Inflammatory Responses and Barrier Function of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization. In this article, Orlova and colleagues show that hiPSC-ECs have similar features to primary ECs but also show some differences. hiPSC-ECs exhibited higher barrier function, lower expression of pro-inflammatory adhesive receptors, and more stringent stromal cell requirements. Importantly, healthy control CD31+ hiPSC-ECs showed high consistency between different batches and lines, forming a good basis for disease modeling applications

Quantitative Analysis of Intracellular Ca2+ Release and Contraction in hiPSC-Derived Vascular Smooth Muscle Cells

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 Image