ETV2 mediated differentiation of human pluripotent stem cells results in functional endothelial cells for engineering advanced vascularized microphysiological models

Patient-specific microphysiological models, exemplified by organs-on-a-chip and organoids, have become a valuable tool for broad applications, revolutionizing biomedical research. However, limitations persist, with functional vasculature being a significant challenge. Generating functional human induced pluripotent stem cell (h-iPSC) derived endothelial cells (h-iECs) represent an urgent need. With the discovery of ETV2’s determinant role in specifying EC lineages during differentiation, researchers have adopted techniques involving ETV2 overexpression to produce h-iECs more efficiently and consistently. However, the capacity of these cells to form functional vasculatures has not yet been thoroughly investigated. Here, we generated multiple h-iPSC lines with inducible ETV2 expression, and subsequently differentiated them into h-iECs, which were validated functionally and by key endothelial markers and RNA-seq analysis. These cells are capable of self-organizing into stable microvascular networks (MVNs) in a microfluidic chip reproducibly, forming lumenized and functional vessels that mimic the in vivo capillary bed in both morphology and function – a result not achieved using h-iECs differentiated with conventional two-step methods using the same h-iPSC lines. Furthermore, complex microphysiological models featuring perfusable vasculature were also successfully developed using ETV2 activated h-iECs, demonstrated with vascularized tumor and blood-brain barrier (BBB) models. Additionally, by pooling genetically engineered h-iPSCs with inducible ETV2, we effectively employed an orthogonally induced differentiation approach to enhance vascularization of an organoid model. Our methodology opens avenues in precision medicine, leading to personalized microphysiological models with perfusable vasculature for various applications.