High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells

Abstract

Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo^® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.

Keywords: angiogenesis; endothelial cells; high-throughput; iPSC-derived cells.

Fig. 1.

Assay breakdown of toxicity screening in iPSC-derived ECs and HUVECs. In this study, we present a multidimensional comparison of high-throughput in vitro assays for assessing the alterations in vascularization (angiogenesis assays in both Geltrex™ and SP-105 hydrogel) and cell viability (Hoechst Nuclei Content, Viable Cell Staining with Calcein AM and ATP content with CellTiter-Glo^®) caused by chemicals. EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; iPSC, induced pluripotent stem cell.

Fig. 2.

Representative images for HUVECs (A, B) and iPSC-derived ECs (C, D) grown on different extracellular matrices [Geltrex™ (A, C) and SP-105 hydrogel (B, D)] with treatment of vehicle (0.5% DMSO), 100 nM nocodazole, and 15 μM suramin from 16 to 24 h. DMSO, dimethyl sulfoxide.

Fig. 3.

Assessment of angiogenesis using HUVECs (A, B) and iPSC-derived ECs (C, D) on both Geltrex™ (A, C) and SP-105 hydrogel (B, D) extracellular matrices treated with angiogenic inhibitors. Concentration–response plots for ECs treated with suramin (red), SU5402 (blue), nocodazole (orange), and TAB (green). Concentration–response graphs shown were prepared in GraphPad using a normalized nonlinear fit. Data points in each concentration–response plot represent the average ± SEM of three separate experiments. SEM, standard error of the mean; TAB, tetraoctylammonium bromide.

Fig. 4.

Assessment of cytotoxic effects of select agents on HUVECs (A, B) and iPSC-derived ECs (C, D) after 24-h chemical treatment with (A, C) and without (B, D) cytokine exposure. Reproducibility of concentration–response plots for histamine (blue) and chloroquine (red) for the three cytotoxic endpoints each represented by the different shapes. Concentration–response graphs shown were prepared in GraphPad using a normalized nonlinear fit. Each line represents the mean ± SEM for a single experiment with three replicates.