Journal: Frontiers in Cell and Developmental Biology

Article Title: Establishment of a novel amyotrophic lateral sclerosis patient ( TARDBP N345K/+ )-derived brain microvascular endothelial cell model reveals defective Wnt/β-catenin signaling: investigating diffusion barrier dysfunction and immune cell interaction

doi: 10.3389/fcell.2024.1357204

Figure Lengend Snippet: ALS patient-derived EECM-BMEC-like cells exhibit tight junction discontinuation. EECM-BMEC-like cells were cultivated onto a 0.4 um pore size Transwell filter for 6 days. Junctions were stained for VE-cadherin, claudin-5, or occludin (red), and nuclei were stained with DAPI (blue). Yellow arrowheads indicate visible discontinuations of the claudin-5 and occludin. Each staining represents results from at least five independent experiments, each performed with five separate differentiation batches. Scale bar = 50 μm.

Article Snippet: In brief, EECM-BMEC-like cells were seeded onto 0.4 μm pore Transwell filter inserts (Corning 3,401, 12 wells 0.4 μm pore size, PC membrane, Corning, United States) with a consistent density of 1.12 × 10 5 cells per 500 μL and cultured for 6 days.

Techniques: Derivative Assay, Pore Size, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Establishment of a novel amyotrophic lateral sclerosis patient ( TARDBP N345K/+ )-derived brain microvascular endothelial cell model reveals defective Wnt/β-catenin signaling: investigating diffusion barrier dysfunction and immune cell interaction

doi: 10.3389/fcell.2024.1357204

Figure Lengend Snippet: EECM-BMEC-like cells derived from ALS patients exhibited impaired diffusion barrier properties. EECM-BMEC-like cells derived from HCs (black), ALS clone (red), or gene-editing clones (orange) were grown on 0.4 μm pore size Transwell filters for 6 days to form a confluent monolayer in monoculture. Permeability was measured on day 6. The permeability was calculated from the fluorescence intensity of 0.37 kDa sodium fluorescein (NaFl) across the EECM-BMEC-like cell monolayers. Each symbol showed the permeability of independent filters with at least three independent differentiations, each performed in at least triplicate. Data were shown as mean ± SD. Statistical analysis: unpaired t -test. P-values are indicated in the corresponding figures.

Article Snippet: In brief, EECM-BMEC-like cells were seeded onto 0.4 μm pore Transwell filter inserts (Corning 3,401, 12 wells 0.4 μm pore size, PC membrane, Corning, United States) with a consistent density of 1.12 × 10 5 cells per 500 μL and cultured for 6 days.

Techniques: Derivative Assay, Diffusion-based Assay, Clone Assay, Pore Size, Permeability, Fluorescence

Journal: Human Molecular Genetics

Article Title: iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration

doi: 10.1093/hmg/dds469

Figure Lengend Snippet: Differentiation of BD and control hiPSCs into functional RPE cells. (A) Photographs and light microscopic images of RPE derived from BD and control hiPSCs plated on transwell inserts showed characteristic RPE morphology and pigmentation (Scale bars: upper panel = 1 mm, lower panel = 100 µm). (B) Transmission electron microscopic images of hiPSC-RPE showed the presence of apical microvilli, intracellular pigment and tight junctions. (C) Apical localization of EZRIN (red) by immunocytochemistry confirmed the proper polarization of hiPSC-RPE (blue: nuclei). (D) Immunocytochemistry for ZO-1 showed correct morphology and tight junction formation in hiPSC-RPE. (E) RT–PCR and (F) western blot analysis revealed the expression of characteristic RPE genes and proteins, respectively. (G) Transepithelial resistance (TER) measurements in hiPSC-RPE were comparable to that of human fetal RPE (hfRPE) (dotted red line demarcates proposed functional threshold) (39). Scale bars: B = 1 µm, C = 5 µm, D = 25µm.

Article Snippet: Large patches of pigmented RPE cells were micro-dissected, dissociated with trypsin-EDTA (0.05%) and plated onto laminin-coated transwell inserts (Corning Costar, 3460-Clear, 0.4 mm pores, 12 mm inner diameter, polyester membranes).

Techniques: Functional Assay, Derivative Assay, Transmission Assay, Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing