Morphology and differentiation status of birth and one-year repeat WD-PNEC cultures.

Cultures were monitored by (A) phase-contrast microscopy (magnification x20) or (B) confocal microscopy after staining for β-tubulin (ciliated cell marker) (red), Muc5Ac (goblet cell marker) (green), or nuclei (DAPI) (blue). For (B), square panels represent en face images, whereas rectangular panels represent orthogonal sections, with the apical side at the top (magnification x63, with x1.5 digital zoom, bar = 20 μm). (C) Transwell cultures from birth and 1-year donors (n = 8 each) were fixed and stained for ciliated, goblet and total DAPI+ cells. Images from 5 fields/Transwell were taken at x60 magnification and ciliated, goblet and total DAPI+ cell numbers were counted using fluorescent microscopy. The % ciliated and goblet cells were determined relative to total DAPI+ cell numbers. (D) Representative preterm and term Transwell cultures (newborn term n = 9, preterm n = 10, one-year term n = 9, preterm n = 6 donors) were trypsinised, contents fixed onto slides by smearing or use of cytospin funnels, as described, and stained for ciliated (anti–β-tubulin), goblet (anti-Muc5Ac), and total DAPI+ cells. Ciliated and goblet cell numbers were expressed as mean (±SD) % proportion of total DAPI+ cell numbers for each cohort. *** unpaired t-test, p<0.0001. (E) Transwell cultures were trypsinized and total cell count performed by trypan blue exclusion using a haemocytometer. Total cell numbers are presented as mean (±SD) for newborn (n = 17) and 1-year cohorts (n = 15), p = 0.0087 (unpaired t-test of difference in means). (F) Combined total goblet cell numbers were determined for trypsinised representative Transwell cultures. Data plotted for newborn (n = 17) and 1-year (n = 15) cohorts as mean (±SD) for each culture cohort. ***unpaired t-test of difference in means, p = 0.0005.