Journal: Fluids and barriers of the CNS

Article Title: Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

doi: 10.1186/s12987-023-00437-0

Figure Lengend Snippet: Fig. 1 Schematic detailing mESC-D3 differentiation strategy to generate brain-endothelial-like cells and transwell BBB model in vitro. Schematic diagram of mESC-D3 directed monolayer differentiation protocol via initial mesodermal induction and subsequent endothelial cell differentiation and maturation. Representative phase contrast images illustrating morphological changes accompanying the various stages (Days) of differentiation

Article Snippet: To induce endothelial differentiation and maturation, the medium was switched to complete Endothelial Media (EM): Mouse Brain Endothelial Cell Culture Serum Free Media (Celprogen, Torrance, California), 5% Fetal Bovine Serum (Hyclone, Logan, Utah), 5 ng/ ml bFGF (Thermo Fisher Scientific, Waltham, Massachusetts) and 10 mM all-trans Retinoic Acid (RA; Sigma-Aldrich, St. Louis, Missouri) on day 6.

Techniques: In Vitro, Cell Differentiation

Journal: Fluids and barriers of the CNS

Article Title: Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

doi: 10.1186/s12987-023-00437-0

Figure Lengend Snippet: Fig. 2 Differentiation of mESC-D3 into mouse brain endothelial like-cells (mBECs). Flow cytometry analysis assessing transitional differentiation stages of endothelial differentiation. A Brachyury expression was assessed after 24 h of mesodermal induction with 5 ng/ml BMP4 and 30 ng/ ml VEGF and B endothelial progenitor Flk1 expression was assessed after 5 days in endothelial differentiation media. C–F GLUT1, CD31, VE-Cadherin and CLAUDIN 5 expression was assessed at the end of the differentiation period (Day 8). Red = unstained controls. G Temporal GLUT1 expression was assessed during the entire differentiation period. Increasing GLUT1 expression is shown with MFI (inset) at Day 1, 3, 5 and 7. The terminally differentiated mBECs exhibit cobblestone monolayer morphology and stained positive for key H endothelial (CD31, green) and (I–K) BBB-specific tight junction proteins (Claudin 5, Occludin and ZO-1; all green). Hoechst counterstain (blue); Scale bar = 20 µm. L In the presence of VEGF, the mBEC formed vascular–like structures in Matrigel assay within 24 h. Green = CFDA staining; Scale bar = 100 µm. M Following stimulation with inflammatory cytokines, the mBECs expressed immune adhesion molecule VCAM-1. Blue = unstained, Purple = non-stimulated; Orange = TNF-α and INF-γ stimulated. Representative images shown of 5 independent differentiations

Article Snippet: To induce endothelial differentiation and maturation, the medium was switched to complete Endothelial Media (EM): Mouse Brain Endothelial Cell Culture Serum Free Media (Celprogen, Torrance, California), 5% Fetal Bovine Serum (Hyclone, Logan, Utah), 5 ng/ ml bFGF (Thermo Fisher Scientific, Waltham, Massachusetts) and 10 mM all-trans Retinoic Acid (RA; Sigma-Aldrich, St. Louis, Missouri) on day 6.

Techniques: Flow Cytometry, Expressing, Staining, Matrigel Assay

Journal: Fluids and barriers of the CNS

Article Title: Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

doi: 10.1186/s12987-023-00437-0

Figure Lengend Snippet: Fig. 4 Comparative RNASeq analysis of genes expression profiles in mBEC, bEnd.3 and pmBECs. Heatmaps depicting log2 transformed transcript abundances of A endothelial, B Wnt signaling, C tight junction D BBB receptor/transporters gene expression profiles in mBEC, immortalized bEnd.3 and primary mouse brain endothelial cells (pmBECs). Green = low expression; Red = high expression. Results are from three independent differentiations

Article Snippet: To induce endothelial differentiation and maturation, the medium was switched to complete Endothelial Media (EM): Mouse Brain Endothelial Cell Culture Serum Free Media (Celprogen, Torrance, California), 5% Fetal Bovine Serum (Hyclone, Logan, Utah), 5 ng/ ml bFGF (Thermo Fisher Scientific, Waltham, Massachusetts) and 10 mM all-trans Retinoic Acid (RA; Sigma-Aldrich, St. Louis, Missouri) on day 6.

