| Summary: | Summary: Vascular smooth muscle cells (vSMCs) are highly heterogeneous across different vascular beds. This is partly dictated by their developmental origin but also their position in the vascular tree, reflected in their differential responses to vasoactive agonists depending on which arteriolar or venular segment they are located. Functional assays are necessary to capture this heterogeneity in vitro since there are no markers that distinguish subtypes. Here we describe methods for determining real-time intracellular Ca2+ release and contraction in vSMCs of neural crest origin differentiated from human induced pluripotent stem cells using multiple protocols, and compare these with primary human brain vascular pericytes and smooth muscle cells. Open-source software was adapted for automated high-density analysis of Ca2+-release kinetics and contraction by tracking individual cells. Simultaneous measurements on hundreds of cells revealed heterogeneity in responses to vasoconstrictors that would likely be overlooked using manual low-throughput assays or marker expression. : In this article, Orlova and colleagues describe methods for real-time intracellular Ca2+ release and contraction in vascular smooth muscle cells differentiated from human induced pluripotent stem cells. Open-source software adapted for automated high-density analysis and simultaneous measurements of hundreds of cells revealed heterogeneity in responses to vasoconstrictors that would likely be overlooked using standard low-throughput assays or marker expression. Keywords: human induced pluripotent stem cells (hiPSCs), vascular smooth muscle cells (vSMCs), neural crest-derived vascular smooth muscle cells (NC-SMCs), real-time intracellular Ca2+ release in vSMCs, microfluidics, contraction, cell tracking, automated image analysis, CellProfiler, LC_Pro plugin for ImageJ
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