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Fig. 1 | Fluids and Barriers of the CNS

Fig. 1

From: Commentary on human pluripotent stem cell-based blood–brain barrier models

Fig. 1

VE-Cadherin expression in H9 and H9-CDH5-eGFP hESC lines. a VE-cadherin immunocytochemistry of BMEC-like cells differentiated from the H9-CDH5-eGFP reporter using the RA-enhanced differentiation protocol [5] (UM-BMEC) and the chemically-defined, directed differentiation protocol [6] (D-BMEC). Scale bars: 100 μm. b Flow cytometry analysis of eGFP fluorescence in undifferentiated hESCs, UM-BMECs, D-BMECs, and generic hPSC-derived ECs [33] (Generic EC). Representative contour plots from biological triplicates showing eGFP expression and forward scatter (FSC) in the H9 hESC line (blue) and H9-CDH5-eGFP hESC line (red). Example gating strategy is shown in Additional file 1: Figure S1. c Quantification of eGFP geometric mean fluorescence intensity from flow cytometry analysis of biological triplicates of cells as described in b. Data are plotted as mean ± s.d. P-values: Student’s unpaired t-test. d Western blot for VE-cadherin, GFP, and β-actin expression in hESCs, UM-BMECs, D-BMECs, and generic ECs. Bands shown are from representative biological triplicates from the H9 hESC line (blue) and H9-CDH5-eGFP hESC line (red). Full western blots are shown in Additional file 1: Figure S2. Green arrows indicate the VE-cadherin-eGFP fusion protein bands. e Quantification of VE-cadherin-eGFP fusion protein abundance from anti-VE-cadherin Western blot analysis for samples as described in d. Data from three biological triplicates from a representative differentiation are shown. p-values: Student’s unpaired t-test. f eGFP fluorescence of undifferentiated H9-CDH5-eGFP hESCs and BMEC-like cells differentiated using the accelerated, chemically-defined serum free differentiation protocol [18] (A-BMEC). Scale bars: 100 μm

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