Techniques: Expressing, Transformation Assay, Gene Expression

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Schematic of the proposed mechanism by which interleukin (IL)-18 amplifies macrophage (Mφ) M2 polarization and its morphological alteration, leading to excessive angiogenesis. IL-18 amplifies IL-10-induced increases in the production of osteopontin (OPN) and thrombin as soluble mediators derived from Mφ, yielding the generation of thrombin-cleaved form of OPN (Thr-OPN). Subsequently, Thr-OPN binds to integrins α4/α9 receptors on Mφ, which in turn augments M2 polarization of Mφ with higher expression of CD163 and its morphological alteration. Furthermore, CD163 may be responsible for mediating the direct cell–cell interaction between these Mφs and endothelial cells, ultimately resulting in the excessive angiogenesis.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: Derivative Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Characteristic behavior of macrophages (Mφs) during angiogenesis. (A) Representative series of time-lapse images at 4 h intervals from 0 to 16 h extracted from Video S1 which shows live-cell imaging of Matrigel tube formation assay where endothelial cells (green) and Mφs [interleukin (IL)-10 + IL-18] (red) were cocultured. Scale bar represents 50 µm. (B) Higher magnification images of white rectangle region in panel (A) were reconstructed from 4 h 00 min to 7 h 00 min in Video S5 in Supplementary Material. The time elapsed after starting the movie is indicated in hours:minutes in the bottom left of each panel. White arrowheads highlight the characteristic behavior of Mφ (IL-10 + IL-18) as well as the cell–cell interaction with endothelium in respective image. Scale bar represents 10 µm.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: Live Cell Imaging, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Ultrastructural analysis of cell–cell interaction between macrophages (Mφs) [interleukin (IL)-10 + IL-18] and endothelia. SEM images were obtained at 4 h after coculture of b.End5 with Mφs (IL-10 + IL-18) on Matrigel. Lower images are magnified regions from red rectangles in the corresponding upper panels. Magnification and scale bars: (A) upper, ×1.0 K, 50 µm; Lower, ×5.0 K, 10 µm; (B) upper, ×1.0 K, 50 µm; lower, ×2.0 K, 20 µm; (C) upper, ×1.0 K, 50 µm; lower, ×8.0 K, 5 µm; (D) upper, ×1.0 K, 50 µm; lower, ×3.0 K, 10 µm; (E) upper, ×1.0 K, 50 µm; lower, ×5.0 K, 10 µm; (F) upper, ×1.0 K, 50 µm; lower, ×4.0 K, 10 µm; (G) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (H) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (I) upper, ×1.0 K, 50 µm; lower, ×3.5 K, 10 µm; respectively. Black or white arrows indicate Mφs or endothelial cells, respectively. Red arrowheads indicate pseudopodia of Mφs interacting with endothelial cells.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia Inhibits Cell Cycle Progression and Cell Proliferation in Brain Microvascular Endothelial Cells via the miR-212-3p/MCM2 Axis

doi: 10.3390/ijms24032788

Figure Lengend Snippet: Functional analysis of differentially expressed genes (DEGs) in bEnd.3 cells under 24 h hypoxia. ( A ) Volcano map of differentially expressed genes between the control and hypoxia conditions. ( B ) Heatmap of the top upregulated and downregulated genes. ( C ) Gene ontology (GO) term analysis of differentially expressed genes. ( D ) Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. ( E ) Top 10 upregulated pathways from gene set enrichment analysis (GSEA). ( F ) Top 10 downregulated pathways from GSEA.

Article Snippet: The mouse brain microvascular endothelial bEnd.3 cell line was obtained from iCell (Shanghai, China).

Techniques: Functional Assay