Journal: Scientific Reports

Article Title: A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis

doi: 10.1038/s41598-018-19522-8

Figure Lengend Snippet: Differentiation of AF-iPSCs into induced brain endothelial cells (i-BECs). ( A ) Schematic of AF- iPSC BEC directed differentiation process depicting the transitional stages of i-BEC monolayer differentiation. ( B ) Representative immunofluorescence depicting temporal GLUT1 expression during pre-differentiation and endothelial differentiation and specification in KOEB and EM medium, respectively. Days (d) in each differentiation medium are shown. ( C ) Flow cytometry histograms depicting the increase in mean fluorescence intensity of GLUT1 expression during different stages of differentiation (3D KOEB, blue; 3D EM, green and 10D EM, red). Grey histogram depicts unstained cells. ( D ) Quantitation of mean fluorescence intensity (arbitrary units, AU) of GLUT1 expression is shown during KOEB 3D and EM 10D differentiation timeframe (mean ± SD, n = 3). Results are representative of at least two independent differentiations. ( E ) Cropped gel electrophoresis of RT-PCR products for transcripts encoding Wnt-β-catenin signaling intermediates involved in BBB specification such as: FZD4 , FZD6 , FZD7 , FST and STRA6 in i-BECs and HBMECs. ( F ) Ubiquitous expression of SOX17, a Wnt-β-catenin downstream signaling target, in i-BECs and HBMECs. Nuclei counterstained with Hoechst. Scale bar, 20 µm.

Article Snippet: Cells were blocked with 3% BSA in PBS for 30 min at room temperature and incubated with Human Glut1 Alexa Fluor 700-conjugated Antibody (R&D Systems) at a dilution of 5 μl/1.0 × 10 6 cells in 800 μl of 1% BSA in PBS and 2 mM EDTA for 1 hr.

Techniques: Immunofluorescence, Expressing, Flow Cytometry, Fluorescence, Quantitation Assay, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis

doi: 10.1038/s41598-018-19522-8

Figure Lengend Snippet: BBB and endothelial specific gene expression in i-BECs. ( A ) A comparative gene microarray analysis comparing the individual gene intensity signals (arbitrary units) of 434- BBB and endothelium-specific genes between HBMEC (y-axis) and i-BECs (x-axis). Scatter plot demonstrates a similar expression profile between both cell types, specifically in ABC transporters (green squares), tight junctions (purple triangles) and SLC transporters (red diamonds) transcripts (Pearson Correlation, r 2 = 0.86, p = 0.001). ( B ) Table compiling a list of differentially expressed transcripts in i-BECs when compared to HBMECs. Statistical significant was determined using Student’s T-Test, (* p ≤ 0.05) and a 1.35 fold change in ratio was used to generate the gene list. ( C ) Representative immunofluorescence staining of i-BECs for vWF, Claudin-5, CD31 (PECAM-1), Occludin, ZO-1, GLUT1 and P-gp. HBMECs were used as a positive control. All characterizations were carried out at day 21 of EM differentiation. Nuclei counterstained with Hoechst. Scale bar, 20 µm.

Article Snippet: Cells were blocked with 3% BSA in PBS for 30 min at room temperature and incubated with Human Glut1 Alexa Fluor 700-conjugated Antibody (R&D Systems) at a dilution of 5 μl/1.0 × 10 6 cells in 800 μl of 1% BSA in PBS and 2 mM EDTA for 1 hr.

Techniques: Gene Expression, Microarray, Expressing, Immunofluorescence, Staining, Positive Control