Introduction: Pluripotent stem cells can differentiate into all cell types of the human body and therefore hold great promise for regenerative medicine. Interestingly, human pluripotent stem cells (hPSCs) have a unique adhesive signature that accounts for their extremely low and selective adhesion properties compared to differentiated cells. In addition, recent studies have demonstrated the importance of ECM stiffness and Rho-ROCK-myosin signalling in stem cell biology. These observations highlight the role of adhesion, contraction and integrin signalling in the maintenance of pluripotency. However, the organization, composition and significance of focal adhesions, the cell’s mechanotransducing units, and their actin linkage have remained unstudied in hPSC colonies.
Our research: We aim to characterize in detail the structure, distribution and function of focal adhesions and their connection to the actin cytoskeleton in human induced pluripotent colonies (hiPSCs). Furthermore, we study how the cytoskeletal network mediates traction stress and influences adhesion and colony morphology in pluripotency and differentiation. In order to achieve our goals we study hiPSC colonies with high resolution imaging techniques such as total fluorescence microscopy (TIRF), Interferometric photoactivation and localization microscopy (iPALM) (Janelia Research Campus), stimulated emission depletion (STED) microscopy and traction force microscopy (TFM).
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Prominent focal adhesions and actin fence direct sharp-edge colony morphology of human pluripotent stem cell colonies. Left panel: Phase contrast of two human induced pluripotent stem cell colonies plated on vitronectin. Right panel: Total internal reflection fluorescence microscopy (TIRF) image stained with focal adhesion marker paxillin and F-actin. DAPI in mid plane shows number of cells in the colony.