Abstract
SummaryVascular 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.
Highlights
Vascular smooth muscle cell dysfunction is associated with many diseases ranging from atherosclerosis and hypertension to cerebrovascular disorders (Owens et al, 2004; Sinha and Santoro, 2018)
We developed functional assays and an automated quantification framework for intracellular Ca2+ release and contraction in Vascular smooth muscle cells (vSMCs). vSMCs were differentiated via neural crest intermediates from three independent healthy Human induced pluripotent stem cells (hiPSCs) lines, using various protocols based on previously published methods (Cheung et al, 2012; Dash et al, 2016; Granata et al, 2017; Wanjare et al, 2013, 2014)
Quantitative Assessment of Contraction in NCCs into vSMCs (NC-SMCs) and Primary vSMCs To assess vSMC contraction, we developed an automated pipeline that included primary object identification and object tracking based on pixel intensity (Figure 4A and Video S2)
Summary
Vascular smooth muscle cell (vSMC) dysfunction is associated with many diseases ranging from atherosclerosis and hypertension to cerebrovascular disorders (Owens et al, 2004; Sinha and Santoro, 2018). Human induced pluripotent stem cells (hiPSCs) have been shown to be an excellent source of vSMCs of various developmental origins (Cheung et al, 2014, 2012; Granata et al, 2017), presenting new opportunities for disease modeling and drug discovery using patient-specific cells. VSMCs in situ exhibit rapid intracellular Ca2+ release in response to vasoconstrictors. Cultured vSMCs exhibit profound heterogeneity in their responses to vasoconstrictors. Intracellular Ca2+ release is typically measured as a low-throughput assessment of selected regions of interest that may not represent the whole cell population and its intrinsic heterogeneity. High-throughput, robust, and standardized assays that accurately assess vSMC functionality would be of value in monitoring drug responses and disease phenotypes
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