Human embryonic stem cells (hEScs) are derived from embryos early in development before their fate has been determined, and can differentiate into all of the cell types of the body. By exploiting the potential of hESCs to differentiate into multiple lineages medicine stands to benefit enormously. To do so requires a comprehensive understanding of the optimal conditions to grow and differentiate these cells without inducing tumors. What is clear is that the physical, three dimensional (3D) microenvironment in which the hESCs reside, regulates directly or indirectly their tissue-specific differentiation. hESc make physical contact with both an insoluble extracellular scaffold termed the extracellular matrix (ECM) and to other cells through specific proteins (receptors) on their own surface. These interactions are responsible for the structural integrity of the body and are at the center of structural transformations that characterize embryogenesis. While this structural role has long been appreciated it has only recently been shown that these points of contact can be the focus of transmission of mechanical forces originating within and outside the cell and that these forces can be converted into the more familiar signals that influence cell fate decisions. A major goal of our work is to define how these cell derived and externally transmitted forces might regulate hESC behavior. Our hypothesis is that these mechanical forces alter hESC fate by regulating the activity of enzymes called RhoGTPases, that are strongly implicated in ESC behavior. During this past year we have optimized the preparation and culture of hESc on a two dimensional (2D) synthetic matrices of defined composition and stiffness that recapitulate the range of mechanical environments hESC experience during embryogenesis as well as those unnaturally stiff mechanical environments that are currently used for their propagation. For these studies we used inert acrylamide gels cross linked with an ECM molecule (laminin) important in early embryonic development. On 2D surfaces feedback loops originating from cell derived contractile forces sense the mechanical “give” of the surface, and dynamically change their organizational and signaling state both at the single cell level and the multicellular level. Using these 2D gels we have defined the range of mechanical environments in which hESc exhibit mechanosensitivity. We have also shown that this causes changes in their external and internal organizational states across a range of scales from the subcellular (becoming stiffer on stiffer substrates), to the cellular (spreading more on stiffer substrates) to the multicellular (compacting under enhanced cell-cell adhesion on softer substrates). Surprisingly, we have found that the standard (very stiff) substrates that are used routinely for maintenance of a non-differentiated state (pluripotent self renewal) are mechanically suboptimal: hESc have higher rates of cell death and lower rates of growth than on surfaces orders of magnitude softer (operationally termed mid range). At the softest end, although we have found that hESc largely maintain pluripotential self renewal they show signs of either low level differentiation or a move towards a state poised to differentiate. This suggests that precise control of the mechanical environment is an important parameter in the establishment of safe and effective propagation of hESc for regenerative medicine and might be exploited for directed differentiation. To complement these studies we are also developing approaches to imparting external mechanical forces to hESc growing in a 3D context. We have both established novel and robust protocols for the efficient encapsulation of hESc in 3D deformable hyaluronic acid (HA) hydrogels and shown that they support pluripotent self renewal and constructed a bioreactor that will impart oscillatory and static compressive loads to hESCs in these gels. We anticipate that these studies will further illustrate the role(s) by which mechanical forces influence hESC fate and provide additional insight into the underlying molecular mechanisms.