Year 4 (NCE)
In this grant we found that the softer the surface upon which human embryonic stem cells (hESc) were grown the greater the efficiency of differentiation to mesoderm, from which arises heart and skeletal muscle among others. This was due in part to the indirect permissive effect of soft surfaces in the resulting (cyto)architecture hESc and in part to as-yet-incompletely-understood mechanisms. Soft surfaces permitted hESc to reform cell-to-cell contacts called Adherens Junctions (AJ) , consisting of the proteins E-cadherin, β-catenin and p120-catenin, more efficiently. During differentiation this results in more β-catenin (activation of mesoderm differentiation) and more p120-catenin (derepression of mesoderm differentiation) that drive the process. We also found that wnt proteins synthesized and secreted by hESc in are required and more are made (and less wnt inhibitors) by hESc on softer surfaces. The combined effect is robust mesoderm differentiation. In related work still in progress, we explored the effect of cell mechanical properties on mesodermal differentiation of hESc adhered to soft surfaces in mm scale colonies. These display patterned development of mesoderm at the colony margins, which we visualized microscopically using genome editing technologies to tag the early mesoderm marker T. We also measured the forces exerted by these colonies and showed that the margins are the sites of the highest exerted cell-generated forces suggesting that they might predispose cells that in these domains to mesoderm differentiation. To this end we patterned hESc colonies in arbitrary shapes and showed that these forces can be manipulated at the margins: for ex., high at angular vertices and lower on straight edges. While this work continues and challenges remain there appears to be a predisposition of high traction domains on the margins of these shapes to subsequently differentiate into mesoderm.