ASSIGNMENT STARTS ON 1 JULY
Unraveling Mechanotransduction in Single Cell Microgels
Unraveling the mechanotransduction mechanism of direct on-cell crosslinked hydrogels.
A cell’s behavior is intimately controlled through continuous interactions with its microenvironment. For example, (stem) cells sense microelasticity by deforming themselves and the extracellular matrix, and respond to these stimuli via mechanotransduction pathways that control migration, proliferation, apoptosis, metabolism, and differentiation.[1,2] To recapitulate these biological events in engineered tissues, biomaterials have typically been endowed with cell adhesive moieties that specifically bind to cell adhesion molecules. In particular, the integrin binding tripeptide arginine-glycine-aspartic acid (RGD) has been widely explored to this end. Other, less frequently explored strategies, have been based on modifying biomaterials with, amongst others, CAM binding antibodies or homing-CAM (CD44) binding hyaluronic acids. However, none of these strategies explored the covalent coupling of cells and extracellular matrix. We hypothesized that tethering a non-adhesive biomaterial directly onto cells using a cytocompatible enzymatic crosslinking approach would readily support cell/biomaterial mechanotransduction in a novel, unique, and RGD-independent manner. Our group has developed a tyramine-functionalized polymer that could be simultaneously (i) enzymatically crosslinked to form a hydrogel and (ii) enzymatically co-crosslinked with the tyrosines of the cellular membrane. We are currently investigating whether this ‘Direct On-cell CrosslinKing’ (DOCKING) technology would enable the mechanotransduction between cells and biomaterials in a novel RGD-independent manner. To this end, we leverage an advanced microfluidic system to encapsulate individual mesenchymal stem cells and study the mechanotransduction of the enzymatically crosslinked microgels to the stem cells with single cell resolution. Using cytocompatible enzymatic post-curing, the microgels can be stiffened on demand within a physiological relevant range (~5-50 kPa) to control stem cell lineage commitment. Using on-demand stiffening, we reveal that early-stage biomechanical stimuli are crucial to program long-term stem cell fate.
In this project, the student aims to identify the biological mechanism through which DOCKING regulates stem cell differentiation. Specifically, the student will perform multilineage differentiation experiments of single-cell microgels in the presence of various potent chemical inhibitors that act on, for example, the cell cytoskeleton or mechanosensitive ion channels.
- Produce cell-sized microgels using droplet microfluidics
- Encapsulate single stem cells in soft and stiff microgels using droplet microfluidics
- Differentiate single cell microgels towards the osteogenic and adipogenic lineage
- Perform biochemical blocking experiments to investigate DOCKING-mediated mechanotransduction
The student will learn several state-of-the art techniques, including droplet microfluidics, enzymatic hydrogel crosslinking, (3D) stem cell culture, immunohistochemistry, and confocal fluorescence imaging and apply this skill set in a top-level institute to (partially) unravel a yet unstudied cell mechanotransduction mechanism.
 Discher, D.E., et al., T. Science, 2005. 310(5751): p. 1139-43
 Huebsch, N., et al., Nat Mater, 2010. 9(6): p. 518-26
 Hersel, U., et al., Biomaterials, 2003. 24(24): p. 4385-415
 Rafat, M., et al., Biomaterials, 2012. 33(15): p. 3880-6
 Kim, Y. et al., Mol Cancer Res, 2014. 12(10): p. 1416-29
 Kamperman, T., PhD Thesis, University of Twente, 2018. p. 174.