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Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays
Citation:
Belair, D., M. Schwartz, T. Knudsen, AND W. Murphy. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays. Acta Biomaterialia. Elsevier B.V., Amsterdam, Netherlands, 39:12-24, (2016).
Impact/Purpose:
Blood vessel formation (angiogenesis) is a critical process in development and a target for environmental chemical disruption. This study is developing synthetic extracellular matrices to support in vitro angiogenesis assays with human endothelial cells, which will optimize the in vitro conditions for profiling environmental chemicals. It tests a subset of 38 toxCast chemicals for effects on the initial stage the angiogenic cycle involving sprouting of pre-existing vasculature in vitro.
Description:
Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds that inhibited iPSC-EC sprouting and five compounds that were overtly cytotoxic to iPSC-ECs. The chemically-defined iPSC-EC sprouting model (iSM) is thus amenable to enhanced-throughput screening of small molecular libraries for effects on angiogenic sprouting and iPSC-EC toxicity assessment.
