Figure 1

Effect of day 0 hPSC density on differentiation efficiency and yield of brain microvascular endothelial cells. a Schematic representation of BMEC differentiation protocol. hPSCs were seeded as singularized cells at day −3 and induced to differentiate at day 0. Cells were differentiated for 6 days in UM followed by 2 days in EC media (10 μM RA treatment was included for all reported data unless otherwise noted) and subcultured onto collagen/fibronectin matrix. UM unconditioned medium, EC endothelial cell, RA retinoic acid, C/F collagen/fibronectin. b Brightfield images of IMR90-4 BMEC differentiation. Recorded densities are the day 0 hPSC starting densities. i Brightfield images of IMR90-4 iPSCs at day 0, following 3 days of expansion in pluripotency media. Scale bars 500 μm. ii Brightfield images after 8 days differentiation. Scale bars 500 μm. iii Unmagnified images of differentiated cells in 6-well tissue-culture plate following 8 days of differentiation. Scale bars 5 mm. c i and ii Expression of VE-cadherin (red) and nestin (green) in IMR90-4 cells differentiated for 8 days. iii Higher magnification images of VE-cadherin+ EC colonies. Scale bars: i and ii 500 μm, iii 100 μm. d Quantification of VE-cadherin and nestin expression in IMR90-4 iPSCs. i Quantification via flow cytometry of percent VE-cadherin+ and nestin+ following 8 days of differentiation. VE-cadherin and nestin flow cytometry were compared to appropriate mouse IgG control antibody. Values are mean ± SD of three independent differentiations. Statistical significance was calculated via Student’s unpaired t test (*p < 0.05). ii Representative flow cytometry dot plots from medium density culture. Gates represent expression above mouse IgG control. e Quantification of VE-cadherin yield per input hPSC as a function of the day 0 hPSC starting density. IMR90-4 iPSCs were differentiated for 8 days, and the yield was calculated as the % VE-cadherin+ as measured by flow cytometry multiplied by total cell number at 8 days and normalized to the day 0 hPSC starting density. Values are the mean ± SD of two biological replicates from a single differentiation. The experiment was repeated for two independent differentiations to verify reported trends.