Product Manager, MEA Systems Axion BioSystems Atlanta, Georgia, United States
Abstract: Induced pluripotent stem cells (iPSCs) are widely used in studies of embryonic development, disease modelling, and tissue engineering. A promising application for iPSCs is the generation of endothelial cells, critical for forming blood vessels within larger tissue constructs to ensure oxygen and nutrient delivery. Effective differentiation of iPSCs into mature, functional endothelial cells presents several challenges, requiring monitoring of the differentiation process and recapitulating complex vascular networks. This study presents a novel non-invasive workflow for analysis and functional testing of iPSC-derived endothelial cells (id-ECs). First, iPSCs (StemCell Technologies) were differentiated into endothelial cells using the suppliers’ 14-day protocol. Morphological changes (e.g. size and shape) were monitored using the Omni live-cell imager. Endothelial differentiation was verified via fluorescence staining of the endothelial marker CD31 and the absence of stem cell markers SSEA-4 and TRA-1-60. Functional assessment of id-ECs was performed to validate important endothelial behavior like migration (scratch assay), tubule formation and barrier integrity ( transendothelial electrical resistance (TEER)). The actin polymerization inhibitor Cytochalasin D (CytoD) was added at various concentrations to validate functionality. Brightfield imaging of the scratch assay using the Omni showed 100% wound closure by id-ECs within 24 hours. Scratch closure was reduced 59% and 86% by 500 nM and 1000 nM CytoD, respectively. Interconnected tubular networks, characterized by long tubules and large mesh sizes were formed in the tubule formation assay. The addition of CytoD disrupted this in a dose-dependent manner, reducing tubule length and mesh integrity from CytoD concentrations as low as 50 nM. TEER measurements with the Maestro Z revealed that CytoD reduced barrier integrity, with barrier index values (TEER normalized to confluency) dropping by 34% (500 nM) and 69% (1000 nM), correlating with tight junction disruption. This non-invasive workflow combines live-cell imaging and real-time impedance measurements for efficient monitoring and functional validation of id-ECs, supporting advancements in vascularized tissue engineering and iPSC-based regenerative medicine.
