SummarySeveral 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.

Summary Chromosomal translocations are prevalent among soft tissue tumors, including those of the vasculature such as pseudomyogenic hemangioendothelioma (PHE). PHE shows endothelial cell (EC) features and has a tumor-specific t(7;19)(q22;q13) SERPINE1-FOSB translocation, but is difficult to study as no primary tumor cell lines have yet been derived. Here, we engineer the PHE chromosomal translocation into human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 and differentiate these into ECs (hiPSC-ECs) to address this. Comparison of parental with PHE hiPSC-ECs shows (1) elevated expression of FOSB, (2) higher proliferation and more tube formation but lower endothelial barrier function, (3) invasive growth and abnormal vessel formation in mice after transplantation, and (4) specific transcriptome alterations reflecting PHE and indicating PI3K-Akt and MAPK signaling pathways as possible therapeutic targets. The modified hiPSC-ECs thus recapitulate functional features of PHE and demonstrate how these translocation models can be used to understand tumorigenic mechanisms and identify therapeutic targets.

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Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow. Video Link The video component of this article can be found at https://www.jove.com/video/58678/ This protocol is useful for the assessment of inflammatory responses in hiPSC-ECs in drug screening, disease modeling and personalized regenerative medicine.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.