Integrins and forces in mesenchymal stem cell differentiation

Cells sense and respond to their microenvironment by integrating physical and biochemical cues to regulate cell function. Adhesion of cells to extracellular matrix (ECM) impacts diverse physiological processes, including cell cycle, migration, differentiation, and contractility. Cell-ECM adhesion is chiefly mediated by transmembrane integrin receptors, which are implicated in mechanosensing and bidirectional transmission of mechanical force. Reducing integrin-mediated adhesion affects cellular geometry and influences both stem cell fate and cytoskeletal tension. Similar effects on cell-ECM adhesion and cell function have been observed in cells attached to ECM on soft substrates or elastic microposts. It is unclear, however, whether integrin activation is sufficient to drive these cell behaviors. In this thesis, I have examined the role of mechanoadhesive signals in stem cell fate as well as the specific role of integrin activation in the generation of cell traction forces. For the first project, I hypothesized that mesenchymal stem cells (MSCs) from different tissues would exhibit the same behavioral responses to mechanoadhesive stimuli. Utilizing MSCs derived from either human bone marrow or periosteum, I analyzed the effects of varying cell seeding density, micropatterned cell shape, and cytoskeletal tension on MSC proliferation and differentiation in vitro. This study demonstrated the dependence of MSC differentiation on the investigated biophysical parameters with subtle cell-type differences in differentiation efficiency. The first project illustrated the need to understand the complex interplay of adhesion, cell shape, and cytoskeletal tension. Thus for the second project, I hypothesized that integrin activation would directly impact cytoskeletal tension and the associated cell traction forces on ECM. To measure these cell generated forces, I fabricated dense arrays of elastomeric microposts to serve as substrates on which cells could adhere and produce forces that deflect the underlying microposts. I found that activation of β1 integrin by either increased fibronectin density or manganese promotes fibroblast contractility in addition to cell spreading and focal adhesion growth. Additionally, this force generation is mediated by Rho kinase and myosin activity but not β3 integrin. This newly described regulation of cytoskeletal tension by integrin activation suggests a more complex link between integrins, forces, and cellular function than previously appreciated