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.

Cardiovascular Effects of Polychlorinated Biphenyls and Their Major Metabolites

“…The culture conditions of iCell ® -endothelial cells were as reported elsewhere (Iwata et al 2017). Briefly, T75 flasks were coated with a fibronectin solution (3 lg=cm 2 ) followed by incubation for 1-2 h. Cells were thawed for 3 min in a 37°C water bath.…”

“…Thawed cells were added to maintenance medium made of VascuLife VEGF Medium Complete Kit, excluding the supplied FBS, and iCell ® Endothelial Cells Medium Supplement. The final formulation of the maintenance medium is detailed in (Iwata et al 2017). The aliquoted fibronectin solution was aspirated from the T75 flasks, and cells were seeded at 1:0 × 10 4 cells=cm 2 .…”

“…Similar to the cultivation of iCell ® -endothelial cells, HUVEC maintenance and experimentation are detailed elsewhere (Iwata et al 2017). Briefly, HUVECs were thawed, seeded, and grown in T75 tissue culture flasks in Medium 199 with EGM™-2 BulletKits™.…”

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

“…This has limited their utility in disease modeling and drug discovery, particularly where isogenic controls for patient lines are not available since it may be difficult to distinguish line-to-line “noise” from true, disease-related phenotypes. Furthermore, widely available human umbilical vein ECs (HUVECs) are often used in preference to hiPSC-ECs in bioassays since they are perceived as more robust, but functional comparisons are rarely made ( Iwata et al., 2017 ). Exceptionally, we showed that the ability of HUVECs to integrate into the developing vasculature (in zebrafish) is inferior to that of hiPSC-ECs ( Orlova et al., 2014a ).…”

High Yield of Adult Oligodendrocyte Lineage Cells Obtained from Meningeal Biopsy

“…The development of a successful protocol for OPC/oligodendrocyte lineage cell culture of adult origin could provide a useful tool for the in vitro screening and testing of drugs able to influence the biology and remyelinating potential of OPCs ( Allen et al, 2005 ; Soldatow et al, 2013 ; Kerman et al, 2015 ). Different strategies are now under investigation for in vitro drug screening, as the use of primary cultures, including OPCs ( Merrill, 2008 ; Gonzalez et al, 2016 ; Lariosa-Willingham et al, 2016 ), iPSCs ( Iwata et al, 2017 ; Rana et al, 2017 ), organoids ( Vrij et al, 2016 ; Pino et al, 2017 ), spheroids ( Sarkar et al, 2017 ; Sirenko et al, 2017 ), and bioprinted 3D tissues ( Chang et al, 2010 ; Massa et al, 2017 ). However, none of these methods is able to provide adult patient-specific oligodendrocytes without major in vitro transformation.…”