Poster V1 - Building Predictive Models of Stem Cell Fate From Curvature-Induced Cytoskeletal Changes

Introduction: Human mesenchymal stem cells (hMSC) are sensitive to subcellular surface topographical features, such as nanofiber curvature, which cause changes to cell morphology and behavior. These responses to surface architecture result from signaling induced by mechanosensitive complexes, such as focal adhesions, adherens junctions, and the cell cortex, in contact with the surface and other cells. Control over downstream signaling from these centers of mechanical sensing allow for potential control over cell fate. However, the complexes responsible for sensing and communicating the stimuli that trigger these cascades are elaborate and highly dynamic, features which make experimental characterization of complexes themselves extremely difficult. However, it is beneficial to identify how surface features affect traits of each of these mechanosensitive complexes (i.e. focal adhesion size), as these traits can be easily determined through straightforward assays and also may hold predictive power when combined with other traits to create hMSC response profiles to surface features. To determine what aspects of cell mechanical state have predictive power for cell fate, we first characterized cell mechanical state through three mechanosensitive complexes (cell cortex, focal adhesions, adherens junctions) on an array of nanotopographies with unique curvatures. We then investigated which mesenchymal lineages were promoted on each curvature. With these two large datasets, we can perform a wide variety of data analyses including determining corelated cell responses, identifying traits of significance, and building predictive models.

Materials and

Methods: Glass coverslips coated with poly(2-hydroxyethyl methacrylate) and electrospun poly(methyl methacrylate) (PMMA) topographies from nanoislands to nanofibers and PMMA-coated glass coverslips (control) were seeded with HMSCs. Cells were lysed at 24 hours, 7- and 14-days. Relative amounts of cytoskeleton proteins actin, tubulin, and α-actinin and mechanical proteins vinculin, cadherin, and ezrin were measured by western blotting.  Images of membranes were analyzed in ImageJ. In addition, cells were immunostained at the same timepoints for the aforementioned proteins plus the nucleoskeletal protein lamin A/C.  CellProfiler was used to extract parameters such as size, texture, colocalization, etc. from images. Protein abundance and image-extracted parameters are used to describe cell mechanical state and comprise inputs for a classical model.

Results, Conclusions, and Discussions: hMSCs exhibited spread, spindle-like morphology, developing increasing aspect ratios as fiber diameter increases. Focal adhesions (marked by vinculin) appear at 24 hours, whereas adherens junctions (marked by cadherin) do not form until 7 days.  However, it is interesting to note that cadherin is visible at 24 hours as diffuse staining in the cytoplasm and nucleus. This diffuse staining is present even in cells treated with a buffer used to remove soluble cytoplasmic and nuclear proteins, which indicates that this cadherin is strongly bound to some structure inside of the cell before it arrives at adherens junctions, although this structure is unknown. Vinculin abundance increases with proliferation, as does ezrin abundance.  Ezrin also appears to be mechanically engaged in the cortex across the entire cell on most surfaces. Details regarding the mechanical state on each surface curvature will be described in more detail as more data processing occurs. At present, we have evidence which aligns with previous data from our lab that cells are sensitive to changes in curvature.

Acknowledgements (Optional): I would like to thank my PI Dr. Justin Brown for all of his help in developing and conducting my PhD thesis work in this abstract.