The main goal of this proposal is to induce and detect in real time and at single-cell level the differentiation of human mesenchymal stem cells using mechanical loads in 2D and 3D conditions. This proposal integrates disciplines from nanotechnology, bioengineering and cell and molecular biology. First we propose to develop a method to track the differentiation of stem cells in real time and at the single cell level using cytoskeletal organization of actin, microtubules and intermediate filaments as a suitable cell biomarker. We will then establish mechanical loading protocols to induce, via direct force application onto cells, the first stages of stem cell differentiation towards specific cell lineages. We will apply cyclic tensile strain and compression to stem cells in 2D and 3D conditions, and track their differentiation status in real time using the cytoskeletal biomarkers that we will have identified before. The results of this proposal will have implications for the field of stem cell mechanobiology in particular, and some of the techniques developed will also contribute to the wider field of directed stem cell differentiation.