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Figure 3 | Fluids and Barriers of the CNS

Figure 3

From: Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells

Figure 3

Comparison of BBB phenotypic characteristics between medium- and high-density cultures. All experiments were performed on purified IMR90-4-derived BMECs at day 10 of differentiation. a Immunostaining of PECAM, VE-cadherin, claudin-5, occludin, Glut-1, P-glycoprotein, MRP-1, and BCRP. DAPI overlay is included on P-glycoprotein and BCRP images. Scale bars 50 μm. b Flow cytometry histograms of VE-cadherin, claudin-5, occludin, P-glycoprotein, MRP-1, and BCRP. Appropriate mouse IgG control is included for each marker. The difference in expression as assessed by the geometric means of the labeled populations was not greater than 20% between medium- and high-density cultures for any marker in at least two independent differentiations. c Efflux transporter inhibition as measured via intracellular accumulation of either rhodamine 123 (i) or DCFDA (ii). Inhibitor-treated samples were independently normalized to each respective non-inhibitor-treated control sample. Statistical significance was calculated using the Student’s unpaired t test (***p ≤ 0.001, ****p ≤ 0.0001). Values are mean ± SD of three replicates from a single differentiation, and experiments were repeated for two additional independent differentiations for verification of reported trends.

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