Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration and embryogenesis in the stem cell niche. Tissue homeostasis involves the interaction of multipotent cells with nanoscaled topographical features in their ECM to regulate aspects of cell behavior. Synthetic nanostructures can drive specific cell differentiation, but the sensing mechanisms for nanocues remain poorly understood. Here, we report that nanotopography-induced human mesenchymal stem cell (hMSC) differentiation through cell mechanotransduction is modulated by the integrin-activated focal adhesion kinase (FAK). On nanogratings with 250 nm line width on polydimethylsiloxane, hMSCs developed aligned stress fibers and showed an upregulation of neurogenic and myogenic differentiation markers. The observed cellular focal adhesions within these cells were also significantly smaller and more elongated on the nanogratings compared to microgratings or unpatterned control. In addition, our mechanistic study confirmed that this regulation was dependent upon actomyosin contractility, suggesting a direct force-dependent mechanism. The topography-induced differentiation was observed on different ECM compositions but the response was not indicative of a direct ECM-induced hMSC differentiation pathway. FAK phosphorylation was required for topography-induced hMSC differentiation while FAK overexpression overruled the topographical cues in determining cell lineage bias. The results indicated that FAK activity had a direct impact on topography-induced gene expression, and that this effect of FAK was independent of cell shape. These findings suggest that hMSC sense and transduce nanotopographical signals through focal adhesions and actomyosin cytoskeleton contractility to induce differential gene expression.

Through mechanotransduction, cells can sense physical cues from the extracellular environment and convert them into internal signals that affect various cellular functions. For example, human mesenchymal stem cells (hMSCs) cultured on topographical gratings have been shown to elongate and differentiate to different extents depending on grating width. Using a combination of experiments and mathematical modeling, the physical parameters of substrate topography that direct cell elongation were determined. On a variety of topographical gratings with different grating widths, heights and rigidity, elongation of hMSCs was measured and a monotonic increase was observed for grating aspect ratio (crosssectional height to line-width ratio) between 0.035 and 2. The elongation was also dependent on the grating substrate rigidity over a range of 0.18-1.43 MPa. A mathematical model was developed to explain our observations by relating cell elongation to the anisotropic deformation of the gratings and how this anisotropy depends on the aspect ratio and rigidity of the gratings. Our model was in good agreement with the experimental data for the range of grating aspect ratio and substrate rigidity studied. In addition, we also showed that the percentage of aligned cells, which had a strong linear correlation with elongation for slightly elongated cells, saturated toward 100 % at higher level of cell elongation. Our results may be useful in designing gratings to elicit specific cellular responses that may depend on the extent of cell elongation.