Human iPSC-derived endothelial cell responses to fluid flow-induced shear stress: arterial-venous identity and comparisons to primary endothelial cell types

Soos, Agnes 1, 2;  Fitzsimmons, Ross E. 1, 2; Santerre, J Paul 1, 2, 3; Simmons, Craig A. 1, 2, 4

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 
2. Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research; 
3. Faculty of Dentistry, University of Toronto; 
4. Department of Mechanical and Industrial Engineering, University of Toronto

Human induced pluripotent stem cells (iPSC) are a promising source of endothelial cells (EC) for tissue engineering and regenerative medicine. While iPSC-EC have demonstrated functional capabilities analogous to mature EC in static culture, they remain largely unstudied in physiological conditions involving fluid flow. Hence, we evaluated the response of iPSC-EC (iCell-EC; Cellular Dynamics International) to extended shear conditioning. Comparisons were made to human primary EC types sourced from umbilical veins (HUVEC) and iliac arteries (HIAEC).

Cultures were grown to confluence on fibronectin-coated glass slides and conditioned with 15 dynes/cm2 shear stress for 48 hours using a closed-loop parallel plate flow setup. iPSC-EC morphology was similar to primary EC, with typical cobblestone appearance in static culture and elongation and alignment in the direction of fluid flow. Immunofluorescent staining was used to evaluate expression of key endothelial markers; in both static and sheared cultures, CD31 and VE-cadherin expression was similar for all EC types, however, vWF expression was significantly lower in iPSC-EC than in mature EC (p<0.01).

Shear stress was also found to impact the arterial-venous (A-V) specification of iPSC-EC. Shear promoted venous specification with a 1.7±0.18-fold increase in Coup-TFII expression (p<0.001) and downregulation of key arterial genes (EfnB2: 0.64±0.14; Hey1: 0.39±0.39; Dll4: 0.27±0.09 ; p<0.05 for all) relative to static controls. A similar response was observed in sheared HUVEC with a 2.3±0.26-fold increase in venous Coup-TFII (p<0.05), whereas HIAEC favoured arterial specification, exhibiting a 3.9±0.65-fold increase in EfnB2 (p<0.001) and no significant change in Coup-TFII levels. All measures were compared against un-sheared, static cultures.

To assess angiogenic sprouting capabilities, cells were seeded on microcarrier beads and embedded in hydrogels. By day 7, iPSC-EC had significantly more sprouts per bead compared to HUVEC and HIAEC (p<0.005 for both), and longer sprout lengths compared to HIAEC (p<0.0001). 

Currently, we are examining the mechanisms behind the A-V shear response (to better understand A-V differentiation processes) and completing comparisons to microvascular EC (to evaluate micro- versus macro-vascular features of these cells). These will enable us to better situate iPSC-EC in the spectrum of endothelial heterogeneity and will inform potential applications of these cells for tissue engineering.