(567b) Engineering/Controlling Microenvironments for Cardiovascular Differentiation of Human Embryonic Stem Cells

Numerous studies provide evidence that hydrodynamic shear stress can intricately regulate vascular function. During embryonic development shear stress has a morphogenetic function in cardiovascular development. Recent studies further demonstrate the molecular basis of the observed effects of shear stress on cardiovascular differentiation of mouse embryonic stem cells (ESCs) and the interaction with other culture factors (e.g. growth factors, serum concentration etc.). Controlled studies of the effects and mechanisms of hydrodynamic shear individually and interactively with other factors, are of particular interest for situations where hydrodynamic shear is implied in vivo, most notably for vascular differentiation of human ESCs. We previously demonstrated that vascular cells could be derived from hESCs under strict two-dimensional (2D) culture conditions.

Here we expand upon that work using a novel shear stress device developed in our laboratory. A unique feature of this device is that cells are cultured in a standard tissue culture treated 6-well plate, thereby avoiding any problems related to surface attachment. Exposing adherent vascular derivatives of hESCs at different developmental stages to shear (0 to 15 dyne/cm²) had a dramatic effect on derivation and maturation. Using this high-throughput system, embryonic derivatives were easily compared to various mature vascular cell arrangement and angiogenesis potential under shear stress conditions. Next, we considered the shear response of hESCs cultured in the three-dimensional environment (3D) of a hydrogel. A precise channel in the hydrogel was created by photocrosslinking (hyaluronic acid) or setting (collagen) the hydrogel around a rod in a specially designed chamber. Once the rod was removed, medium flow through the channel exposed cells to defined shear stress, the value of which was easily calculated from Hagen-Poiseuille relation. Both encapsulation of cells within the hydrogel, as well as seeding cells into already formed channel, were considered. Finally, a microchannel microfluidic device was designed and tested to study the response of individual cells to shear, electrical stimuli, and cytokine signaling.