Stem cell mechanoadaptation Part B - Microtubule stabilization and substrate compliance effects on cytoskeletal remodeling

Stem cells adapt to their local mechanical environment by rearranging their cytoskeleton, which underpins the evolution of their shape and fate, as well as the emergence of tissue structure and function. Here we report on the second part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling and resulting changes in morphology and mechanical properties of cells, their nuclei, akin to mechanical testing of the most basic living and adapting unit of life, in situ in model tissue templates. We probed the native and PAX-exposed (inhibiting cytoskeleton tubulin depolymerization) stem cells cytoskeletal adaptation capacity on substrates of different compliance (exerting local tension on cells) and in combination with exposure to local compression effected with increased target seeding densities (5000 cells/cm2 - Low Density, LD; 15,000 cells/cm2, High Density, HD). On 10 and 100 kPa gels, cells seeded at both LD and cells proliferated to HD exhibited bulk moduli that nearly matched those of their respective substrates, hence exhibiting a greater increase in Youngs Modulus after microtubule stabilization than cells cultured on glass. Culture on compliant substrates also reduced the PAX-mediated F-actin and microtubule concentration increase. On gels, F-actin alignment decreased as more randomly oriented, short actin crosslinks were observed, representing emergent adaptation to the compliant substrate, mediated through myosin II contractility. We conclude that stem cell adaptation to compliant substrates facilitates the accommodation of larger loads from the PAX-stabilized polymerizing microtubule, which in turn exerts a larger effect in determining cells capacity to stiffen and remodel the cytoskeleton. Taken as a whole, these studies establish correlations between cytoskeleton and physical and mechanical parameters of stem cells that progress our understanding of the dynamic cytoskeleton, as well as shape changes in cells and their nuclei, culminating in emergent tissue development and healing. Significance StatementStem cells adapt to their dynamic environment by means of cytoskeleton rearrangements - underpinning the emergence of tissue structure-function relationship; this represents a current gap in knowledge that needs to be addressed, to better target tissue neogenesis and healing in context of regenerative medicine. We introduced compression via increasing seeding density and tension via compliant substrates to create tissue templates, while stabilizing microtubules. We found that mechanical and biophysical cues exert a greater effect in modulating cytoskeletal adaptation than exogenous chemical agents targeting the cytoskeleton, thus counterbalancing the concentration-dependent effect on cell physical and mechanical properties. We further found that stem cells with stabilized microtubules are sensitive to a range of substrate stiffness and seeding density that allowed cells or multicellular constructs to broaden their capacity to adapt their mechanical properties.