature of the ECM in a 3D environment. Importantly, such a pseudo 3D environment has provided a convenient platform to analyze cell migration using microscopy techniques that do not require confocality. This has given novel insight about the molecular mechanisms of how cells perceive and migrate in 3D versus 2D environments. Comparable results have also been observed during cell migration on similar patterns at the nanometer scale. In this study, we sought to understand the molecular mechanisms of how neurons respond to matrix nanotopography during the process of neurite outgrowth. For that purpose, we explored in detail neuronal morphology and morphodynamics on nanopatterns.