The developmental potential of pluripotent stem cells is influenced by their local cellular microenvironment. To better understand the role of vascular endothelial growth factor (VEGFA) in the embryonic cellular microenvironment, we synthesized an artificial stem cell niche wherein VEGFA was immobilized in an agarose hydrogel. Agarose was first modified with coumarin-protected thiols. Upon exposure to ultra-violet excitation, the coumarin groups were cleaved leaving reactive thiols to couple with maleimide-activated VEGFA. Mouse embryonic stem cells (ESC) aggregates were encapsulated in VEGFA immobilized agarose and cultured for 7 days as free-floating aggregates under serum-free conditions. Encapsulated aggregates were assessed for their capacity to give rise to blood progenitor cells. In the presence of bone morphogenetic protein-4 (BMP-4), cells exposed to immobilized VEGFA upregulated mesodermal markers, brachyury and VEGF receptor 2 (T+VEGFR2+) by day 4, and expressed CD34 and CD41 (CD34+CD41+) on day 7. It was found that immobilized VEGFA treatment was more efficient at inducing blood progenitors (including colony forming cells) on a per molecule basis than soluble VEGFA. This work demonstrates the use of functionalized hydrogels to guide encapsulated ESCs toward blood progenitor cells and introduces a tool capable of recapitulating aspects of the embryonic microenvironment.
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