(726e) Wnt5a Conjugated Poly(ethylene glycol) - Gelatin Composite for Vascularized Tissue Engineering

Embryonic stem cell (ESC) differentiation holds enormous potential for generating a renewable source of cells for regenerative medicine. However, directed in vitro differentiation of ESC has proven to be a challenging task.  In early embryonic development, a tightly controlled series of cell-cell, cell-soluble factor and cell-matrix interactions create a microenvironment that provides a coordinated set of cues to developing cells. Wnt signaling molecules are among the most important cues that control key developmental events.  Recently we have used microscale technologies to demonstrate the role of noncanonical Wnt11 in cardiogenesis and Wnt5a in directing endothelial cell differentiation of ESCs. Here, we propose to fabricate Wnt5a conjugated poly(ethylene glycol) vinyl sulfone (PEGVS)-gelatin composite to engineer vascular tissues by controlling ESCs differentiation. We fabricated photo cross-linked PEGVS-gelatin hydrogels with tunable mechanical and degradation properties by controlling the amount of gelatin. We then conjugated Wnt5A protein to 4-arm PEGVS via Michael-type addition utilizing the cysteine groups of the Wnt5a protein and analyzed the conjugation using matrix-assisted laser desorption/ionization (MALDI). The results indicated that one or two Wnt5a molecules were conjugated on the PEGVS arms. We further confirmed the conjugation by labeling the Wnt5a with a florescent molecule and observing the release kinetics. The biofunctionality of the Wnt5a conjugated hydrogels was evaluated by monitoring protein and gene expression of the encapsulated EBs. The immunocytochemistry results indicated that conjugation of Wnt5a to copolymer network results in higher expression of CD31 and higher sprouting compared to the control (without Wnt5a). Additionally, it was observed that CD31 was evenly distributed at the cell-cell junction and the expression pattern was similar to the fully mature endothelial cells. Furthermore, the endothelial differentiated of EBs was determined by monitoring the gene expression using quantitative polymerase chain reaction. The results indicated that conjugation of Wnt5a to PEGVS-Gelatin hydrogels up-regulated Wnt5a and CD31 gene expression compared to the control. Overall, we were able to successfully design Wnt5a conjugated PEGVS-gelatin hydrogels and able to modulate ESC signaling microenvironment based on noncanonical Wnt5a pathway for potential application in vascularized tissue engineering.