Journal: The EMBO Journal

Article Title: Long‐term expanding human airway organoids for disease modeling

doi: 10.15252/embj.2018100300

Figure Lengend Snippet: Box‐and‐whisker plot showing concentration‐dependent forskolin‐induced swelling of AOs in the absence and presence of CFTR inhibitors CFTRinh‐172 and GlyH101. Upon CFTR inhibition, swelling is noticeably decreased but not absent. Shown are pooled data from three different AO lines used in each of three independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. AUC, area under the curve. Box‐and‐whisker plot showing concentration‐dependent E act ‐induced swelling of AOs, but not rectal organoids (black outlines). Forskolin causes swelling in both organoid types (gray outlines). Shown are pooled data from three different AO and two different rectal organoid lines used in three to four independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. Swelling was linear for 2 h for AOs, but only 1 h for rectal organoids. See Appendix Fig S3A for respective time course plots. Representative histological sections of periodic acid–Schiff (PAS)‐stained organoids from a CF patient with CFTR F508del/F508del mutation. Note the thick layer of PAS‐positive polysaccharides apically lining the airway epithelium. Rectal organoids were generated from rectal biopsies; AOs were generated from broncho‐alveolar lavages (BALs). Scale bars equal 50 μm. See Appendix Fig S3B for PAS‐stained wild‐type and CFTR R334W/R334W organoid sections. Box‐and‐whisker plot showing swelling assays of several CF patient AO lines carrying the indicated CFTR mutations (G542X is a premature stop associated with severe disease and no functional CFTR protein; F508del is the most common CFTR mutation in subjects with CF and severely reduces apical trafficking and function, leading to severe disease (high sweat chloride, high pancreas insufficiency, high pseudomonas infection rate); R334W is a milder CFTR mutation associated as indicated by lower pseudomonas infection rates and pancreas sufficiency with reduced ion channel conductivity, normal apical expression, and some residual function). Forskolin‐induced swelling rarely exceeds vehicle controls in CF AOs, but increases in the presence of the CFTR modulating drugs VX‐770 and VX‐809. E act ‐induced swelling exceeds forskolin‐induced swelling to a similar extent as pre‐treatment with VX‐770 and VX‐809 in four out of five CF AO lines. In contrast, BAL‐derived WT AOs swell following forskolin stimulation independent of the presence of VX‐770 and VX‐809. Of note, E act ‐induced swelling is not observed in this AO line. Shown are pooled data of four to five independent experiments (CF AOs) or two independent experiments (WT AOs). Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. See Appendix Fig S3C for selected time course plots. Whole‐mounted immunofluorescence analysis of BAL AO‐derived ALI cultures. After 3 weeks of differentiation, ALI cultures show ciliated airway epithelium (green, β‐tubulin). Counterstained are the actin cytoskeleton (red) and nuclei (blue). Transepithelial electrical measurements of BAL AO‐derived ALI cultures. Shown are equivalent current ( I eq ) traces of a CFTR wt/wt (gray) and CFTR F508del/F508del donor, treated with vehicle (blue) or VX‐809 and VX‐770 (red). Inhibition of the epithelial sodium channel (ENaC) with benzamil was observed in all traces (declining I eq ). Forskolin increased the current in CFTR wt/wt and CFTR F508del/F50del donor treated with VX‐809 and VX‐770, but not in the vehicle treated. A decline was observed with CFTRinh‐172 in all examined conditions. The bar graphs quantify the peak forskolin activated‐ and CFTRinh‐172 inhibited‐currents. Shown are pooled data of four independent measurements for each donor. Results are shown as mean ± s.d.

Article Snippet: Calcein green (3 μM, Invitrogen) was added 1 h prior to stimulation with forskolin (Selleck Chemicals), E act (Calbiochem), or DMSO (Sigma) at the indicated concentrations.

Techniques: Whisker Assay, Concentration Assay, Inhibition, Staining, Mutagenesis, Generated, Functional Assay, Infection, Expressing, Derivative Assay, Immunofluorescence

Journal: The EMBO Journal

Article Title: Long‐term expanding human airway organoids for disease modeling

doi: 10.15252/embj.2018100300

Figure Lengend Snippet:

Article Snippet: Calcein green (3 μM, Invitrogen) was added 1 h prior to stimulation with forskolin (Selleck Chemicals), E act (Calbiochem), or DMSO (Sigma) at the indicated concentrations.

Techniques: Recombinant, Sequencing, Red Blood Cell Lysis, Staining, Cell Recovery, Software, Imaging, Inverted Microscopy, Microscopy, Light Microscopy, Viability Assay, Reverse Transcription, Purification, Plasmid Preparation