Determination of critical shear stress for maturation of human pluripotent stem cell derived endothelial cells towards an arterial subtype.

Human pluripotent stem cell derived endothelial cells (hPSC-EC) present an attractive alternative to primary EC sources for vascular grafting but there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arterio-venous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses is still not well understood. Here, we employed a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of 6 parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4-15 dyne/cm2 . We found that hPSC-ECs required up to 40 hours of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications. This article is protected by copyright. All rights reserved.